Toward a brighter constellation: multiorgan neuroimaging of neural and vascular dynamics in the spinal cord and brain.

Significance: Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. Aim: We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. Approach: We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). Results: We describe "universal" implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the "BLmini," which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. Conclusions: The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord-brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

Efficient opto- and chemogenetic control in a single molecule driven by FRET-modified bioluminescence.

Significance: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions: We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

A New Highly Efficient Molecule for Both Optogenetic and Chemogenetic Control Driven by FRET Amplification of BioLuminescence.

SIGNIFICANCE: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AIM: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. APPROACH: We developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). RESULTS: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. CONCLUSIONS: We report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

Bicistronic Expression of a High-Performance Calcium Indicator and Opsin for All-Optical Stimulation and Imaging at Cellular Resolution.

State-of-the-art all-optical systems promise unprecedented access to neural activity in vivo, using multiphoton optogenetics to allow simultaneous imaging and control of activity in selected neurons at cellular resolution. However, to achieve wide use of all-optical stimulation and imaging, simple strategies are needed to robustly and stably express opsins and indicators in the same cells. Here, we describe a bicistronic adeno-associated virus (AAV) that expresses both the fast and bright calcium indicator jGCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, stChrimsonR stimulation with two-photon holography in the visual cortex of mice drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and stable. Cells expressing this bicistronic construct show responses to both photostimulation and visual stimulation that are similar to responses measured from cells expressing the same opsin and indicator via separate viruses. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging.

Towards a Brighter Constellation: Multi-Organ Neuroimaging of Neural and Vascular Dynamics in the Spinal Cord and Brain.

Significance: Pain is comprised of a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. Aim: Here, we aimed to develop and validate new tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations were targeted to developing novel imaging hardware that addresses the many challenges of multi-site imaging. The second key set of innovations were targeted to enabling bioluminescent imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity and decreased resolution due to scattering of excitation signals. Approach: We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for bioluminescent imaging, and developed a novel modified miniscope optimized for these signals (BLmini). Results: Here, we describe novel 'universal' implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of bioluminescent signals in both foci, and a new miniscope, the 'BLmini,' which has reduced weight, cost and form-factor relative to standard wearable miniscopes. Conclusions: The combination of 3D printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a new coalition of methods for understanding spinal cord-brain interactions. This work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

Selective control of synaptically-connected circuit elements by all-optical synapses.

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control in vivo.

Miniaturized Devices for Bioluminescence Imaging in Freely Behaving Animals.

In vivo fluorescence miniature microscopy has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non- homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescence provides an alternative kind of activity-dependent light indicator. Bioluminescent calcium indicators do not require light input, instead generating photons through chemiluminescence. As such, limitations inherent to the requirement for light presentation are eliminated. Further, bioluminescent indicators also do not require excitation light optics: the removal of these components should make a lighter and lower cost microscope with fewer assembly parts. While there has been significant recent progress in making brighter and faster bioluminescence indicators, the advances in imaging hardware have not yet been realized. A hardware challenge is that despite potentially higher signal-to-noise of bioluminescence, the signal strength is lower than that of fluorescence. An open question we address in this report is whether fluorescent miniature microscopes can be rendered sensitive enough to detect bioluminescence. We demonstrate this possibility in vitro and in vivo by implementing optimizations of the UCLA fluorescent miniscope v3.2. These optimizations yielded a miniscope (BLmini) which is 22% lighter in weight, has 45% fewer components, is up to 58% less expensive, offers up to 15 times stronger signal and is sensitive enough to capture spatiotemporal dynamics of bioluminescence in the brain with a signal-to-noise ratio of 34 dB.

The BioLuminescent-OptoGenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex.

BioLuminescent (BL) light production can modulate neural activity and behavior through co-expressed OptoGenetic (OG) elements, an approach termed "BL-OG." Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and nontoxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically nonsensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

Ocular Component Development during Infancy and Early Childhood.

