Optogenetic effectors and sensors enable the interrogation of complex biological signaling networks at the molecular level in vivo (Akerboom et al., 2013; Fenno et al., 2011; Gengyo-Ando et al., 2017; Kotlikoff, 2007; Tsien, 2003). The power of these molecular tools is optimized in mammalian systems by genetic lineage specification and combinatorial strategies. The former provides the ability to activate or determine the responses of individual cellular components of a complex network, whereas the latter, using spectrally compatible optogenetic tools, enables the dissection of the network by activation of defined elements and simultaneous interrogation of individual system component responses, thereby significantly enhancing the ability to understand functional networks. Although simple in concept, the usefulness of optogenetic tools in mammalian systems requires a parsimonious allelic strategy that minimizes experimental crosses, while supporting robust and consistent sensor/effector expression. For example, while DNA recombination strategies can achieve lineage specificity by crossbreeding Cre recombinase and floxed responder mouse lines (Zariwala et al., 2012), the attendant allelic requirements render this approach impractical for combinatorial experiments using multiple optogenetic tools. Such experiments are facilitated by the design and assembly of sets of individual monoallelic lines that strategically place spectrally distinct optogenetic effectors and sensors in interacting cell lineages, allowing simple crosses to probe cell-cell signaling. Here, we report a marked expansion of the mammalian optogenetic toolbox through the development of multiple mouse lines designed for combinatorial experiments within the cardiovascular system. Interacting vascular, neural, and muscular tissues are targeted through a variety of genetic strategies, expression specificity confirmed, and examples of their use provided.

We report the generation of CHROMus (Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling) mice, 21 lines of optogenetic sensor and effector mice, in which the expression of the transgene is lineage specified (Table 1) and demonstrate the utility of these lines for in vivo and ex vivo imaging. The optogenetic effector proteins we selected for this toolbox were optoα1AR (Airan et al., 2009), optoß2AR (Airan et al., 2009), and CatCh2 (Zhang et al., 2006) for light activation of InsP₃ or cAMP secondary messenger pathways or Ca²⁺ membrane channel, respectively. Each of the open reading frames for the optogenetic proteins were linked to an internal ribosome entry sequence (IRES) element and bacterial LacZ ORF for simple screening. The fluorescent GECI proteins, GCaMP and RCaMP, in reporter lines were derived from circularly permutated EGFP and RFP constructs for in vivo, real-time reporting of cellular Ca²⁺ signaling, as shown in Figure 1—figure supplement 1.

CHROMus mice can be used directly as individual optogenetic effector or fluorescent reporter lines to study real-time response to light activation of the target tissue or to study cellular or subcellular Ca²⁺ signaling in response to various physiological stimuli. However, the system provides the novel opportunity to create novel effector/reporter mouse combinations to study the complex network of physiological signals and responses, as illustrated in Figure 1. Here, activation of the CatCh2 protein expressed in sinoatrial (SA) nodal pacemaker cells triggers cardiac conduction, as monitored by GCaMP8 fluorescence in ventricular myocytes. Other combinatorial preparations from the CHROMus toolbox enable the evaluation of cellular function in mouse lines that simulate human disease. We have constructed 11 mouse lines expressing various optogenetic effector proteins in seven different lineages and 10 expressing fluorescent GECI reporter proteins in seven lineages across a wide range of tissues, organs, and systems (Table 1).

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Cardiac conduction system optogenetic mice

The potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 protein (HCN4) underlies the pacemaker current in the SA node, controlling heart rate and heart rhythm (DiFrancesco, 1993; Moosmang et al., 2001). For light-activated control of heart rhythm, we constructed a Hcn4^BAC-CatCh2_IRES_lacZ mouse line, where the expression of the optoeffector CatCh2 and a marker protein (LacZ) are under transcriptional control of the Hcn4 locus in a BAC (Table 1), thus directing expression to the cells of the cardiac conductance system. X-gal staining of adult Hcn4^BAC-CatCh2_IRES_LacZ mice demonstrated specific staining in cells of the cardiac conductance system, including the SA node region in the right atrium, the atrioventricular junction, and the ventricular conducting network (Figure 2A). LacZ-expressing cells were observed at the base of the right carotid vein where it joins the right atrium (Figure 2—figure supplement 1A) and along the right carotid vein to the base of the common carotid vein (Figure 2—figure supplement 1B). Expression was also documented in embryonic (E10.5) and neonatal (PN4) hearts in the atrium and the ventricular conducting system (Figure 2B).

