We thank B. Conklin (University of California, San Francisco) for providing the Gqi5 and Gqs5 cDNAs, A. Schweitzer for assistance with the electronics, N. Taylor for assistance with screening of clones, Ian Glaaser and Robert Rifkin for proof reading, and Olivier Griesbeck for TN-XXL. This work was supported by research grants through the US National Institute on Drug Abuse (NIDA) (DA029706; DA037170), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) (EB003832), Hoffman-La Roche (88610A) and the &#34;Neuroscience Related to Drugs of Abuse&#34; training grant through NIDA (DA007315).

<ol>
	<li>Day, J. C., Kornecook, T. J., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+vivo.+microdialysis+to+the+study+of+cholinergic+systems.">Application of in vivo. microdialysis to the study of cholinergic systems.</a> Methods. 23, 21-39 (2001).</li><li>Robinson, D. L., Venton, B. J., Heien, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+subsecond+dopamine+release+with+fast-scan+cyclic+voltammetry+in+vivo.">Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo.</a> Clin Chem. 49, 1763-1773 (2003).</li><li>Liang, R., Broussard, G. J., Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Chemical+Neurotransmission+with+Genetically+Encoded+Fluorescent+Sensors.">Imaging Chemical Neurotransmission with Genetically Encoded Fluorescent Sensors.</a> ACS Chem Neurosci. , (2015).</li><li>Okubo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+extrasynaptic+glutamate+dynamics+in+the+brain.">Imaging extrasynaptic glutamate dynamics in the brain.</a> Proc. Natl. Acad. Sci. USA. 107, 6526-6531 (2010).</li><li>Marvin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+fluorescent+probe+for+visualizing+glutamate+neurotransmission.">An optimized fluorescent probe for visualizing glutamate neurotransmission.</a> Nat Methods. 10, 162-170 (2013).</li><li>Nguyen, Q. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+biosensor+for+neurotransmitter+release+and+in+situ+receptor+activity.">An in vivo biosensor for neurotransmitter release and in situ receptor activity.</a> Nat Neurosci. 13, 127-132 (2010).</li><li>Muller, A., Joseph, V., Slesinger, P. A., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+reporters+reveal+in+vivo+dynamics+of+dopamine+and+norepinephrine+release+in+murine+cortex.">Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex.</a> Nat Methods. 11, 1245-1252 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J Vis Exp. , e3998 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. , (2009).</li><li>Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., Bourne, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+three+amino+acids+switches+receptor+specificity+of+Gq&#945;+to+that+of+Gj&#945;.">Substitution of three amino acids switches receptor specificity of Gq&#945; to that of Gj&#945;.</a> Nature. 363, 274-276 (1993).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1, 3166-3173 (2006).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J Vis Exp. , (2012).</li><li>Drew, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nat Methods. 7, 981-984 (2010).</li><li>Shih, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> J Cereb Blood Flow Metab. 32, 1277-1309 (2012).</li><li>Yamauchi, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+ligand-gated+ion+channel+receptors+with+genetically+encoded+Ca2++sensors.">Characterizing ligand-gated ion channel receptors with genetically encoded Ca2+ sensors.</a> PLoS One. 6, e16519 (2011).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J. Neurosci. 32, 13819-13840 (2012).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Cui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+activation+of+striatal+direct+and+indirect+pathways+during+action+initiation.">Concurrent activation of striatal direct and indirect pathways during action initiation.</a> Nature. 494, 238-242 (2013).</li></ol>

The authors have nothing to disclose.

The creation of CNiFERs provides an innovative and unique strategy for optically measuring release of neurotransmitters in the brain in vivo. CNiFERs are ideally suited for measuring extrasynaptic release, i.e., volume conduction, for neurotransmitters. Importantly, each CNiFER possesses the properties of the native GPCR, providing a physiological optical measurement of the changes in levels of neurotransmitters in the brain. To date, CNiFERs have been created for detecting acetylcholine (M1-CNiFER)<sup...