SIGNIFICANCE: The study fills an important gap by providing a longitudinal description of development of the major structural and optical components of the human eye from 3 months to nearly 7 years of age. Normative development data may provide insights into mechanisms for emmetropization and guidance on intraocular lens power calculation. PURPOSE: The purpose of this study was to describe the pattern of development of refractive error and the ocular components from infancy through early childhood. METHODS: Cycloplegic retinoscopy (cyclopentolate 1%), keratophakometry, and ultrasonography were performed longitudinally on between 162 and 293 normal birth weight infants at 0.25, 0.75, 1.5, 3, 4.5, and 6.5 years of age. RESULTS: Refractive error and most ocular components displayed an early exponential phase of rapid development during the first 1 to 2 years of life followed by a slower quadratic phase. Anterior and vitreous chamber depths, axial length, and crystalline lens radii increased at every visit. The crystalline lens thinned throughout the ages studied. The power of the cornea showed an early decrease, then stabilized, whereas the crystalline lens showed more robust decreases in power. The crystalline lens refractive index followed a polynomial growth and decay model, with an early increase followed by a decrease starting at 1 to 2 years of age. Refractive error became less hyperopic and then was relatively stable after 1 to 2 years of age. Axial lengths increased by 3.35 ± 0.64 mm between ages 0.25 and 6.5 years, showed uniform rates of growth across the range of initial values, and were correlated with initial axial lengths (r = 0.44, P < .001). CONCLUSIONS: Early ocular optical and structural development appears to be biphasic, with emmetropization occurring within the first 2 years of infancy during a rapid exponential phase. A more stable refractive error follows during a slower quadratic phase of growth when axial elongation is compensated primarily by changes in crystalline lens power.

Accommodation, acuity, and their relationship to emmetropization in infants.

PURPOSE: To evaluate the relationship between accommodation, visual acuity, and emmetropization in human infancy. METHODS: Defocus at distance and near (57 cm) was assessed using Mohindra and dynamic retinoscopy, respectively, in 262 normal birthweight infants at 3, 9, and 18 months of age. Preferential looking provided acuity data at the same ages. The spherical equivalent refractive error was measured by cycloplegic retinoscopy (cyclopentolate 1%). RESULTS: Univariate linear regression analyses showed no associations between the change in refractive error and defocus at distance or near. Change in refractive error was linearly related to the accommodative response at distance (R = 0.17, p < 0.0001) and near (R = 0.13, p < 0.0001). The ten subjects with the poorest emmetropization relative to the change predicted by the linear effects of their refractive error had higher average levels of hyperopic defocus at distance and near (p < 0.043). Logistic regression showed a decrease in the odds of reaching +2.00 diopter or less hyperopia by 18 months with increasing levels of hyperopia at 3 months, or if Mohindra retinoscopy was myopic combined with acuity better than the median level of 1.25 logMAR [area under the receiver operating characteristic curve = 0.78 (95% CI = 0.68 to 0.88)]. CONCLUSIONS: The level of cycloplegic refractive error was the best single factor for predicting emmetropization by 18 months of age, with smaller contributions from visual acuity and Mohindra retinoscopy. The lack of correlation between defocus and change in refractive error does not support a simple model of emmetropization in response to the level of hyperopic defocus. Infants were capable of maintaining accurate average levels of accommodation across a range of moderate hyperopic refractive errors at 3 months of age. The association between the change in refractive error and accommodative response suggests that accommodation is a plausible visual signal for emmetropization.

Axial growth and changes in lenticular and corneal power during emmetropization in infants.

PURPOSE: To evaluate the contribution made by the ocular components to the emmetropization of spherical equivalent refractive error in human infants between 3 and 9 months of age. METHODS: Keratophakometry in two meridians was performed on 222 normal-birthweight infant subjects at 3 and 9 months of age. The spherical equivalent refractive error was measured by cycloplegic retinoscopy (cyclopentolate 1%). Anterior chamber depth, lens thickness, and vitreous chamber depth were measured by A-scan ultrasonography over the closed eyelid. RESULTS: Both the mean and SD for spherical equivalent refractive error decreased between 3 and 9 months of age (+2.16 +/- 1.30 D at 3 months; +1.36 +/- 1.06 D at 9 months; P < 0.0001, for the change in both mean and SD). Average ocular component change was characterized by increases in axial length, thinning, and flattening of the crystalline lens, increases in lens equivalent refractive index, and decreases in lens and corneal power. Initial refractive error was associated in a nonlinear manner with the change in refractive error (R(2) = 0.41; P < 0.0001) and with axial growth (R(2) = 0.082; P = 0.0005). Reduction in hyperopia correlated significantly with increases in axial length (R(2) = 0.16; P < 0.0001), but not with changes in corneal and lenticular power. Decreases in lenticular and corneal power were associated with axial elongation (R(2) = 0.40, R(2) = 0.12, respectively; both P < 0.0001). CONCLUSIONS: Modulation in the amount of axial growth in relation to initial refractive error appeared to be the most influential factor in emmetropization of spherical equivalent refractive error. The associations between initial refractive error, subsequent axial growth, and change in refractive error were consistent with a visual basis for emmetropization. The cornea and crystalline lens lost substantial amounts of dioptric power in this phase of growth, but neither appeared to play a significant role in emmetropization.

Repeatability of corneal topography measurement in keratoconus with the TMS-1.

PURPOSE: The purpose of this study was to report the test-retest variability of simulated indices derived from the TMS-1 topography instrument (Tomey Technology, Waltham, MA) in keratoconus subjects enrolled in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. METHODS: Four images were taken at an initial visit and at a repeat visit several weeks later. From these images, 17 indices were simulated from published formulas. Mixed-model analysis was used on test-retest data from the TMS-1 videokeratography instrument during the baseline year. This analysis yields estimates of within- and between-visit variability. RESULTS: Repeatability analysis revealed that within-visit standard errors were 1.0 to 5.9 times greater in keratoconus eyes than in normal controls when two images were analyzed from each visit. These values changed only slightly when more images were used. The ratio of between-visit standard errors of the indices were nearly equally greater than normal controls for (0.9-4.6 and 0.9-4.3) two images per eye and all images per eye, respectively. CONCLUSIONS: These results suggest that the repeatability of simulated indices derived from TMS-1 topography in keratoconus subjects is poorer than in normal controls.

Refractive astigmatism and the toricity of ocular components in human infants.

PURPOSE: Many studies have characterized astigmatism in infancy, but few have been longitudinal or contained ocular component data. This study characterized the frequency, orientation, and longitudinal change with age of infant astigmatism. Additional factors investigated were the influence of early astigmatism on emmetropization and its relation to corneal and lenticular toricity. METHODS: Three hundred two infants were enrolled in the study. Of these, 298 provided data for at least one visit at 3 +/- 1 months, 9 +/- 1 months, 18 +/- 2 months, and 36 +/- 3 months. Testing included cycloplegic retinoscopy (cyclopentolate 1%), video-based keratophakometry, and ultrasonography over the closed eyelid. RESULTS: Astigmatism > or =1.00 DC was common at 3 months of age (41.6%) but decreased in prevalence to 4.1% by 36 months (p < 0.0001). The most common orientation was with-the-rule at 3 months (37.0% compared with 2.7% for against-the-rule) but against-the-rule at 36 months (3.2% compared with 0.9% for with-the-rule). Most of the change in the average value of the horizontal/vertical component of astigmatism (J0) occurred between 3 and 9 months (-0.26 +/- 0.36 D; p < 0.0001) with no significant change between 9 and 36 months (-0.05 +/- 0.36 D; p=0.09). Spherical equivalent refractive error was not correlated with J0 at 3 and 9 months (R=0.002, p=0.48 and R=0.001, p=0.56, respectively). The two were only weakly correlated at 18 and 36 months (R=0.06 for each age, p <0.0001, p=0.0002, respectively). Changes in spherical equivalent between 3 and 9 months were unrelated to either the initial value of J0 (partial R for J0=0.0001; p=0.85) or the change in J0 (partial R for change in J0=0.0031; p=0.31). Across all the ages, corneal toricity was with-the-rule, and lenticular toricity was against-the-rule (produced by the toricity of the posterior lens surface). The cornea and anterior lens surface became more spherical with age, contributing to the shift away from with-the-rule refractive astigmatism. Toricity of all the refractive surfaces became less variable with age. CONCLUSIONS: Consistent with many reports, astigmatism was common in early infancy but decreased in prevalence with age, particularly when with-the-rule in orientation. The reduction in percentage of infants with astigmatism appeared to be caused by decreases in the toricity of the cornea and the anterior lens combined with decreases in the variability of corneal and lenticular surfaces. Astigmatism in infancy appeared to be unrelated to emmetropization of spherical equivalent refractive error.

Refractive error and ethnicity in children.

OBJECTIVE: To report the baseline prevalence of refractive error in the study population. DESIGN: A multicenter, longitudinal, observational study of refractive error and ocular development in children from 4 ethnic groups. PATIENTS AND METHODS: The study population included 2523 children (534 African American, 491 Asian, 463 Hispanic, and 1035 white) in grades 1 to 8 (age, 5-17 years). Myopia was defined as -0.75 diopters (D) or more and hyperopia as +1.25 D or more in each principal meridian, and astigmatism was defined as at least a 1.00-D difference between the 2 principal meridians (cycloplegic autorefraction). RESULTS: Overall, 9.2% of the children were myopic, 12.8% were hyperopic, and 28.4% were astigmatic. There were significant differences in the refractive error prevalences as a function of ethnicity (chi2, P<.001), even after controlling for age and sex (polychotomous logistic regression, P<.001). For myopia, Asians had the highest prevalence (18.5%), followed by Hispanics (13.2%). Whites had the lowest prevalence of myopia (4.4%), which was not significantly different from African Americans (6.6%). For hyperopia, whites had the highest prevalence (19.3%), followed by Hispanics (12.7%). Asians had the lowest prevalence of hyperopia (6.3%) and were not significantly different from African Americans (6.4%). For astigmatism, Asians and Hispanics had the highest prevalences (33.6% and 36.9%, respectively) and did not differ from each other (P =.17). African Americans had the lowest prevalence of astigmatism (20.0%), followed by whites (26.4%). CONCLUSION: There were significant differences in the prevalence of refractive errors among ethnic groups, even after controlling for age and sex (P<.001).

Ocular component data in schoolchildren as a function of age and gender.

PURPOSE: To describe the refractive error and ocular components of a large group of school-aged children as a function of age and gender. METHODS: In this report, we describe the refractive error and ocular components of 2583 school-aged children (49.3% girls, overall mean [+/-SD] age 10.0 +/- 2.3). Measurement methods included cycloplegic autorefraction, autokeratometry, videophakometry, and A-scan ultrasonography. For statistical comparisons across gender and age, a critical point of alpha = 0.005 was used to assess significance because of the large sample size and the large number of comparisons made. RESULTS: Of these 2583 children, 10.1% were myopic (-0.75 D or more myopia in both meridians), and 8.6% were hyperopic (+1.25 D or more hyperopia in both meridians). As would be expected, there was a significant effect of age on refractive error (spherical equivalent, p < 0.0001), toward less hyperopia/more myopia. There was no significant difference in the average refractive error between girls and boys (p = 0.0192). Girls had steeper corneas than boys (0.74 D steeper in the vertical meridian and 0.63 D steeper in the horizontal meridian, p < 0.0001). There were no significant differences in corneal power with age (p = 0.16). Both older age and male gender were significantly associated with deeper anterior chambers (p < 0.0001 for both). The crystalline lens showed significant thinning with age (p < 0.0001), however, there was no significant difference in the lens thickness between girls and boys (p = 0.66). Both Gullstrand lens power and calculated lens power showed significant effects of age and gender (p < 0.0001 for both). Girls, on average, had Gullstrand lens powers that were 0.28 D steeper and calculated lens powers that were 0.80 D more powerful than boys. Axial length also showed significant effects of age and gender (p < 0.0001 for both). Girls' eyes were, on average, 0.32 mm shorter than those of boys. CONCLUSIONS: These cross-sectional data show a general pattern of ocular growth, no change in corneal power, and crystalline lens thinning and flattening between the ages of 6 and 14 years. Girls tended to have steeper corneas, stronger crystalline lenses, and shorter eyes compared with boys.