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To confirm optical control of heart rhythm, we stimulated the SA node area with 473 nm light in vivo while recording the ECG. As shown in Figure 2C, cardiac conduction was driven with stimulation between 2 Hz and 4 Hz, with dropouts at higher frequencies. Heart rhythm returned to normal following optical stimulation (Figure 2—figure supplement 1C), and illumination of off-target tissue (left ventricle) did not result in optical rhythm control (Figure 2—figure supplement 1D). Pacing was maintained with laser pulse lengths varying between 15 and 70 ms (data not shown). The increase in laser-stimulated R-wave duration is shown as a widening of the R-wave in the R-wave-centered average of ECG for stimulated and unstimulated beats and the extended PR interval in the P-wave-centered average of ECG sections (Figure 2D and F). ECG parameters indicated that R-wave duration and PR interval increased significantly, relative to the autonomously activated cardiac cycle (Figure 2E and G), likely indicating a more peripheral activation of SA nodal conducting tissue.

To demonstrate the feasibility of combining optogenetic effector and sensor lines, we crossed Hcn4^BAC-CatCh2_IRES_LacZ and Myh6-GCaMP8 mice. Pacing by laser stimulation resulted in coupling between optical stimulation and GCaMP8 fluorescence, as shown in Figure 2H and Figure 2—video 1. Differences in effector and sensor kinetics must be considered at high stimulation rates, as previously reported (Tallini et al., 2006a). Hcn4^BAC-CatCh2 mice could also be mated to Acta2^BAC-GCaMP_GR (Figure 4) and Cdh5^BAC-GCaMP8 lines (Figure 6) to study smooth muscle or endothelial cell responses to the cardiac pacing. GCaMP_GR (for green/red) is a ratiometric fusion protein consisting of an enhanced GCaMP sequence fused to mCherry (Shui et al., 2014).

For the study of the development of the cardiac conduction system and disturbances of conduction in disease states, we developed a Hcn4^BAC-GCaMP8 mouse line. GCaMP8 is expressed in the sinus venosus (Figure 2I, left panels) of the embryonic heart and in the SA nodal area at the junction of right atrium and right carotid vein in the adult heart, extending down to the base of the common carotid artery (Figure 2I, right panel), similar to the expression pattern in Hcn4^BAC-CatCh2 mice (Figure 2—figure supplement 1B). In developing embryos, spontaneous GCaMP8 signaling was observed in the heart at embryonic day (E) 8.5, E10.5, and E13.5, as well as at postnatal day (PN) 4 (Figure 2—figure supplement 2A). Conduction of the GCaMP8 fluorescence signal was observed moving from the atrium to the ventricle in the E13.5 embryo (Figure 2—video 2). In the adult heart, GCaMP8 signaling was observed prior to spontaneous ventricular contractions (Figure 2—figure supplement 2B, Figure 2—video 3).

Cardiac myocyte optogenetic mice

We constructed two optogenetic lines designed to study Ca²⁺ signaling and electrical communication between heart cells using a minimal promoter for MYH6 protein (Gulick et al., 1991). In order to optically initiate cardiac dysrhythmias at virtually any surface region of the heart, we developed the Myh6-CatCh2_IRES_lacZ strain, which expresses the CatCh2 and LacZ transgenes in cardiomyocytes. X-gal-stained cryosections of the heart showed widespread staining of cardiomyocytes throughout the heart (Figure 3A). Laser irradiation (473 nm) of the left ventricle enabled the stimulation of ventricular premature activations at lower frequencies and complete ventricular pacing at 8 Hz; above 8 Hz, pacing was lost but irregular ventricular initiation could be produced, consistent with the refractory period of ventricular myocytes (Figure 3B). As expected, laser stimulation of the left ventricle from a control wildtype mouse did not result in pacing (Figure 3—figure supplement 1). As predicted for ventricular activation, the ECG showed markedly enhanced QRS duration without corresponding P-waves, as visualized in an R-wave-centered average of ECG sections for stimulated and unstimulated beats in three mice (Figure 3C). Differences in ECG morphology among animals at baseline, as well as unsuccessful stimulation for some beats, account for high variability and abnormal morphology in the averaged trace. The mean R-wave duration significantly increased upon laser stimulation compared to the native unstimulated heartbeat in all three mice examined (Figure 3D). Higher magnification ECG traces of autonomous heart activation following ventricular laser stimulation show typical widened QRS complexes (red) followed by return to normal heart activation (Figure 3E and F).

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We also created the Myh6-GCaMP8 line to enable visualization of in vivo, real-time, cardiac Ca²⁺ responses under different experimental stimuli and disease conditions. Cryosections from the heart revealed widespread GCaMP8 fluorescence and immunoreactivity in hearts from this line (Figure 3G). To obtain high-resolution intravital images of a beating heart, we injected Texas Red-conjugated dextran retro-orbitally to label the vasculature and acquired two-channel, two-photon images of the left ventricle (Figure 3H). GCaMP8 Ca²⁺ fluorescence was observed in cardiomyocytes in the stabilized ventricular free wall during the normal cardiac cycle, coincident with heart contractions. The vascular dextran fluorescence signal reports only motion-related fluorescence changes (Figure 3I, Figure 3—video 1).

Smooth muscle optogenetic mice

We constructed optogenetic effector and sensor BAC transgenic mice using the α-Actin2 gene locus, which is selectively expressed in smooth muscle (Mack and Owens, 1999). Sensor lines expressing GCaMP_GR protein (modified GCaMP3 fused to mCherry; Shui et al., 2014) or RCaMP1.07 (Ohkura et al., 2012a) fluorescent Ca² reporters and effector lines expressing the optoα1AR (Airan et al., 2009), optoß2AR (Airan et al., 2009), or CatCh2 (Zhang et al., 2006) proteins were developed under smooth muscle-specific transcriptional control.

We engineered three recombinant BAC constructs to drive expression of red or green-red sensors in smooth muscle cells (Figure 1—figure supplement 1). The Acta2^BAC-RCaMP1.07 (Bethge et al., 2017) line expresses the sensor selectively in arterial smooth muscle (Figure 4—figure supplement 1A, upper panels). In vivo imaging revealed cyclic fluorescence increases associated with contractions of the right atrium (Figure 4—figure supplement 1A, lower panel; Figure 4—video 1). When used with optoeffector mice, the line eliminates spectral overlap with excitation of GFP-based sensors.

Acta2^BAC-GCaMP_GR mice provide the advantage of ratiometric Ca²⁺ determination, as well as ease of expression determination in vivo. As shown in brain surface vessel images (Figure 4A, upper panel), the GCaMP_GR fusion protein is strongly expressed in the arterial system extending to the arteriolar-capillary junctional cells, but not in the capillary network. To further enhance the intensity of red fluorescence, we created a second dual-color sensor line using the same transcriptional strategy, but substituting mVermilion, a novel monomeric mCherry variant with a twofold increase in brightness but equivalent excitation and emission profiles. Acta2^BAC-GCaMP8.1_mVermilion mice displayed a similar expression pattern (Figure 4A, lower panel). In vivo experiments with these lines indicated robust smooth muscle Ca²⁺-dependent fluorescence. Contractions of blood vessels, airways, and gastrointestinal organs demonstrated increases in green fluorescence during muscle contraction. As shown in Figure 4—figure supplement 1B and Figure 4—video 2, in vivo fluorescent imaging of spontaneous contractions in the large intestine showed a wave of GCaMP signaling in the longitudinal muscles that was coupled to contractions, whereas mCherry fluorescence remained constant (data not shown).

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We constructed three effector lines for smooth muscle optical activation using the Acta2 BAC (RP23-370F21; Table 1) and examined the expression of the LacZ protein located downstream of the optogenetic effector coding sequence in different tissues from Acta2^BAC-Optoα1AR_IRES_lacZ, Acta2^BAC-Optoß2AR_IRES_lacZ and Acta2^BAC-CatCh2_IRES_lacZ mice by X-gal staining. The staining data show widespread expression of all three constructs in the brain arterial system (Figure 4B), including the arteriolar/capillary junction. LacZ activity was also observed in arteries supplying the diaphragm, smooth muscles surrounding coronary blood vessels, the bladder, bronchiolar and vascular smooth muscles in lung, and other tissues such as the small intestines, kidney. and uterus (data not shown). X-gal staining of a non-transgenic littermate did not detect LacZ activity (Figure 4—figure supplement 1C).

As receptor-mediated activation of phospholipase C or adenylyl cyclase (with associated increases in InsP₃ or cAMP) mediates smooth muscle contraction and relaxation, respectively, we examined constriction or dilation of isolated cerebral and mesenteric arteries in response to optical activation of the chimeric Optoα1AR and Optoß2AR proteins. Brief exposure of arteries isolated from Acta2^BAC-Optoα1AR_IRES_lacZ mice to 405 nm light resulted in vasoconstriction (Figure 5A, left panel), while light-activated arteries from Acta2^BAC-Optoß2AR_IRES_lacZ lines underwent slow dilations (Figure 5A, middle panel; Figure 5—video 1), demonstrating functional expression of the proteins and expected physiological responses. The activation of channelrhodopsin in arteries isolated from Acta2^BAC-CatCh2_IRES_lacZ mouse resulted in rapid constriction (Figure 5A, right panel; Figure 5—video 2). Laser stimulation of arteries from control wildtype mice did not result in vasodilation or vaso constriction similar to the transgenic arteries (Figure 5—figure supplement 1). Optoeffector coupling was demonstrated at a cellular level in whole-cell, voltage-clamped arterial myocytes. We first examined coupling through the known linkage between cAMP/protein kinase A signaling and voltage-gated potassium channels in arterial myocytes (Johnson et al., 2009; Luykenaar and Welsh, 2007; Nelson and Quayle, 1995; Wellman et al., 2001). Macroscopic I_K currents from single voltage-clamped myocytes isolated from male mesenteric and mid-cerebral arteries were recorded before and after photoactivation of Optoß2AR. I_K (composed of Kv1, Kv2.1, and BK currents) was activated at 10 s intervals by the application of a 500 ms step depolarization from the holding potential of –70 mV to +60 mV. Activation of the Optoß2AR protein by 488 nm light increased the amplitude of I_K 10 s after the exposure, reaching a higher, relatively stable plateau approximately 80 s after photoactivation (Figure 5B). Although the dispersion of I_K amplitudes increased about 150 s after activation of Optoß2AR, I_K remained higher than under control conditions for at least 500 s. Exposure to a second and third pulse of light had limited additional effects, suggesting that the initial Optoß2AR-induced potentiation of I_K was near maximal. In five similar experiments in mesenteric myocytes, Optoß2AR photoactivation increased I_K 1.15 ± 0.04 fold at +60 mV, and 1.23 ± 0.05 fold in cerebral arterial myocytes.