To fully understand how neurons communicate in the brain, it is necessary to have a method to measure the release of neurotransmitters in vivo. There are several well-established techniques for measuring neurotransmitters in vivo. One commonly used technique is microdialysis, in which a cannula is inserted into the brain and a small volume of cerebrospinal fluid is collected and analyzed using high-performance liquid chromatography and electrochemical detection1. Microdialysis has a spatial resolution on the order of a few diameters of the probe, e.g., ~0.5 mm for a 200 &#956;m diameter microprobe. The temporal resolution of this technique, however, is slow due to sampling intervals that typically last ~5 min or longer1. Moreover, analyses are not made in real-time. Another technique is fast scanning cyclic voltammetry (FSCV), which uses a carbon-fiber probe that is inserted into the brain. FSCV has excellent temporal resolution (subsecond), high sensitivity (nanomolar), and spatial resolution with probe&#160;diameters of 5 to 30 &#956;m. However, FSCV is limited to transmitters that produce a characteristic oxidation and reduction profile with voltage on a carbon potentiometric probe2.
A third technique to measure neurotransmitters is directly through genetically-encoded neurotransmitter (NT) biosensors3. With this method, a fusion protein is created that contains a ligand-binding domain for a transmitter coupled to a fluorescence resonance energy transfer (FRET)-based pair of fluorophores4 or a permutated GFP5. Unlike the previous two methods, these biosensors are genetically encoded and expressed on the surface of a host cell, such as a neuron, through the production of transgenic animals or acutely with the use of viral agents to infect cells. To date, genetically-encoded biosensors have been only developed for detecting glutamate and GABA3-5. Limitations with these techniques have been the low sensitivity, in the nM range,&#160;and the inability to expand the detection to the large number of transmitters, e.g., classical neurotransmitters, neuropeptides and neuromodulators, which signal through G protein-coupled receptors (GPCRs). In fact, there are nearly 800 GPCRs in the human genome.
To address these shortfalls, we have developed an innovative tool to optically measure release of any neurotransmitter that signals through a GPCR. CNiFERs (cell-based neurotransmitter fluorescent engineered reporters) are clonal HEK293 cells engineered to express a specific GPCR that, when stimulated, triggers an increase in intracellular [Ca2+] that is detected by a genetically encoded FRET-based Ca2+ sensor, TN-XXL. Thus, CNiFERs transform neurotransmitter receptor binding into a change in fluorescence, providing a direct and real-time optical read-out of local neurotransmitter activity. By utilizing the native receptor for a given neurotransmitter, CNiFERs retain the chemical specificity, affinity and temporal dynamics of the endogenously expressed receptors. To date, we have created three types of CNiFERs, one for detecting acetylcholine using the M1 receptor, one for detecting dopamine using the D2 receptor, and one for detecting norepinephrine using the &#945;1a receptor6,7. The CNiFER technology is readily expandable and scalable, making it amenable to any type of GPCR. In this JoVE article, we describe and illustrate the methodology to design, realize, and test in vivo CNiFERs for any application.

<table><tbody><tr><td>pCDH-CMV-MCS-EF1-Puro&amp;nbsp;</td><td>System Biosciences</td><td>CD510B-1</td><td>Cloning: for generating lentivirus</td></tr><tr><td>12 x 75 *BD Falcon High Clarity Polypropylene Round Bottom Test Tube</td><td>BD Biosciences</td><td>352063</td><td>FACS</td></tr><tr><td>BD 40 um Falcon cell strainers</td><td>BD Biosciences</td><td>352340</td><td>FACS</td></tr><tr><td>0.05% Trypsin EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200056</td><td>FACS</td></tr><tr><td>96 Well Plate, flat bottom, clear&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>3596</td><td>FACS</td></tr><tr><td>96 well cell culture plates&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>CLS3997</td><td>Flexstation</td></tr><tr><td>Optilux black clear bottom&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>3603</td><td>Flexstation</td></tr><tr><td>Flexstation pipet tips</td><td>Molecular Devices</td><td>9000-0911</td><td>Flexstation</td></tr><tr><td>Acetylcholine Chloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2661</td><td>Flexstation</td></tr><tr><td>Norepinephrine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A7256</td><td>Flexstation</td></tr><tr><td>Dopamine Hydrochloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>PHR1090</td><td>Flexstation</td></tr><tr><td>GABA&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2129</td><td>Flexstation</td></tr><tr><td>Histamine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H7125</td><td>Flexstation</td></tr><tr><td>Glutamate&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>49621</td><td>Flexstation</td></tr><tr><td>Epinephrine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E4642</td><td>Flexstation</td></tr><tr><td>Somatostatin&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S1763</td><td>Flexstation</td></tr><tr><td>5HT&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H9523</td><td>Flexstation</td></tr><tr><td>VIP&amp;nbsp;</td><td>Alpha Diagnostics Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>SP-69627</td><td>Flexstation</td></tr><tr><td>Orexin A</td><td>Alpha Diagnostics Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>12-p-01</td><td>Flexstation</td></tr><tr><td>Substance P&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S6883</td><td>Flexstation</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>Flexstation</td></tr><tr><td>Melatonin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M5250C</td><td>Flexstation</td></tr><tr><td>Fluorescence Plate Reader &amp;amp; software</td><td>Molecular Devices</td><td>Flexstation 3</td><td>Flexstation</td></tr><tr><td>&amp;nbsp;DMEM (high glucose) with Glutamax&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>10569-010</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Fetal bovine serum</td><td>Life Technologies</td><td>10082-139</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Pen/Strep&amp;nbsp; antibiotics</td><td>Life Technologies</td><td>15140-122</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Puromycin&amp;nbsp;&amp;nbsp;</td><td>InvivoGen</td><td>ant-pr-1</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F0895</td><td>Tissue culture</td></tr><tr><td>CoolCell LX Alcohol-free controlled-rate cell freezing box</td><td>Bioexpress</td><td>D-3508)</td><td>Tissue culture</td></tr><tr><td>cyanoacrylate glue&amp;nbsp;</td><td>Loctite</td><td>Loctite no. 495</td><td>surgery and stereotaxic injection</td></tr><tr><td>plastic paraffin film&amp;nbsp;</td><td>VWR</td><td>Parafilm&amp;reg;</td><td>surgery and stereotaxic injection</td></tr><tr><td>Nanoinjector</td><td>Drummond</td><td>3-000-204</td><td>surgery and stereotaxic injection</td></tr><tr><td>Glass electrodes</td><td>Drummond</td><td>3-000-203G</td><td>surgery and stereotaxic injection</td></tr><tr><td>hand held drill</td><td>OSADA</td><td>Exl-M40</td><td>surgery and stereotaxic injection</td></tr><tr><td>Burrs for drill</td><td>Fine Scientific</td><td>19007-05; 19007-07)</td><td>surgery and stereotaxic injection</td></tr><tr><td>Sterilizing bath</td><td>FST</td><td>18000-45, Hot Bead Sterilizer</td><td>surgery and stereotaxic injection</td></tr><tr><td>isoflurane chamber/mask</td><td>Highland Medical Equipment</td><td>564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm</td><td>surgery and stereotaxic injection</td></tr><tr><td>3D scope with arm</td><td>Zeiss</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>fiber optic light</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Betadine&amp;nbsp;</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>70 % (v/v) isopropyl alcohol</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Povidone-Iodine Prep Pads</td><td>dynarex</td><td>1108</td><td>surgery and stereotaxic injection</td></tr><tr><td>NaCl 0.9% (injection, USP, 918610)</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>CYCLOSPORINE (INJECTION, USP)</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Buprenex (injection) buprenorphine (0.03 &amp;mu;g per g rodent)</td><td>Sigma-Aldrich</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Ophthalmic ointment&amp;nbsp;</td><td>Akorn</td><td>NDC 17478-235-35</td><td>surgery and stereotaxic injection</td></tr><tr><td>Surgifoam</td><td>Ethicon</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Grip dental cement</td><td>Dentsply</td><td>#675571, 675572</td><td>surgery and stereotaxic injection</td></tr><tr><td>Instant SuperGlue&amp;nbsp;</td><td>NDindustries</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>LOCTITE 4041</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>METABOND</td><td>C&amp;amp;B</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>no. 0 cover glass</td><td>Fisher</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>stereotaxic frame&amp;nbsp;</td><td>Kopf</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Rectal probe and heating pad</td><td>FHC</td><td>40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe</td><td>surgery and stereotaxic injection</td></tr><tr><td>optical breadboard for imaging</td><td>Thorlabs</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Mineral oil</td><td>Fisher</td><td>S55667</td><td>surgery and stereotaxic injection</td></tr><tr><td>Kwik-Cast (Silicone elastomer)</td><td>World Precision Instruments</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Suture&amp;nbsp;&amp;nbsp;</td><td>Ethicon</td><td>18&amp;rsquo;&amp;rsquo;, 1667, 4-0</td><td>surgery and stereotaxic injection</td></tr><tr><td>Scissors</td><td>Fine Scientific Tools</td><td>91500-09, 15018-10</td><td>surgery and stereotaxic injection</td></tr><tr><td>Forcepts</td><td>Fine Scientific Tools</td><td>11252-30; #55, 11295-51; Grafe, 11050-10</td><td>surgery and stereotaxic injection</td></tr><tr><td>Student Halsted-Mosquito Hemostats</td><td>Fine Scientific Tools</td><td>91308-12</td><td>surgery and stereotaxic injection</td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>Fine Scientific Tools</td><td>18000-00</td><td>surgery and stereotaxic injection</td></tr><tr><td>Hot Bead Sterilizers</td><td>Fine Scientific Tools</td><td>18000-45</td><td>surgery and stereotaxic injection</td></tr><tr><td>Instrument Case with Silicone Mat</td><td>Fine Scientific Tools</td><td>20311-21</td><td>surgery and stereotaxic injection</td></tr><tr><td>Plastic Sterilization Containers with Silicone Mat</td><td>Fine Scientific Tools</td><td>20810-01</td><td>surgery and stereotaxic injection</td></tr><tr><td>2P fixed-stage fluorescence scope for &lt;em&gt;in vivo&lt;/em&gt; imaging</td><td>Olympus</td><td>FV1200 MPE</td><td>in vivo imaging</td></tr><tr><td>Multiphoton laser</td><td>SpectraPhysics</td><td>Mai Tai DeepSee</td><td>in vivo imaging</td></tr><tr><td>Green Laser</td><td>Olympus</td><td>473 nm Laser</td><td>in vivo imaging</td></tr><tr><td>xy translational base</td><td>Scientifica</td><td>MMBP</td><td>in vivo imaging</td></tr><tr><td>FRET filter cube for YFP and CFP</td><td>Olympus</td><td></td><td>in vivo imaging</td></tr><tr><td>10x and 40x water immersion objectives</td><td>Olympus</td><td></td><td>in vivo imaging</td></tr><tr><td>air table</td><td>Newport</td><td></td><td>in vivo imaging</td></tr><tr><td>custom built light-tight cage</td><td>Thorlab</td><td></td><td>in vivo imaging</td></tr></tbody></table>

construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

A CNiFER is derived from a human embryonic kidney (HEK293) cell that is engineered to stably express at least two proteins: a specific G-protein coupled receptor (GPCR) and a genetically encoded [Ca2+] sensor, TN-XXL. TN-XXL undergoes fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins, eCFP and Citrine, respectively, in response to Ca2+ ions6,15. Activation of GPCRs that couple to endogenous Gq G proteins ...

Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

construction-cell-based-neurotransmitter-fluorescent-engineered

Research

JoVE Journal

Neuroscience

13.1K Views.  Icahn School of Medicine at Mount Sinai. We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Video: Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Developmental Biology

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Biology

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Summary

Explore More Videos

Chapters in this video

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells