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.

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			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenex (INJECTION) buprenorphine (0.03 &#956;g per g rodent)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic ointment&#160;</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:429px;">NDC 17478-235-35</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgifoam</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grip dental cement</td>
			<td style="width:191px;">Dentsply</td>
			<td style="width:429px;">#675571, 675572</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instant SuperGlue&#160;</td>
			<td style="width:191px;">NDindustries</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LOCTITE 4041</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">METABOND</td>
			<td style="width:191px;">C&#38;B</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">no. 0 cover glass</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stereotaxic frame&#160;</td>
			<td style="width:191px;">Kopf</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Rectal probe and heating pad</td>
			<td style="width:191px;">FHC</td>
			<td style="width:429px;">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 height="20">
			<td height="20" style="height:20px;">optical breadboard for imaging</td>
			<td style="width:191px;">Thorlabs</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mineral oil</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;">S55667</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kwik-Cast (Silicone elastomer)</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suture&#160;&#160;</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;">18&#8217;&#8217;, 1667, 4-0</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91500-09, 15018-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forcepts</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">11252-30; #55, 11295-51; Grafe, 11050-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Student Halsted-Mosquito Hemostats</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91308-12</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small Vessel Cauterizer Kit</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-00</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot Bead Sterilizers</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-45</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instrument Case with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20311-21</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Sterilization Containers with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20810-01</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:387px;">2P fixed-stage fluorescence scope for in vivo imaging</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">FV1200 MPE</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Multiphoton laser</td>
			<td style="width:191px;">SpectraPhysics</td>
			<td style="width:429px;">Mai Tai DeepSee</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Green Laser</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">473 nm Laser</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">xy translation base</td>
			<td style="width:191px;">Scientifica</td>
			<td style="width:429px;">MMBP</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">FRET filter cube for YFP and CFP</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">25-X water immersion objective</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">air table</td>
			<td style="width:191px;">Newport</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">custom built light-tight cage</td>
			<td style="width:191px;">Thorlab</td>
			<td style="width:429px;"></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

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.