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Similarly, CatCh2 optical activation was examined in cerebral and mesenteric arteries by recording membrane currents at –70 mV before and after illumination (488 nm, 50 ms pulses at 1 Hz). As shown in Figure 5C, optical stimulation repeatedly evoked fast inward currents that terminated as soon as the laser was turned off. In the representative cerebral myocyte shown, the amplitude of the evoked current was about 75 pA, and subsequent light pulses evoked stable I_Cat with an amplitude of 68 ± 1 pA, suggesting that under our experimental conditions there is a relatively small level of inactivation of CatCh2 channels (Figure 5C, upper panel). Longer light pulses produced a larger inward current (∼125 pA) that quickly (∼100 ms) decayed to a lower amplitude (∼35 pA) plateau, remaining relatively constant as long as the laser was turned on. This I_Cat quickly terminated (<100 ms) after the laser was turned off. Notably, a second 488 nm light pulse to the same cell evoked an I_Cat that lacked the high-amplitude transient component of the first light pulse but had the amplitude and kinetics of the steady-state I_Cat in the first pulse. Similar observations were made in isolated mesenteric myocytes (Figure 5B, middle panels). The activation of cerebral myocytes resulted in higher currents compared to mesenteric arteries and repeated pulses resulted in dampened initial inward current (Figure 5B, bottom panels). Thus, Acta2 optoeffector lines express functional chimeric receptors and channel proteins, enabling optical modulation of smooth muscle signaling in a variety of experimental conditions.

Endothelial optogenetic mice

To enable studies of the interactions between endothelium and surrounding perivascular cells, we created three BAC transgenic optogenetic effector and one fluorescent reporter mouse lines using the endothelial cell-specific Cdh5 gene locus. As with smooth muscle, Optoα1AR, Optoß2AR, and CatCh2 cDNAs and LacZ ORF were targeted to the Cdh5 initiation codon, with an IRES and the LacZ sequence downstream of the optoeffectors. X-gal staining revealed wide expression in endothelial cells of the heart (Figure 6A), brain (Figure 6—figure supplement 1A), and other tissues (Figure 6—figure supplement 1B). Similarly, GCaMP8-selective expression in endothelial cells was achieved in Cdh5^BAC-GCaMP8 mice (Figure 6B, Figure 6—figure supplement 1C). The endothelial GCaMP8 signal was sufficiently bright to be observed in coronary microvasculature using intravital two-photon microscopy (Figure 6C).

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To confirm functional expression of optoeffector proteins, we deployed several analyses. First, we examined endothelium-induced vasodilation that occurs secondary to the release of Ca²⁺ and synthesis of NO in EC (Straub et al., 2014). Optical activation of femoral arteries in Cdh5^BAC-Optoα1AR_IRES_lacZ mice in vivo resulted in marked vasodilation, whereas laser stimulation alone caused a slight vasoconstriction in control arteries (Figure 7A). Similar results were obtained in cremaster arteries, but not in control arteries (Figure 7—figure supplement 1). We also examined the propagation of arterial dilation that occurs following muscarinic stimulation (Tallini et al., 2007) and is difficult to separate from agonist diffusion (Welsh and Segal, 1998). Laser stimulation triggered bidirectional arterial dilation at points distal to the laser beam, with a delay in the onset of vasodilation and attenuation of the amplitude (Figure 7B). Vasodilation was triggered at 32.2 mW, but not at lower laser powers (Figure 7C). Photoactivation of chimeric ß2 adrenergic receptors in Optoß2AR mice also triggered vasodilation (Figure 7D). Interestingly, ß2AR-mediated dilation did not result in conducted dilation distal to the point of laser stimulation as observed in Optoα1AR mice.

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Other optogenetic lines

The gap junction protein connexin40 (Gja5) is expressed in specialized cells of the cardiac conduction system and in arterial endothelial cells, and expression in embryonic myocytes is critical for heart function prior to development of a mature conduction system (Delorme et al., 1995). To facilitate improved studies of embryonic and adult cardiac conduction defects, as well as physiological vasomotor control, we created a Gja5^BAC-GCaMP_GR mouse line. The ratiometric green-red fusion protein is expressed in arterial endothelia from the kidney, the ventricular conductance system and atrial myocytes (Figure 8A), and in arterial endothelial cells (Figure 8—figure supplement 1A). To extend studies to the sympathetic nervous system, we used the dopamine ß-hydroxylase locus to target optogenetic effectors to peripheral neurons and adrenal medulla cells (reviewed in Gonzalez-Lopez and Vrana, 2020). X-gal staining of adult brain and adrenal glands of Dbh^BAC-CatCh2_IRES_lacZ demonstrated the expression of LacZ in the locus coeruleus (Figure 8B) and adrenal medulla (Figure 8—figure supplement 1B). BAC DNA from the aldehyde dehydrogenase family 1 L1 (Aldh1l1) gene locus was similarly used to target glial cells. Aldh1l1^BAC-Optoα1AR_IRES_lacZ mouse express in cerebellar (Figure 8C) and olfactory regions in the brain and the kidney (Figure 8—figure supplement 1C). The expression of Optoα1AR protein in immune cells was targeted using a BAC containing the Lck gene to facilitate studies on InsP₃ activation in T-lymphocytes. X-gal staining for LacZ expression indicates optoeffector expression in the spleen (Figure 8D). Mice expressing GCaMP8.1 protein in Foxj1⁺ cells were developed using BAC RP23-294J17 (Table 1). Immunostaining analysis with anti-GFP antibody indicated GCaMP8.1 expression in the bronchioles, trachea, and the testis (Figure 8E). We constructed the Sftpc^BAC-GCaMP8 mouse to target reporter expression in type II alveolar cells. Anti-GFP antibody analysis of lung sections indicated wide distribution of GCaMP8 expression (Figure 8—figure supplement 1D).

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We have generated 21 lines of optogenetic sensor and effector mice, in which the expression of the optogene is lineage specified in cardiovascular and related cell lineages (Table 1), and sensor/effector pairs can be combined through simple, monoallelic crosses. More broadly, the lines facilitate the combination of two technologies, each with enormous potential for use in the dissection of signaling processes in complex multicellular systems: genetically encoded optical sensors (Kotlikoff, 2007; Nakai and Ohkura, 2003; Tsien, 2003) and effectors (Fenno et al., 2011). Individually, these technologies have advanced our understanding of physiological in vivo processes; combined, biallelic effector/reporter mice constitute a novel platform for the activation and detection of cellular signaling in vivo. These new mouse lines comprise a publicly available molecular toolbox that enables investigators to generate physiologically relevant mono- or multiallelic transgenic mice tailored to their research interests, without the need for requisite technical expertise and financial commitment associated with constructing transgenic mice. The monoallelic nature of these lines also facilitates crosses with mouse models of cardiovascular disease. All mice are available as part of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) and The Jackson Laboratory.

Previous approaches have developed, or exploited, available Cre recombinase driver mice to confer tissue-specific expression of fluorescent markers or optogenetic sensors downstream of LoxP-STOP-LoxP sequences (Madisen et al., 2010; Zariwala et al., 2012). While this genetic strategy has the advantage of using previously characterized Cre lines and strong promoter sequences to drive optogenetic protein expression, there are several important drawbacks to this approach. These include variable penetration of the Cre-mediated recombination at LoxP sites within the target cells, limited specificity of non-BAC Cre promoters (Madisen et al., 2010), and the inefficient process of producing biallelic mice for experiments. The latter drawback excludes, for practical purposes, one of the most important advantages of the use of optogenetic mice – crosses with genetic strains or mutant lines that mimic human disease. The use of Cre/Lox approaches for such experiments requires complicated and inefficient breeding strategies. An additional drawback of a Cre/Lox approach relates to the requirement for constant, high transcriptional throughput in order to achieve sufficient optical sensitivity and observable fluorescent intensity. Reported optogenetic responder lines are often driven by non-specific minimal promoters and may not have sufficient transcriptional strength in all lineages sufficient for real-time optical experiments. The strains reported here provide direct, high-level tissue-specific expression of optogenetic proteins and enable simple mating strategies for the development of biallelic mice. Each line has been analyzed for expression and lines with off-target or low levels of expression have been eliminated, and each line has been bred for multiple generations.

On the other hand, the lines reported here result from the random integration of large DNA elements into the mouse genome in a stable manner, whereas many (but not all) Cre-driver and LoxP flanked responder lines result from targeted integration within a known locus. Thus a drawback of CHROMus lines is the unknown site of integration, which could result in integration site-dependent effects on the transcription of other genes, or impact the transcriptional fidelity of the inserted optogenetic cDNA due to positional effects. While we have verified the insertion of BAC or cDNA sequences in these lines, and confirmed the viable breeding of the reported lines, these effects cannot be completely known and thus the lines should be used where they provide functional value, but may not be useful in all circumstances. One example is the Sftpc^BAC-GCaMP8 line, which expresses the GCaMP8 protein in type II alveolar cells, but a subset of type II cells did not express the reporter protein. Also, we noted a lack of X-gal staining in the central nervous system of Hcn4^BAC-CatCh mice, but this expression pattern has been previously reported for HCN4 mRNA and protein (Kouranova et al., 2008). Finally, we note that laser stimulation of the left ventricle in the Hcn4^BAC-CatCh mouse failed to induce pacing.

Separate targeting of cardiac conducting cells and myocytes enabled light-activated heart activation, and we demonstrate activation of the heart either through stimulation of cells within the SA node (Figure 2) or extra-conduction system activation equivalent to premature ventricular contractions (Figure 3). By crossing nodal optoeffector (Hcn4^BAC-CatCh2_IRES_LacZ) and myocyte sensor (Myh6-GCaMP8 strains) mice, we demonstrate the feasibility of combinatorial optogenetics and report the ability to activate and detect heart signaling (Figure 2H, Figure 2—video 2). We also demonstrate the ability to selectively examine cardiac conduction using Hcn4^BAC-GCaMP8 mice and their robust signaling of conduction events in embryonic, neonatal, and adult hearts (Figure 2, Figure 2—figure supplement 2). The cardiac GCaMP8 sensor lines represent several improvements as they exploit recent structure-based improvements in circular permutation of GFP (Ohkura et al., 2012b; Tian et al., 2009), resulting in higher brightness and dynamic range. Conduction failures result in the implantation of more than 200,000 pacemakers annually in the United States alone (Mond and Proclemer, 2011), and the ability to selectively initiate cardiac conduction at local sites within the heart (optoeffector lines), either through activation of specialized conducting cells or myocytes, and to monitor activation of the conduction system (optosensor lines), confers significant experimental advantages both in understanding conduction system failure and strategies to develop biological pacemakers (Meyers et al., 2016; Nyns et al., 2019; Vedantham, 2015). Similar approaches can be envisioned for studies of heart failure, a major cause of mortality worldwide (Bui et al., 2011). Two areas of specific value would be the understanding of cardiomyopathy-induced arrhythmias (Asimaki et al., 2014) and cell and gene-based approaches to therapy (Nakamura and Murry, 2019).

The development of mouse lines with robust expression of optical effectors and sensors in smooth muscle and endothelial cells provides similar experimental advantages for vascular biology. We report the first mice conferring lineage-specific expression of sensors that allow light-activated depolarization through CatCh2 channels or release of intracellular Ca²⁺ through Optoα1AR, chimeric α-adrenoceptors, as well as relaxation of smooth muscle through photoactivation of Optoß2AR, chimeric ß2 receptors. Interestingly, we observed rhythmic endothelial cell signaling during the cardiac cycle, enabling dissection of mechanisms that regulate blood flow, matching perfusion to metabolic demand (Duncker and Bache, 2008). Similar approaches have led to important advances in the understanding of metabolic vascular coupling in the brain and other arterioles (Gonzales et al., 2020; Sonkusare et al., 2012; Sonkusare et al., 2014), but studies in the heart face additional imaging challenges associated with imaging the beating heart. We used stabilized optical windows and deconvolution approaches to address these challenges (Figures 2 and 3). The creation of lines that enable ratiometric measurements (Acta2^BAC-GCaMP_GR and Acta2^BAC-GCaMP8.1_Vermilion; Figure 4) adds additional experimental advantages in this regard. Similarly, the ability to initiate synthesis of critical smooth muscle second messengers through the expression of light-sensitive chimeric receptor proteins fused to catalytic domains of α1 and ß2 adrenoceptors (Airan et al., 2009) provides important capabilities to probe vascular control mechanisms in smooth muscle tissues, and the ability to control the synthesis in an individual cell type.

We report additional lines that enable studies of sympathetic neurons, glial cells, and type II alveolar cells. These lines show robust, tissue-specific expression of the optoeffector or optosensor proteins, enabling similar studies of neurovascular and lung cell signaling and provide important advantages for the study of neurosecretion, neuromodulation, and excitation/secretion coupling.

Finally, we anticipate that CHROMus mice, either as effector/detector pairs or individually, will have great utility when crossed with existing mouse models of human disease. Such models exist for many cardiovascular, neurovascular, airway, and immune diseases, and the mouse lines reported here provide a direct way to apply powerful optogenetic techniques to the study of pathophysiology and the assessment of treatment.

In summary, BAC transgenic optogenetic effector and sensor lines provide novel experimental platforms for real-time in vivo investigations of molecular signaling in complex physiological systems. The mice are available to experimenters through JAX and represent a tool kit that can advance the use of optogenetics in a wide array of experimental investigations.

Information on all mouse lines and the CHROMus project can be found at https://chromus.vet.cornell.edu. CHROMus mice are available from the Jackson Laboratory or IMSR.

The mouse strains have been deposited with Jackson Laboratory. The source data files for Figures 2, 3, 5, 7 and Figure 7 - figure supplement have been provided.

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Author details

1. Frank K Lee

   Department of Biomedical Sciences, Cornell University, Ithaca, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Jane C Lee

   Department of Biomedical Sciences, Cornell University, Ithaca, United States

   Contribution

   Supervision, Data curation, Conceptualization, Investigation, Methodology, Project administration, Funding acquisition, Resources, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Bo Shui

   Department of Biomedical Sciences, Cornell University, Ithaca, United States

   Contribution

   Methodology, Funding acquisition

   Competing interests

   No competing interests declared

4. Shaun Reining

   Department of Biomedical Sciences, Cornell University, Ithaca, United States

   Contribution

   Funding acquisition

   Competing interests

   No competing interests declared

5. Megan Jibilian

   Department of Biomedical Sciences, Cornell University, Ithaca, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. David M Small

   Department of Biomedical Engineering, Cornell University, Ithaca, United States

   Contribution

   Data curation, Formal analysis, Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

7. Jason S Jones

   Department of Biomedical Engineering, Cornell University, Ithaca, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

8. Nathaniel H Allan-Rahill

   Department of Biomedical Engineering, Cornell University, Ithaca, United States

   Contribution

   Formal analysis, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8090-8339

9. Michael RE Lamont

   Department of Biomedical Engineering, Cornell University, Ithaca, United States

   Contribution

   Formal analysis, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

10. Megan A Rizzo

    Department of Physiology, University of Maryland School of Medicine, Baltimore, United States

    Contribution

    Conceptualization, Funding acquisition, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6528-7768

11. Sendoa Tajada

    Departments of Physiology and Membrane Biology, University of California, Davis School of Medicine, Davis, United States

    Contribution

    Data curation, Investigation, Methodology, Validation, Visualization, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5138-9808

12. Manuel F Navedo

    Department of Pharmacology, University of California, Davis, Davis, United States

    Contribution

    Data curation, Formal analysis, Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6864-6594

13. Luis Fernando Santana

    Departments of Physiology and Membrane Biology, University of California, Davis School of Medicine, Davis, United States

    Contribution

    Supervision, Data curation, Formal analysis, Conceptualization, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4297-8029

14. Nozomi Nishimura

    Department of Biomedical Engineering, Cornell University, Ithaca, United States

    Contribution

    Supervision, Conceptualization, Resources, Visualization, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4342-9416

15. Michael I Kotlikoff

    Department of Biomedical Sciences, Cornell University, Ithaca, United States

    Contribution

    Supervision, Data curation, Conceptualization, Methodology, Project administration, Resources, Validation, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    mik7@cornell.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9879-1899

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