Patent Publication Number: US-2021161383-A1

Title: Chromatic bioluminescence as a cellular level readout system of neural activity

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. provisional patent application No. 62/684,737, filed Jun. 13, 2019, and U.S. provisional patent application No. 62/684,740, filed Jun. 14, 2019, the entire contents of which are hereby incorporated by reference in their entireties. 
    
    
     GOVERNMENT RIGHTS STATEMENT 
     This invention was made with Government support under grant number 1706761 and 1453339 awarded by the National Science Foundation and grant number EY028391 awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing, created on Jun. 1, 2019; the file, in ASCII format, is designated H1482103.txt and is 85.4 KB in size. The file is hereby incorporated by reference in its entirety into the instant application. 
     FIELD OF THE INVENTION 
     The subject matter disclosed herein relates to bioluminescent compounds. More particularly, this disclosure relates to calcium-sensitive bioluminescent indicators for reporting neural activity. 
     BACKGROUND OF THE INVENTION 
     Certain compounds are capable of emitting luminescence in response to changes of various intracellular ion concentrations. For example, some proteins, in combination with particular compounds within a cell, can emit electromagnetic radiation when complexed with intracellular calcium ions. Because depolarization of a neuron concordant with generation of an action potential entails an elevation in intracellular calcium, such compounds can function as indicators of neural activity, emitting fluorescence from neurons firing an action potential. Potential use of such compounds could include, for example, visualization of activity within neurons of a brain or nerve in vivo or visualizing neural activity of slice preparations or other ex vivo applications. 
     Brain-machine interfaces (BMIs) allow direct communication to occur between the brain and an external device. BMIs have the potential to restore sensory or motor function to patients with spinal cord injuries or amputations. Preferably, advantageous features of a BMI system would include (1) minimal invasiveness, (2) long lifetime, (3) provision of high information content, (4) robustness, and (5) portability. Conventional BMI systems are deficient in these features. A particular advantageous feature of a BMI system would be if it could differentiate activity of particular neurons within an observed field of neurons, such as when neurons responsive to one input but not another are anatomically co-mingled with neurons of the converse responsiveness, or that do not respond to either of said stimulus, respond to both, or otherwise respond differently to one or more stimuli than do other neurons located in proximity thereto. Conventional BMI systems do not provide such ability to distinguish between activity of neighboring neurons. 
     A species of jellyfish,  Aequorea victoria,  produces a protein known as aequorin which is a Ca2+ dependent bioluminescent protein. In jellyfish, aequorin exists as a complex with fluorescent protein (e.g., GFP). In the presence of calcium, aequorin is able to act as a catalytic enzyme in a luciferase reaction. Aequorin oxidizes coelenterazine (CTZ), a molecular cofactor found naturally in jellyfish. The energy gained from the oxidation reaction is passed to the neighboring fluorescent protein which (in the case of GFP) releases a green photon in a process known as chemiluminescence resonance energy transfer (CRET). As a result, neural activity can be reported by aequorin as demonstrated in zebrafish and nerves in the legs of mice. Aequorin-fluorescent protein constructs act as calcium indicators because they have good temporal resolution and are bright with low or no background noise. However, although a single color bioluminescent indicator in the brain can report bulk activity, it cannot distinguish the activity between multiple, intermingled neurons because of the temporal overlap between signals generated from different neurons. 
     The present disclosure is directed to overcoming these and other deficiencies in the art. As disclosed further herein, use of bioluminescent proteins can provide significant improvement over conventional BMI systems. 
     SUMMARY OF THE INVENTION 
     In one aspect, provided is a method of detecting neural activity, including inducing neurons of a subject to express at least one of two or more polypeptides each comprising an amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO: 28, or a sequence having at least 90% sequence homology with any one of the foregoing, wherein inducing comprises stimulating interneuronally different relative levels of expression of the two or more polypeptides; applying coelenterazine to the subject; applying a first stimulation of neural activity to the subject; detecting a first spatiotemporal and spectral pattern of electromagnetic radiation emitted by neurons of the subject in response to the first stimulation; recording the first spatiotemporal and spectral pattern of electromagnetic radiation in a computer memory; applying a second stimulation of neural activity to the subject; detecting a second spatiotemporal and spectral pattern of electromagnetic radiation emitted by neurons of the subject in response to the second stimulation; analyzing, using one or more microprocessors, the second spatiotemporal and spectral pattern of electromagnetic radiation to the first spatiotemporal and spectral pattern of electromagnetic radiation, wherein the one or more microprocessors are configured to determine differences and similarities between the second spatiotemporal and spectral pattern of electromagnetic radiation and the first spatiotemporal and spectral pattern of electromagnetic radiation, differences indicate differences in neural activity caused by the first stimulation and the second stimulation, and similarities indicate similarities in neural activity caused by the first stimulation and the second stimulation. 
     In an embodiment, the two or more polypeptides each include a sequence having at least 90%, at least 95%, or 100% sequence homology with SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28. 
     In another embodiment, inducing includes transfecting neurons with at least one of two or more polynucleotides wherein each of the two or more polynucleotides encodes for one of the polypeptides. In an example, a polynucleotide includes a viral vector. In another example, the polynucleotides include a plasmid. 
     In another embodiment, the first stimulation, the second stimulation, or both, are sensory stimulation. In an example, the first stimulation, the second stimulation, or both, are visual stimulation, olfactory stimulation, auditory stimulation, gustatory stimulation, tactile stimulation, proprioceptive stimulation, pain stimulation, or electrical stimulation. In another embodiment, the first stimulation, the second stimulation, or both, are pharmacological stimulation. In another embodiment, the first stimulation, the second stimulation, or both, are electrical neural stimulation. In another embodiment, the subject is a rat, a mouse, a human, or a non-human subject. 
     Also provided is a method of detecting neural activity, including inducing neurons of a subject to express at least one of two or more polypeptides each comprising a fluorescent protein connected to an aequorin by a linker, wherein the amino acid sequence of each of the fluorescent proteins is independently represented by amino acids 1 through 239 of SEQ ID NO:13, amino acids 1 through 239 of SEQ ID NO:13, amino acids 1 through 239 of SEQ ID NO:14, amino acids 1 through 237 of SEQ ID NO:15, amino acids 1 through 237 of SEQ ID NO:16, amino acids 1-239 of SEQ ID NO:26, or amino acids 1-476 of SEQ ID NO:28, the amino acid sequence of each of the linkers is independently represented by amino acids 240 through 256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, or amino acids 477-493 of SEQ ID NO:28, and the amino acid sequences of each of the aequorins is independently represented by amino acids 257 through 448 of SEQ ID NO:13, amino acids 257 through 450 of SEQ ID NO:17, amino acids 257 through 450 of SEQ ID NO:21, amino acids 381-488 of SEQ ID NO:26, or amino acids 494-685 of SEQ ID NO 28.; and applying coelenterazine to the subject; applying a first stimulation of neural activity to the subject; detecting a first spatiotemporal and spectral pattern of electromagnetic radiation emitted by neurons of the subject in response to the first stimulation; recording the first spatiotemporal and spectral pattern of electromagnetic radiation in a computer memory; applying a second stimulation of neural activity to the subject; detecting a second spatiotemporal and spectral pattern of electromagnetic radiation emitted by neurons of the subject in response to the second stimulation; analyzing, using one or more microprocessors, the second spatiotemporal and spectral pattern of electromagnetic radiation to the first spatiotemporal and spectral pattern of electromagnetic radiation, wherein the one or more microprocessors are configured to determine differences and similarities between the second spatiotemporal and spectral pattern of electromagnetic radiation and the first spatiotemporal and spectral pattern of electromagnetic radiation, differences indicate differences in neural activity caused by the first stimulation and the second stimulation, and similarities indicate similarities in neural activity caused by the first stimulation and the second stimulation. 
     In an embodiment, inducing comprises transfecting neurons with at least one of two or more polynucleotides wherein each of the two or more polynucleotides encodes for one of the polypeptides. In an example, a polynucleotide includes a viral vector. In another example, the polynucleotides include a plasmid. In another embodiment, the first stimulation, the second stimulation, or both, are sensory stimulation. In an example, the first stimulation, the second stimulation, or both, are visual stimulation, olfactory stimulation, auditory stimulation, gustatory stimulation, tactile stimulation, proprioceptive stimulation, pain stimulation, or electrical stimulation. In another embodiment, the first stimulation, the second stimulation, or both, are pharmacological stimulation. In yet another embodiment, the first stimulation, the second stimulation, or both, are electrical neural stimulation. In another embodiment, the subject is a rat, a mouse, a human, or a non-human subject. 
     In another embodiment, the method further includes recording the second spatiotemporal and spectral pattern of electromagnetic radiation in a computer memory, wherein analyzing includes comparing the first spatiotemporal and spectral pattern of electromagnetic radiation stored in the computer memory to the second spatiotemporal and spectral pattern of electromagnetic radiation stored in the computer memory. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein: 
         FIG. 1  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for SEQ ID NO:13. 
         FIG. 2A  and  FIG. 2B  show a rise/decay plot in response to saturating calcium for SEQ ID NO:13. 
         FIG. 3  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with a sequence corresponding to SEQ ID NO:14. 
         FIG. 4  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:15. 
         FIG. 5  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:16. 
         FIG. 6A  shows a composite of bioluminescent spectra for polypeptides with amino acid sequences corresponding to SEQ ID NOs:13-16 and 26. 
         FIG. 6B  shows relative light output normalized to protein concentration for SEQ ID NOs:13-16 and 26, expressed as a percentage output of SEQ ID NO:26. 
         FIG. 7A  shows a two-photon excited fluorescence (2-PEF) microscopy image of neurons expressing different bioluminescent protein constructs as disclosed herein and  FIG. 7B  is a 3-dimensional plot showing RGB intensity of neurons shown in  FIG. 7A . 
         FIG. 8A  shows histological data of mouse barrel cortex following transduction of neurons expressing multiple different bioluminescent proteins and  FIG. 8B  shows RGB intensity levels of 15 neurons from  FIG. 8A . 
         FIG. 9  shows a 2-PEF microscopy image of neurons in the mouse cortex expressing different color bioluminescent proteins. 
         FIG. 10  shows stimulus-triggered neural activity in mouse barrel cortex neurons following transfection with AAV vectors driving expression of three different bioluminescent proteins. 
         FIG. 11  shows light emission of three different colors in 1-second epochs before, during, and after shock to the whisker pad with electrodes in two different locations (a and b) in mouse barrel cortex following injection with AAV vectors driving expression of three different bioluminescent proteins. 
         FIG. 12  shows a recording field of cortex of a non-human primate during visualization of fluorescence emitted by a combination of fluorescent proteins. 
         FIG. 13  is a representation of fields of recording at depths of up to 500 um below cortical surface during observation of neural activity. 
         FIGS. 14A  (red),  14 B (yellow),  14 C (green), and  14 D (blue) show number of neurons exhibiting different signal-to-noise ratios of fluorescence at different wavelengths. 
         FIG. 15  shows violin plots of emission detected within given filtered spectra, with ratio of SNR of a cell to the highest SNR measured within given filtered spectrum, where width indicates relative number of cells. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This disclosure relates to compositions, methods, systems, and kits for inducing cells such as neurons to emit detectable electromagnetic radiation, including having wavelengths of visible light, when activated and experiencing elevated calcium levels intracellularly. Compositions disclosed herein could likewise be used for identifying elevations of calcium levels intracellularly in other cells such as myocytes such as cardiomyocytes, or other cell types. Such compositions could also be used for measuring changes in extracellular calcium levels, or in solutions without whole cells. 
     Bioluminescence imaging (BLI) is a form of optical imaging that utilizes the detectable electromagnetic radiation, such as visible light, produced during luciferase-mediated oxidation of substrates to track processes at a molecular level. Molecular imaging with bioluminescence is advantageous because it is both non-invasive and has high signal-to-noise ratios because mammalian tissue has low intrinsic bioluminescence. As disclosed herein, neurons may be manipulated to express bioluminescent proteins that bioluminesce in response to elevated intracellular calcium levels. Furthermore, different species of bioluminescent molecules disclosed herein emit differing wavelengths of electromagnetic radiation. Thus, a neuron expressing one bioluminescent molecule would emit one wavelength of electromagnetic radiation in response to elevated calcium levels, while another neuron expressing a different bioluminescent molecule, or the same bioluminescent molecule in combination with one or more different bioluminescent molecule, would emit electromagnetic radiation of a different frequency upon elevation of intracellular calcium. 
     Surprisingly, as further disclosed herein, inducing expression of different combinations and/or different levels of bioluminescent, calcium-responsive molecules in different neurons gives rise to an exponential increase in the different emission spectra exhibited from cell to cell in response to calcium influx. That is, when inducing the expression of different levels of a bioluminescent molecule and/or different bioluminescent molecules in different combinations from one cell to the next, and or with differing levels of relative expression between bioluminescent molecules expressed per cell, each cell emits at a net frequency reflective of the particular bioluminescent molecule(s) it expresses and at a given level of expression. With, for example, six different bioluminescent markers applied to a field of neurons, at least 3,000 or more distinct emission spectra can be observed from different cells in response to calcium elevations owing to stochastically-determined differences in which bioluminescent molecules become expressed and at what relative levels to one another per cell. The wide variety of differentiable spectra obtained was particularly surprising in view of the failure of other, conventional technologies (e.g., BRAINBOW technology) to generate such diversity when cells are induced to express different marker compounds and/or at different levels. The success of the combinatorial approach undertaken as disclosed herein in generating a wide diversity of emission spectra per cell represents a significant advance over conventional technology that would not have been predicted on the basis of previous attempts with different markers. 
     Thus, multiple bioluminescent molecules may be applied simultaneously across a field of neurons such that neurons express different bioluminescent molecules at relative levels that differs stochastically from neuron to neuron. For example, two, three, four, five, six, or more bioluminescent markers with emission spectra that differ from each other may be applied across a field of neurons, and different neurons express different levels of each of the one, two, three, four, five, six, or more bioluminescent markers compared to other neurons in the field or with which each neuron is spatially comingled. As a result, each neuron would emit in the presence of elevated intracellular calcium at an emission frequency that is a reflection of the combined emission spectrum of each of the one, two, three, four, five, six, or more bioluminescent markers it is expressing at the level at which it is expressing each thereof. With different neurons stochastically expressing different relative levels of the two, three, four, five, or more bioluminescent marks compared to the other neurons within the field, each neuron would fluoresce at a different frequency than the other neurons upon elevations of intracellular calcium. Thus, activity of one neuron as opposed to another could be distinguished not just by the location of the neuron emitting bioluminescence but also by the frequency of bioluminescence it is emitting. 
     As disclosed herein, where cells such as neurons are described as exhibiting different levels of expression of two or more bioluminescent markers such a bioluminescent polypeptides, an example is where a neuron may express none of either of two markers, or some of only one marker, or some of only another marker, or some of any two or more of the two or more markers (such as of up to six or more). Thus, in some examples, a field of neurons may be induced to express from one to six or more bioluminescent proteins. Some neurons of the field may express only one of the six. For example, six different neurons may each express only one of the six markers, and each a different marker than the other of those six. Other neurons may express two or more makers, in any combination. This non-limiting example may include a plurality of neurons wherein each of the plurality of neurons expresses one or more bioluminescent markers having differing amino acid sequences from each other, and a level of expression of a first of the one or more polypeptides relative to a level of expression of a second of the one or more polypeptides in a first neuron (for example, a neuron that expresses some of one marker and none of any other) differs from a level of expression of the first of the one or more polypeptides relative to a level of expression of the second of the one or more polypeptides in a second neuron (which may express some of each of two or more markers, or some of one marker not expressed by the first neuron and some of a marker not expressed in the first neuron). Other neurons of the field may each express more than none of any combination of at least two of the bioluminescent markers, which may also include a first neuron and a second that express different relative levels of two or more bioluminescent markers than each other, or than any neuron that expresses some of only one but not of any other bioluminescent marker. 
     Expression of bioluminescent compounds may be accomplished by any known method. For example, cells can be transfected with nucleotide sequences (DNA, RNA, etc.) that encode for bioluminescent proteins as disclosed herein. As used herein, transfecting, transfected, or transfection includes any process for introducing nucleic acids or proteins into cells, including transduction through viral vector-mediated gene transfer. For example, adeno-associated viral (AAV) vectors may be used to transfect neurons, with different vectors driving expression of genetic material encoding different bioluminescent proteins. AAV vectors derived from a particular serotype or from mixed serotypes may be adopted and used depending on the particular application. If a population of neurons is contacted with a solution or solutions containing different viral vectors such as these, each containing material to drive the expression of different bioluminescent proteins, different cells can be transfected with stochastically different levels of payload from different vectors and thus express relative levels of bioluminescent proteins that differ inter-neuronally. In other examples, different viral vectors may be used, such as lentiviruses, retroviruses, HSV-viral vectors, or other viral vectors known to be effective at driving protein expression in neurons. In some examples, combinations of viral vectors may be used, rather than only one type of vector used to drive expression of all bioluminescent proteins. 
     Other transfection methods may also be used to drive expression, such as lipofection, electroporation, microinjection, gene gun, continuous infusion, and sonication, impalefection, hydrostatic pressure transfection, with genetic material that drives expression of the bioluminescent protein. In other examples, bioluminescent proteins themselves may be introduced into cells rather than genetic material driving their production by cells. All such examples are considered methods of promoting expression of a bioluminescent composition or compositions in cells, and may be used in combination with one another. In other examples, an extracellular bioluminescent compound or compounds may be used such as by addition of a bioluminescent polypeptide or polypeptides directly to a solution, for indicating elevated calcium levels therein. 
     A bioluminescent composition in accordance with the present disclosure may be a polypeptide including a fluorescent protein connected to an aequorin by a linker. For example, the fluorescent protein may be N-terminal to the linker and the aequorin may be C-terminal to the linker. In other examples, the aequorin may be N-terminal and the fluorescent protein may be C-terminal. In some examples, there may not be additional amino acids between a fluorescent protein and a linker, or between a linker and an aequorin, or neither between a fluorescent protein and a linker nor between a linker and an aequorin. Polynucleotides encoding for such polypeptides are also disclosed herein. As would be appreciated, because of the degeneracy of the genetic code, many different polynucleotide sequences could drive expression of a given bioluminescent polypeptide disclosed herein, and all such sequences for all such bioluminescent polypeptides are explicitly included in the present disclosure. Such a polynucleotide may exist in a form convenient for storage or expression within a cell such as a plasmid. Such a plasmid may contain an origin of replication, transcriptional start sites, and/or other structural features that promote replication thereof in a host cell and/or production of and/or translation from a transcript to promote expression of a bioluminescent polypeptide. 
     Examples of fluorescent proteins include mCerulean (represented by amino acids 1-239 of SEQ ID NO:13), eCFP (represented by amino acids 1-239 of SEQ ID NO:14), mTagBFP2 (represented by amino acids 1-237 of SEQ ID NO:15), mTFP1 (represented by amino acids 1-237 of SEQ ID NO:16), eGFP (represented by amino acids 1-239 of SEQ ID NO:26), and tdTomato (represented by amino acids 1-476 of SEQ ID NO:28). Examples of linkers are represented by amino acids 240-256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, and amino acids 477-493 of SEQ ID NO:28. Other examples may include a linker as a portion of an amino acid sequence linking a fluorescent protein to an aequorin wherein the linker has an amino acid sequence that differs from the foregoing examples. In some nonlimiting examples, the linker may be from between 10 and 100 amino acids in length. Examples of aequorins include aequorin from  Aequorea victoria  (represented by amino acids 257-448 of SEQ ID NO:13), obelin from  Obelia longissima  (represented by amino acids 257-450 of SEQ ID NO:17), and aequorin from  Aequorea macrodactyla  (represented by amino acids 257-450 of SEQ ID NO:21), referred to herein as AMac. All combinations of any one of the foregoing fluorescent proteins with any one of the foregoing aequorins connected by a linker are included in the present disclosure. A non-limiting list of examples of such combinations includes polypeptides with amino acid sequences represented by SEQ ID NOs:13-24, 26, and 28. All polynucleotides that encode for any bioluminescent protein disclosed herein is also explicitly included in the present disclosure. A non-limiting list of examples includes polynucleotides with sequences represented by SEQ ID NOs:1-12 (which encode for the bioluminescent proteins with amino acid sequences corresponding to SEQ ID NOs:13-24, respectively), and SEQ ID NOs:25 and 27 (which encode for the bioluminescent proteins with amino acid sequences corresponding to SEQ ID NOs:26 and 28, respectively. 
     In some examples, a bioluminescent polypeptide may include a phosphoprotein that differs by one or more amino acids from mCerulean (as represented by amino acids 1-239 of SEQ ID NO:13), eCFP (as represented by amino acids 1-239 of SEQ ID NO:14), mTagBFP2 (as represented by amino acids 1-237 of SEQ ID NO:15), mTFP1 (represented by amino acids 1-237 of SEQ ID NO:16), eGFP (represented by amino acids 1-239 of SEQ ID NO:26), or tdTomato (represented by amino acids 1-476 of SEQ ID NO:28). For example, a phosphoprotein segment of a bioluminescent protein may differ by up to 10% in amino acid sequence, or by up to 5% in amino acid sequence, or by between 10% and 5% or between 5% and 0% from the foregoing specific examples. A linker segment of a bioluminescent phosphoprotein may also differ by 10%, 5%, between 10% and 5%, or between 5% and 0% from amino acids 240-256 of SEQ ID NO:13, amino acids 240-300 of SEQ ID NO:26, and amino acids 477-493 of SEQ ID NO:28. Furthermore, an aequorin segment of a bioluminescent phosphoprotein may differ in amino acid content by 10%, 5%, between 10% and 5%, or between 5% and 0% from amino acids 257-448 of SEQ ID NO:13, amino acids 257-450 of SEQ ID NO:17, or amino acids 257-450 of SEQ ID NO:21. A bioluminescent protein may differ by 10%, 5%, between 10% and 5%, or between 5% and 0%, from any one of SEQ ID NOs:13-24, 26, or 28. All polynucleotide sequences that may encode for any of the foregoing segments of bioluminescent proteins or bioluminescent proteins are explicitly disclosed herein. For example, a nucleotide sequence may differ by 10%, 5%, between 0% and 5%, or between 5% and 0% in nucleotide sequence from SEQ ID NOs.1-12, 25, or 27. 
     Any fluorescent protein may be linked to any aequorin by any linker disclosed herein. Some neurons may be induced to express a bioluminescent marker of activity with one fluorescent protein linked to a given aequorin by a linker, whereas another neuron may be induced to express a bioluminescent marker of activity with the same fluorescent protein and linker but a different aequorin, or the same linker and aequorin but a different fluorescent protein, or the same fluorescent protein and aequorin but a different linker. In other cases two neurons may be induced to express bioluminescent markers of activity with the same fluorescent protein as each other but a different linker and different aequorin, or the same linker as each other but a different fluorescent protein and a different aequorin, or the same aequorin as each other but a different fluorescent protein and a different linker. All possible combinations of al fluorescent proteins, linkers, and aequorins disclosed herein are explicitly contemplated and included in the present disclosure. In this manner, neurons may be independently induced to express bioluminescent markers of activity that have no, only one, only two, or three of a fluorescent protein, linker, and/or aequorin in common with each other. 
     Neurons may be contacted with a coelenterazine molecule together with a bioluminescent protein or upon induced expression thereof. For example, coelenterazine or 2-deoxycoelenterazine may be applied to neurons transfected with a polynucleotide sequence that drives expression of a bioluminescent protein disclosed herein. In the presence of calcium, such as when intracellular calcium levels are elevated in conjunction with a depolarization event, aequorin may oxidize a coelenterazine molecule or analog thereof (e.g., 2-deoxycoelenterazine), whereupon the energy gained from the oxidation reaction is passed to the fluorescent protein segment of the bioluminescent protein inducing photon release thereby by CRET. Depending on the combination of aequorin and fluorescent protein segments used, and different combinations of expression of various bioluminescent proteins comprising such various segments, different neurons may emit different wavelengths of fluorescence when exposed to elevated calcium levels. Bioluminescent polypeptides or polynucleotides encoding therefor or viral vectors for driving the expression thereof as disclosed herein may be used in neurons taken from a subject of any intended species for ex vivo use or applied in vivo to a nervous system of a subject of any given species of interest. For example, with appropriate promoter sequences and transcriptional features for a given target species, they could be adopted for use in invertebrates or vertebrates. For example, they could be used in insects, fish, amphibians, reptiles, birds, or mammals. For example, they could be used in rodent, ungulate, canine, feline, leporine, porcine, primate, or other species. For example, they could be used in mice, rats, humans, or non-human primates. As disclosed herein, bioluminescent proteins or vectors for driving their expression may be injected or otherwise applied to a nervous system of such species then bioluminescence of the treated area observed in response to stimulatory input known or believed to recruit activity of neurons in such region, or hypothesized or suspected of doing so. 
     For detecting and measuring bioluminescence emitted by neurons expressing a bioluminescent protein as disclosed herein, various known optical imaging devices and techniques may be used. Charge coupled device (CCD) cameras, for example, are imaging modalities that spatially encode incident photons and their intensity into an image. Integrating photon counts on a CCD camera, however, may decrease temporal resolution. To increase temporal resolution to spatially localize neurons, photodetectors (for example, photon counting multiplier tubes (PMTs), photodiodes, or CCD/CMOS detector technologies) may be used. In an example, PMTs convert photonic signals into a current which decreases spatial resolution since it diffusely collects scattered photons. However, by color-coding cells such as neurons with different bioluminescent compositions and/or different combinations of compositions to permit inter-neuronal differentiation of the frequency of electromagnetic radiation emitted in response to elevated intracellular calcium, different color indicators and sampling with spectrally separated PMTs, a unique spectrum of colors that provide spatial resolution is obtained as disclosed herein. With a high sampling rate, PMTs are able to collect photons from hundreds of neurons that may be firing. Recording equipment may be sensitive to a wide range of frequency of electromagnetic radiation, within the visual spectrum, or outside thereof, depending on emission spectra of bioluminescent proteins used for signaling neural activity. 
     Such recording device or system may be connected to a computer for recording and analyzing bioluminescence emitted from a given population or field of neurons being measured. Recordings may be recorded on standard computer media systems and/or analyzed by software for depicting and analyzing activity of neurons within a recording field in response to different types of stimuli (e.g., sensory stimuli, pharmacological stimuli, electrical stimuli, etc.). Visualization and/or recording may also be performed during other events believed, known, or predicted to recruit or involve activation of populations of neurons in different brain regions, such as cortical brain regions. Such examples could include detecting bioluminescence in cortical motor areas during ideation or execution of movement of particular body areas or parts, visual cortical areas during experience or ideation of visual experience, during seizure or ictal events, during specific cognitive tasks, during affective states, including pathological affective states such as anxiety, depression, or mania, during sleep, or other states. 
     Use of bioluminescent proteins as disclosed herein may result in patterns of activity across a field of neurons. Within a given field, an area may be visualized and bioluminescent emission recorded. During a recording session, multiple parameters may be recorded, such as in relation to specific pixels or discrete loci or a recording device within a recorded field. For example, a given pixel or pixels or locus or loci of a recording device within a visualized field may correspond to a single neuron, or several pixels or loci within a visualized field may correspond to a neuron, or a pixel or pixels or loci or loci within a visualized field may correspond to a cluster of neurons or a boundary zone between neurons, etc., or any combination of the foregoing, during a session when intracellular calcium rise induces bioluminescence that may be visualized and recorded. Information specific to each pixel or loci within the field may be individually recorded, e.g. by a computer to which a camera or other visualizing device is connected and to which such device transmits electronic signals determined by activity at given pixels or loci. 
     Over the course of a recording session, each pixel, at different time points, may receive no stimulation, owing to lack of bioluminescence emitted by the neuron, neurons, or area from which it is recording, or may receive some stimulation. Stimulation it does receive may be coded for (1) specific time points during a recording session during which it does receive or is receiving stimulation, (2) wavelength of bioluminescent emission it is receiving at said time points, and (3) intensity of bioluminescent emission of given spectra it is receiving at various time points. Such parameters of input per pixel or loci may be recorded in a session log, such as in a computer readable medium (e.g., flash memory, hard drive, etc.). A session log may contain parameters of pixels or loci of a recording field during a session. A session log may therefore contain a spatial map of activity in that spatial information, represented by a location of a pixel or loci, relative to other pixels or loci, within the field that receives bioluminescent stimulation may be recorded. A session log may also include a temporal map of activity in that temporal information, represented by when pixels or loci did and did not detect bioluminescent stimulation, for pixels or loci may also be recorded. A session log may also include a spectral map of activity in that bioluminescent spectral information, represented by wavelength, intensity, or both, or bioluminescent emission detected at pixels or loci at different time points during a session. A session log may thus include a spatiotemporal and spectral pattern of activity in a field across a session. 
     A spatiotemporal and spectral pattern of bioluminescent emission may thus include a data set indicating what frequencies of bioluminescence were emitted from positions (e.g., specific neurons or neural populations) at a given location or locations within a recording field at time points during a visualization or recording session. Time points may be identified in relation to an onset or offset of a particular stimulation, mental state, cognitive event or process, etc. Stimulation could be sensory (visual, auditory, olfactory, gustatory, tactile, or proprioceptive), chemical or pharmacological (e.g., following administration of chemical compounds to a subject or a subject&#39;s organs such as directly to neural tissue), or electrical stimulation such as directly to the nervous system. Electrical stimulation could also be applied to a subject&#39;s skin. In some examples, a human subject could self-report a mental state or cognitive process experienced during a recording session, or elicited in response to linguistic or other commands or other input given during a recording session. 
     During a recording session, before, during, and after a first stimulus application or state, emission spectra from a recording field, including when and where emissions are emitted and at what frequencies (i.e., spatiotemporal and spectral pattern), may be detected by a camera and a record of such spatiotemporal and spectral signatures recorded in a computer recording medium such as a computer memory, creating a session log containing a spatiotemporal and spectral pattern across a session, as disclosed. Upon exposure to a second stimulation or event (at a different time point from the first stimulation or event), a second spatiotemporal and spectral pattern may be detected. This pattern (e.g., spatiotemporal and spectral pattern) may also be recorded in a computer recording medium such as a computer memory, yielding another session log. Over a stimulus-recording session, or across multiple sessions, numerous such spatiotemporal and spectral patterns emitted in response to various stimulations may be recorded as spatiotemporal and spectral patters of session logs. As a pattern is detected, or after it is recorded, it may be compared with spatial, temporal, and/or spectral information contained in a recorded session log, detected and recorded during presentation of another stimulus or evocation of another state at a different time. The stimuli or state may differ with regard to the type, intensity, duration, or in other attributes. For example, they may be of different sensory modalities, or different types within a sensory modality, when sensory stimuli are used as stimulation events. 
     When spatiotemporal and spectral patterns of emission from session logs are compared, differences therebetween may indicate differences in the population of neurons that respond to the two stimuli that evoked the spatiotemporal and spectral patterns at issue. For example, different regions of a recording field may emit electromagnetic radiation at different times relative to application of a given stimulus, or the same regions but at different times relative to onset or offset of a given stimulus or stimulation session, or different emission spectra may be detected at a given place and time within a field, or all three of these variables may differ from one spatiotemporal and spectral pattern to another. Such differences may indicate differences in neural responsiveness to the stimuli presented. Conversely, where such variables do not differ from each other from one stimulus session to another, similarities in neural responsiveness to the stimuli may be indicated. 
     Detected patterns of neural activity recorded, for example, during applications of different stimuli or stimuli at different times or of different strength or magnitude, or combinations of variations of any of the foregoing, may be stored in a computer readable memory such as a hard drive, mobile storage medium, flash memory, or the like, or in combinations of more than one of the foregoing. Patterns recorded during one recording session may be recorded in the same computer-readable medium as that recorded during a second session, or in a different medium. One or more microprocessors within a computer or computer system may be configured to analyze natural activity patterns observed during different recording sessions and detect differences, similarities, or both therebetween. Similarities (i.e., indicia of activity from different observation sessions whose spatial and emission characteristics overlap with each other) may signify neurons that respond similarly to each other during the two sessions. Conversely, differences (i.e., indicia of activity from different observation sessions whose spatial or emission characteristics do not overlap with each other) may signify neurons that do not respond similarly to each other during the two sessions. 
     The one or more processors may be configured to process instructions for comparing data representing the spatiotemporal and spectral patterns of activity detected during observation sessions when neural activity is observed. In some examples, such data may have been stored in a computer-readable medium after each session and the spatiotemporal and spectral patterns of activity stored thereon analyzed by the one or more microprocessors. In other examples, incoming data representing spatiotemporal and spectral patterns of activity observed during a session may be compared to previously stored data representing spatiotemporal and spectral patterns of activity recorded during a previous session, for real-time comparisons. In either case, a computer system including computer-readable medium and one or more microprocessors would be necessary in order to interpret, analyze, and compare the volume of data generated during an observation session and discern between digitized representations of specific emission signatures emitted at specific loci in a recorded field of cells. Digital representation of minute differences of emission signatures and spatial location thereof, at specific time points during a session, require processing via one or more microprocessors, as does comparisons of observed activity between observation sessions. 
     In some examples, neural activity in response to a modality or example of a given sensory stimulation might be detected and recorded, followed by a second session intended to replicate the neural responsiveness induced by such sensory stimulation. For example, a particular visual stimulation might be applied and a spatiotemporal and spectral pattern of activity within visual cortex detected and recorded. Subsequently, activity of neurons within the visual system may be artificially stimulated such as electrically and the spatiotemporal and spectral response of the same field detected and compared to the spatiotemporal and spectral pattern recorded following application of the visual stimulation. Where the two spatiotemporal and spectral patterns differ from each other, differences in neuronal responsiveness to the stimuli may be indicated whereas similarities may indicate that the second stimulation recapitulated aspects of the visual stimulation. 
     In an example, bioluminescence of visual cortex following induced expression therein of bioluminescent polypeptides as disclosed herein may be visualized and recorded. During stimulation of the visual system such as by reception of light upon a retina, a spatiotemporal and spectral pattern of activity within a field of visual cortex may be detected and recorded. Different types or patterns of visual stimulation as reception of light on different parts of the retina or differently oriented patterns of light on a retina or different types of movement of light stimulation across a retina may be administered across a recording session and captured as a spatiotemporal and spectral pattern of bioluminescent emission in a session log. In a subsequent session, other stimulation of the visual system may be administered. Bioluminescence emitted by the visual cortex may be visualized and recorded during this subsequent session. During such subsequent session, stimulation of pixels or loci within a recording filed at or across time points during the subsequent session, such as in relation to onset or offset of different stimulations of the visual system, by emission of bioluminescence at various frequencies and/or intensities, may result in generation of a spatiotemporal and spectral pattern of activity of the subsequent session. Such spatiotemporal and spectral pattern may be recorded in a session log of the subsequent session. During creation of such session log or after, the two session logs, one of the first session and one of the subsequent session, may be compared by a software system configured to process information from pixels or loci within the recording field during a recording session as recorded in a session log as a spatiotemporal and spectral pattern. Differences and similarities in which neurons or populations of neurons are active and at which relative time points, as exemplified by the spatiotemporal and spectral pattern evoked during a session, may indicate whether different or similar neural populations exhibited evoked activity in response to different stimulations of the visual system, respectively. In this example, the visual system is given as one possible, non-limiting embodiment. As described above, however, similar spatiotemporal and spectral patters may be generated and recorded as a session log in response to any of a variety of stimuli or evoked state according to a particular application of the present disclosure. 
     EXAMPLES 
     The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof. 
       FIG. 1  shows bioluminescent spectra of a polypeptide with an amino acid sequence corresponding to SEQ ID NO:13 sampled in response to saturating calcium (200mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H. 
       FIG. 2A  and  FIG. 2B  show kinetic rise/decay data collected on SFM-400 stopped flow mixer in response to saturating calcium. A polypeptide with an amino acid sequence corresponding to SEQ ID NO:13 was incubated overnight in CTZ-H, buffer exchanged to zero calcium buffer (to remove excess coelenterazine), and rapidly mixed with calcium to elucidate kinetic response ( FIG. 2B  is an axis zoom of  FIG. 2A  over the first 0.5 sec). 
       FIG. 3  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with a sequence corresponding to SEQ ID NO:14. 
       FIG. 4  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:15. 
       FIG. 5  shows bioluminescent spectra sampled in response to saturating calcium (200 mM) at room temperature with 100 ms integration time on Ocean Optics QE65000 after overnight incubation in Coelenterazine-H for a polypeptide with an amino acid sequence corresponding to SEQ ID NO:16. 
       FIG. 6A  shows a composite of bioluminescent spectra for polypeptides with amino acid sequences corresponding to SEQ ID NOs:13-16 and 26, illustrating differentiation of emission spectra amongst these examples. Cells expressing one or combinations of these bioluminescent proteins can be distinguished by their differences in emission spectra in response to calcium elevations. Spectra were sampled in response to saturating Ca2+ (200 mM) at room temperature with 100 ms integration (Ocean Optics QE65000) after overnight incubation with Coelenterazine-H. As shown in  FIG. 6A , bioluminescent proteins as disclosed herein (e.g., those represented by SEQ ID NOs:13-16) showed notable blue-shifting relative to other bioluminescent proteins, such as with a sequence indicated by SEQ ID NO:26. Such blue-shifting was surprising, and not fully explained by Förster or other stable methods in Förster resonance energy transfer pairs. Without being limited to a particular function, one suggested explanation for such unexpected bleu-shifting that different variations in amino acid sequence amongst fusion pairs influences biophysical properties to blue-shift emission spectra. 
       FIG. 6B  Shows relative light output normalized to protein concentration for SEQ ID NOs.13-16 and 26. Light output is calculated as mean gray value per frame collected with Basler AG CMOS camera; average of three trials. Total light collected over entire time course is stated as a (%) relative to SEQ ID NO:26. As disclosed herein, bioluminescent proteins having amino acid sequences represented by, for example, SEQ ID NOs:13-16 showed greater light emission relative to that of SEQ ID NO:26, indicated by their relatively higher areas-under-curve as indicated in  FIG. 6B . 
     Viral Vector Injection for Indicator Expression 
     Individual strains of adeno-associated virus (AAV) viral vectors were packaged with a polynucleotide encoding a single color bioluminescent protein construct. In a sterile surgical preparation, the mouse is fixed onto a stereotax. A drill bit is used to create a burr hole in the skull to expose the surface of the brain. AAV vectors packed with individual polynucleotides encoding a single color bioluminescent protein construct are either individually or mixed with other colors and injected with a glass pipette into the rodent barrel cortex which is 3.5 mm lateral to bregma and 1.5 mm posterior to bregma. A bolus (100 nL to 1000 nL) is injected about 400 μm in depth. The mouse is sutured and following chronic protocols, administered drugs to facilitate healing and prevent inflammation. 
     In Vivo Imaging and Bioluminescence Measurement 
     After approximately three weeks during which time neurons are transduced to express bioluminescent proteins, an acute imaging experiment was performed. In an acute surgical preparation, a craniotomy about ˜3 mm in diameter was performed, centered over the barrel cortex. Coelenterazine, the cofactor required for bioluminescence, is injected with a glass pipette into the barrel cortex, in the same region that the virus was previously injected into. Coelenterazine is also applied topically and gel foam is placed over the surface of the brain to prevent the surface from drying out. In a dark room, coelenterazine is allowed approximately one hour to reconstitute. The mouse is then brought into the imaging facility and an electric stimulator is attached to the whisker pad. Using a non-imaging modality, augmented from optics typically used in 2-photon excitation fluorescence microscopy (2-PEF), light emitted from the rodent brain in response to stimulus is spectrally separated and detected on photomultiplier tubes (PMTs). 
     Tissue Processing and Histology 
     In order to prepare the tissue for histology, the mouse is perfused via a transcardial perfusion. At the conclusion of an imaging experiment, brain tissue is removed and immersed in fixative. The fixed brain is sliced into 70 um slices and mounted onto slides for 1-photon imaging. 
     Results 
     Mixing and co-injecting multiple AAV vectors, each encoding a single color, produced stochastic and varied transduction profile in cortical neurons 
     Both individual and mixes of AAV vectors packaged with different bioluminescent proteins that bioluminesce at different frequencies in response to elevated calcium levels were injected into mouse models to test for expression amounts and patterns. For mixtures of colors, each vector contained 2×10{circumflex over ( )}11GC/mL. Injecting a mixture of colors resulted in individual cells with distinct spectral patterns. Shown in  FIG. 7A  is a two-photon excited fluorescence (2-PEF) microscopy image of mouse cortex showing fluorescence of transfected cells in response to fluorescent stimulation (as opposed to activity-induced calcium increase), demonstrating that different cells emitted different frequencies of florescence due following transfection due to stochastically differential expression in different neurons of bioluminescent proteins.  FIG. 7B  shows a 3D plot of RGB intensity of neurons shown in  FIG. 7A  demonstrating their differential emission levels. 
     In the example in  FIG. 8A , a mouse was co-injected with four different AAV vectors with different bioluminescent proteins and the neurons were allowed 3 weeks to transduce. An acute craniotomy was performed and stimulus triggered bioluminescent data was acquired. The mouse was then perfused and histology was performed to reveal the expression pattern. Neurons were identified as regions with higher intensities. The image shows that neurons are uniquely and stochastically labeled. The ratio of the fluorescence from different color indicators in each cell is displayed in  FIG. 8B . The ratios of three different emission channels measured within neurons varied between cells, with several cells showing distinct patterns in color. 
     Neural Activity-Dependent Bioluminescence in a Seizure Model. 
     Applying pentylenetetrazole (PTZ) topically induces a seizure in the mouse cortex that can be recorded optically, demonstrating for the first time that the mouse brain can fluoresce from stimulated neural activity. PTZ causes chemically induced seizures. During seizure activity, the bioluminescent protein constructs released photons that were collected by the PMTs in the presence of coelenterazine. For the first time, it was shown that the rodent brain could emit in vivo detectable bioluminescence from neural activity. 
     Somatosensory Stimulation-Evoked Neural Activity Reported by Multicolor Bioluminescence 
     In another anesthetized mouse, a stimulation of the whisker pad produced light emissions at multiple wavelengths. Approximately three weeks after the virus injection, an acute craniotomy was performed on the mouse to reveal the surface of the barrel cortex, where three AAV vectors driving expression of different bioluminescent protein had been injected. A 2-PEF microscopy image of neurons in the mouse cortex expressing different color bioluminescent proteins is shown in  FIG. 9 . Coelenterazine was injected and allowed to reconstitute within the brain at which point an electric stimulator was attached to the mouse whisker pad. Stimulating the whisker pad at 10 Hz resulted in neural activity in the barrel cortex, as indicated by bioluminescent signals collected. Across a number of trials, the same stimulation of the whisker pad resulted in emissions of light with similar temporal and spectral shapes ( FIG. 10 ). 
     Relocating the Location of Stimulus on the Whisker Pad Resulted in Different Patterns of Neural Activity that was Repeatable 
     For measuring stimulation-evoked activity, stimulus electrodes were initially placed on one area of the whisker pad. The stimulus electrodes were then moved to a slightly different position on the whisker pad and the same shock stimulus was applied again. In both instances, when the intensity of each emission channel is plotted on a separate axis, the emission from one second epochs before, after, and during the stimulus resulted in clearly different color combinations. As expected, moving the area of stimulation on the whisker pad resulted in different spectral and temporal light emissions. Shown in  FIG. 11  is light emission in 1-second epochs before, during, and after shock to the whisker pad with electrodes in two different locations (a and b). Each trace is a stimulus triggered average of 5 trials where the intensity in the three different emission channels is plotted as a single point and sequential points are connected. Moving the area of stimulus (a) to another area of stimulus (b) affected the patterns of neural activity in the barrel cortex. 
     Methods 
     Surgical Procedure for Burr Hole Injections 
     A drill is mounted into a micromanipulator over the field. A micropipette loaded with a viral vector for driving expression of bioluminescent polypeptide is loaded into a micromanipulator. Mice are anesthetized under 5% isoflurane at the site of the drill and maintained at 1.5-2% isoflurane in 100% oxygen for the duration of the surgery. Mice are injected with ketoprofen (2 mg/mL at 2.5 uL/g of mouse) and dextamethasone (0.1 mg/mL at 2 uL/g of mouse) subcutaneously. Then mice are injected with atropine sulfate (0.15 mg/mL at 3.3 uL/g of mouse) intramuscularly. Mice are then mounted on a stereotax for intracranial injection. Bupivacaine (0.125%) may be injected subcutaneously over the incision site. 
     Holes are drilled in the exposed skull at the pre-determined stereotaxic coordinates to expose barrel cortex. Viral vectors are then applied to the exposed cortical tissue via micropipette, at a depth of, for example, ˜500 um deep. Mice are then sutured and recovered from anesthesia. Transduction is allowed to occur for the next 14-21 days before imaging. 
     Imaging and recording of bioluminescence is performed 21 days after injections to allow for expression of the bioluminescent protein. Skin over the injection site is removed and skull exposed. An approximately 4 mm diameter circle is drilled around the injection site and the skull flap removed. Gel foam is wet with coelenterazine and placed over surface of the brain to reconstitute for at least an hour, prior to imaging. Electric stimulus is then attached to the whisker pad region of the mouse and bioluminescence detected by PMT apparatus in response to various trains of stimulation. 
     Histology 
     Mice were perfused transcardially immediately following imaging session with 1×PBS and 4% Paraformaldehyde (PFA). Brains were removed and stored in 4% PFA for an additional 24 hours. Brains were transferred into 30% sucrose for at least 24 hours, or until the brain is saturated with the 30% sucrose, then repeated with 30% sucrose. Slices at 70 um thickness were taken and stored on Superfrost cover glass and imaged underneath 1-photo microscope and images captured with a fluorescent camera. 
     Non-Human Primate Transfections 
     The ability to discern multiple independent fluorescent signature emitted by cortical neurons in a field transfected with a plurality of vectors driving expression of fluorescent proteins was determined. The visual cortex of a macaque was injected with a combination vectors for driving expression of bioluminescent markers as disclosed herein. Rather than calcium-induced fluorescence, which requires coelenterazine infusion, the fluorescent protein portion of transfected constructs was detected using two-photon microscopy. Results confirmed that simultaneous transfection with a combination of markers permits detection of a large number of discernable signatures. 
     Injection Methods 
     Convection enhanced delivery (CED) was used to keep indfusate within the cortex rather than to allow the fluid to return back up the outside surface of the of the injection pipette&#39;s track. The AAVs contained multi-color fluorescent proteins, randomly mixed, and each injection site was precisely positioned, within ˜0.5 mm of the target region, to optimize cortical filling with just 4-9 injections within a 3.14 cm2 region. The bolus was delivered at a rate of 1-5 μL/min to maintain high-dispersal while preventing pressure-induced tissue damage. A canula was positioned above the cortex, which targeted the positioning of each CED tube injection, 2 mm below the surface of the cortex. 
     Viral Solution Content 
     In the macaque whose data from primary visual cortex (VI) is show in  FIGS. 12-15 , the figure on monkey primary visual cortex (V1), we made four 40-50 μL injections of a mixed solution containing five multi-color fluorescent proteins were made. 
     Imaging Methods 
     Cells were imaged with a Bruker Ultima IV two-photon imaging microscope driven by a MaiTai HP DeepSee Ti:Saph laser at a wavelength of 830 nm. We captured aAn entire 2-cm diameter imaging window (3.14 cm2 area) was captured by tiling the window with 49 (7×7) grid of 500 μm deep z-stack scans (each stack 2.8 mm on a side), as shown in  FIGS. 12 and 13 . The z-stacks were computationally stitched into a single volume to visualize the ultra-large imaging window. 61,017 cells were imaged. 
     Analysis Methods 
     Cellular analyses were performed using Imaris software. Using the Spot Detection feature identified potential cell bodies were identified by the size of the image intensity regions. Background was calculated as an average of the mean intensity of pixels between spots. Each pixel was imaged with four photo-multiplier tubes simultaneously, each with a different color filter (Red, Yellow, Green, Blue), to determine a differential colorimetric measurement of each cell&#39;s emission spectra. Signal-to-noise ratio (SNR) was calculated as the quotient of the difference between the mean intensity of each candidate cell and the background mean intensity divided by the standard deviation of the background. SNR was calculated independently in each of the four acquisition channels. Cells were identified from the candidates that achieved an SNR of one of more in at least one channel. 
     Each cell had an array of the four colors and the profile was established of differentiable colors in each cell in bins of 3 SNRs, to ensure clear differentiability. Binning with 3 SNR resolution is a commonly used and arbitrary technique to distinguish levels of signal in a transmission channel in the field of engineering. By utilizing 3 SNR resolution here, at least 4,752 different colors of cells with our labelling methods (i.e., 9 red 3 SNR bins times 11 yellow 3 SNR bins times 8 green 3 SNR bins times 6 blue 3 SNR bins), as shown in  FIGS. 14A-14D and 15 . Other ways of differentiating the colorimetry may result in higher spectral content (cluster analysis, etc). The unit “Peak SNR” represents is the SNR of a brightest pixel in each identified cell (in a given channel) divided by the maximum SNR for that channel. 
     Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow.