This work was supported by a Simons Foundation Bridge to Independence Award (SFARI Award #387972; J.J.Y.), a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (J.J.Y.), a Research Fellowship from the Alfred P. Sloan Foundation (J.J.Y.), and research grants from the Angelman Syndrome Foundation (J.J.Y.), the Whitehall Foundation (J.J.Y.), and the NIMH (R01MH122786; J.J.Y.).

1. Mutagenesis cloning to generate&#160;&#160;Ube3a variants
<ol>
	<li>Clone all Ube3a variants into the pCIG2 plasmid (Figure 1A), a bicistronic vector that contains a chicken-&#946;-actin promoter and an internal ribosome entry site (IRES) for EGFP expression19. Full-length Ube3a constructs contain an N-terminal Myc-tag sequence and are cloned into pCIG2 using a 5&#39; SacI site and a 3&#39; XmaI site. In addition, naturally occurring EcoRI, EcoRV, and PstI sites within the Ube3a coding sequence are also used to subclone fragments. 
	NOTE: De-identified variants for Ube3a can be obtained through the literature and in publicly accessible repositories such as the ClinVar database1. Additional unreported variants can be obtained through personal communication with medical centers.</li>
	<li>Use an adapted two-step megaprimer mutagenesis method20 to introduce mutations into the Ube3a reading frame. In the first step, design the mutagenic oligonucleotide that contains the desired mutation (the example shown in this protocol is to generate a UBE3A Q588E variant) flanked by at least 30 base pairs (bp) of homology 5&#8242; and 3&#8242; to the mutation (Figure 1B and Table 1).</li>
	<li>Use the mutagenic oligonucleotide along with a complementary oligonucleotide for the first round PCR to generate a 200-400 bp megaprimer containing the mutation (Figure 1C; Table 2 and Table 3). Use the entire 50 &#956;L of the PCR reaction volume for agarose gel electrophoresis using a 1% gel and subsequent gel purification (Table of Materials). Elute the PCR product in 30 &#956;L of deionized H2O (diH2O) for use in the second round PCR step below.</li>
	<li>Set up the second round PCR as indicated (Figure 1C; Table 2 and Table 3). This step will generate larger DNA fragments (typically ~1-1.5 kb in length) containing the mutation and terminal restriction sites suitable for sub-cloning (Figure 1C).</li>
	<li>After completion of the PCR reaction, purify the resulting product using a PCR purification kit (Table of Materials). Elute in 30 &#956;L and digest all of the purified product and the pCIG2 Ube3a WT construct with the appropriate restriction enzymes (the example shows digestion with EcoRI and XmaI).</li>
	<li>After 2 h of digestion at 37 &#176;C, resolve the digestion products through agarose gel electrophoresis (1% gel), and gel-purify the appropriate products. Set up ligations according to the manufacturer&#39;s protocol (Table of Materials), transform the ligation products into a DH10B bacterial strain (Table of Materials), and then plate with ampicillin (100 &#956;g/mL) selection.</li>
	<li>The next day, pick two to four colonies to inoculate 3 mL cultures containing Luria Broth (LB) and 100 &#956;g/mL ampicillin. Let the cultures grow overnight at 37 &#176;C in an orbital incubator with shaking (225 rpm).</li>
	<li>The following day, use 1-1.5 mL of the bacterial culture to miniprep the plasmid DNA (Table of Materials). Save the remaining culture by placing them at 4 &#176;C. Screen the miniprepped plasmids by Sanger sequencing using an oligonucleotide that binds ~200 bp from the desired mutation.</li>
	<li>Use the bacterial cultures saved at 4 &#176;C to re-inoculate 50 mL cultures to midiprep the correct plasmid (Table of Materials). Fully sequence the Ube3a coding sequence to validate the correct sequence of the DNA.</li>
	<li>Create working stocks of Ube3a variant plasmids, as well as the &#946;-catenin activated reporter (BAR)21, TK-Renilla luciferase, ligase-dead Ube3a (UBE3A C820A mutation), and pCIG2 empty vector at a concentration of 100 ng/&#956;L using sterile H2O for the subsequent transfection steps.</li>
</ol>
2. Preparation and transfection of human embryonic kidney 293T (HEK293T) cells
<ol>
	<li>Perform the assays in HEK293T cells grown in Dulbecco&#39;s modified Eagle medium (DMEM) pre-supplemented with glutamine, 10% fetal bovine serum (FBS), and antibiotic-antimycotic agent as described previously17. Grow the cells in a sterile, humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Using a biosafety cabinet, first plate the suspended HEK293T cells onto tissue culture-treated 96-well flat-bottomed plates at a density of 2.2 &#215; 104 cells per well in 100 &#956;L of culture media and allow them to grow overnight.</li>
	<li>On the second day, transfect the cells in a biosafety cabinet with Firefly and Renilla luciferase reporter plasmids (Table 4) and Ube3a plasmids in triplicate. Perform the transfections in 10 &#956;L of total volume per well using a 10:1:8 ratio of plasmids (50 ng of BAR, 5 ng of TK-Renilla, and 40 ng of Ube3a plasmid per well, respectively), as previously described18, and 0.4 &#956;L of transfection reagent (Table 4). 
	NOTE: For each experiment, an empty vector (GFP only) and a plasmid encoding ligase-dead Ube3a are also transfected to serve as negative controls.</li>
	<li>Create a master mix for the transfections for each variant in a 1.5 mL microcentrifuge tube (Table 4) and incubate at room temperature (RT) for 15 min. After the incubation, gently agitate the transfection mixture by tapping the tube, and then add 10 &#956;L of the transfection mixture directly to the existing growth media in the wells.</li>
	<li>Afterward, return the cells to the incubator and allow the plasmids to express for 48 h. Replacement of the growth media is not necessary.</li>
	<li>Monitor the transfection efficiency by GFP fluorescence using a fluorescence microscope. HEK293T cells are efficiently transfected by this method, and &#62;80% of cells should express GFP after 48 h.</li>
</ol>
3. Measurement of luciferase activity
NOTE: Luciferase activity is assessed using a commercially available system that assays both Firefly and Renilla luciferase (Table of Materials) according to the manufacturer&#39;s protocol.
<ol>
	<li>Carefully aspirate the culture media from transfected HEK293T cells, and then wash the cells with cold phosphate-buffered saline (PBS). Aspirate the PBS and lyse the cells by adding 25 &#956;L of non-denaturing lysis buffer and incubate for 15 min with gentle rocking on ice.</li>
	<li>Afterward, add 20 &#956;L of the resulting lysate (no centrifugation is necessary) into a new 96-well assay plate containing 100 &#956;L of a Firefly luciferase substrate reagent. Mix the plate gently by tapping.</li>
	<li>Load the plate onto a plate reader and measure the luminescence using a top detector with a read height of 1 mm and an integration time of 1 s. 
	NOTE: The gain of the detector may need to be adjusted based on the strength of the signal.</li>
	<li>Immediately after reading the Firefly luciferase luminescence, add 100 &#181;L of Renilla luciferase substrate to the wells. This step simultaneously quenches the Firefly luciferase activity while assessing Renilla luciferase activity. Mix the samples by gentle agitation of the plate and measure the luminescence again to assess the Renilla luciferase activity. 
	NOTE: While other plates can be used for this assay, using plates with black frames and white wells will provide the most sensitive results as this amplifies the luciferase signal while minimizing noise.</li>
	<li>Quantify the reporter responses as the Firefly luciferase to Renilla luciferase ratio (Firefly/Renilla), and average the values of the triplicate measurements. Afterward, normalize the Firefly/Renilla value for each variant against the values for wild-type (WT) Ube3a expressing cells to compare the relative activity of variant proteins.</li>
</ol>

<ol>
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M., 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=Variant+interpretation:+Functional+assays+to+the+rescue.">Variant interpretation: Functional assays to the rescue.</a> American Journal of Human Genetics. 101 (3), 315-325 (2017).</li><li>Scheffner, M., Huibregtse, J. M., Vierstra, R. D., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HPV-16+E6+and+E6-AP+complex+functions+as+a+ubiquitin-protein+ligase+in+the+ubiquitination+of+p53.">The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.</a> Cell. 75 (3), 495-505 (1993).</li><li>Albrecht, U., 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=Imprinted+expression+of+the+murine+Angelman+syndrome+gene,+Ube3a,+in+hippocampal+and+Purkinje+neurons.">Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.</a> Nature Genetics. 17 (1), 75-78 (1997).</li><li>Rougeulle, C., Glatt, H., Lalande, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Angelman+syndrome+candidate+gene,+UBE3A/E6-AP,+is+imprinted+in+brain.">The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain.</a> Nature Genetics. 17 (1), 14-15 (1997).</li><li>Vu, T. H., Hoffman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+Angelman+syndrome+gene,+UBE3A,+is+restricted+to+brain.">Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain.</a> Nature Genetics. 17 (1), 12-13 (1997).</li><li>Kishino, T., Lalande, M., Wagstaff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UBE3A/E6-AP+mutations+cause+Angelman+syndrome.">UBE3A/E6-AP mutations cause Angelman syndrome.</a> Nature Genetics. 15 (1), 70-73 (1997).</li><li>Jiang, Y. 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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=The+autism-linked+UBE3A+T485A+mutant+E3+ubiquitin+ligase+activates+the+Wnt/beta-catenin+pathway+by+inhibiting+the+proteasome.">The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/beta-catenin pathway by inhibiting the proteasome.</a> Journal of Biological Chemistry. 292 (30), 12503-12515 (2017).</li><li>Weston, K. 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The authors have nothing to disclose.

The protocol described here provides an efficient and scalable method to assess the enzymatic activity of Ube3a variants. There are several technical details that warrant careful consideration when using this assay. One consideration is the choice of Wnt reporter plasmids used in this assay. The protocol described here specifically uses the &#946;-catenin activated reporter (BAR)21, a reporter that contains a concatemer of 12 T-cell factor (TCF) response elements separated by specifically...

Recent technological advances have made the sequencing of exomes and genomes routine in clinical settings1,2. This has led to the discovery of a large number of genetic variants, including millions of missense variants that typically change one amino acid in a protein. Understanding the functional significance of these variants remains a challenge, and only a small fraction (~2%) of the known missense variants have a clinical interpretation1,3.
A prominent example of this problem is Ube3a, a gene that encodes an E3 ubiquitin ligase that targets substrate proteins for degradation4. Ube3a resides within chromosome 15q11-13 and is expressed exclusively from the maternally inherited allele5,6,7. Observations from disease genetics strongly suggest that insufficient or excessive activity of the UBE3A enzyme causes distinct neurodevelopmental disorders. Deletion of maternal chromosome 15q11-13 causes Angelman syndrome (AS)8, a disorder characterized by severe intellectual disability, motor impairments, seizures, a happy demeanor with frequent smiling, and dysmorphic facial features8,9,10. In contrast, duplication or triplication of maternal chromosome 15q11-13 causes Dup15q syndrome, a heterogeneous condition recognized as one of the most prevalent syndromic forms of autism11,12,13. In addition, there are hundreds of missense variants identified in Ube3a, the majority of which are considered variants of uncertain significance (VUS) as their functional and clinical significance are unknown. Thus, there is considerable interest in developing methods that can empirically assess Ube3a variants to determine whether they contribute to neurodevelopmental disease.
The UBE3A enzyme belongs to the HECT (homologous to E6-AP C-terminus) domain family of E3 ubiquitin ligases that all possess the eponymous HECT domain, which contains the biochemical machinery necessary to accept activated ubiquitin from E2 enzymes and transfer it to substrate proteins14. Historically, the characterization of E3 enzymes has relied on reconstituted in vitro systems that require an ensemble of purified proteins4,15,16. Such methods are slow and labor-intensive and not amenable to assessing the activity of a large number of variants. In previous work, UBE3A was identified to activate the Wnt pathway in HEK293T cells by modulating the function of the proteasome to slow the degradation of &#946;-catenin17. This insight allows the use of Wnt pathway reporters as an efficient and rapid method to identify both loss- and gain-of-function variants of Ube3a18. The protocol below describes in detail a method to generate Ube3a variants as well as a luciferase-based reporter to assess changes in the activity of Ube3a variants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x), phenol red</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Kb DNA ladder</td>
			<td>Lambda Biotech</td>
			<td>M108-S</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Lambda Biotech</td>
			<td>M107</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Buffer for T4 DNA Ligase with 10 mM ATP</td>
			<td>New England BioLabs</td>
			<td>B0202A</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion HF Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0518S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution</td>
			<td>Corning</td>
			<td>30004CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black/White Isoplate-96 Black Frame White Well plate</td>
			<td>PerkinElmer</td>
			<td>6005030</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin Disodium Salt</td>
			<td>Midwest Scientific</td>
			<td>KCC46000-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber&#160; slides</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter&#160;</td>
			<td>life technologies</td>
			<td></td>
			<td>Cell counting machine</td>
		</tr>
		<tr>
			<td>Custom DNA oligos</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Mix</td>
			<td>New England BioLabs</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569044</td>
			<td>Basal medium for supporting the growth of HEK293T cell line</td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF&#160;</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fisherbrand Surface Treated Tissue Culture Dishes</td>
			<td>Fisherbrand</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple (6x)</td>
			<td>New England BioLabs</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High Efficiency ig 10B Chemically Competent Cells</td>
			<td>Intact Genomics</td>
			<td>1011-12</td>
			<td>E. coli DH10B cells</td>
		</tr>
		<tr>
			<td>HiSpeed Plasmid Midi Kit</td>
			<td>Qiagen</td>
			<td>12643</td>
			<td>Midi prep</td>
		</tr>
		<tr>
			<td>pCIG2 plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3 BAR plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion HF DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530L</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>ProFlex 3 x 32 well PCR System</td>
			<td>Applied biosystems by life technologies</td>
			<td></td>
			<td>Thermocycler</td>
		</tr>
		<tr>
			<td>pTK Renilla plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>Mini prep</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (250)</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>Gel purification</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit (250)</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>rCutSmart Buffer</td>
			<td>New England BioLabs</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI-HF</td>
			<td>New England BioLabs</td>
			<td>R3156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Reader</td>
			<td>BioTek</td>
			<td></td>
			<td>&#160;Plate reader runs Gen5 software v3.08 (BioTek)</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202L</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>TAE Buffer, Tris-Acetate-EDTA, 50x Solution, Electrophoresis</td>
			<td>Fisher Scientific</td>
			<td>BP13324</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 wells, Flat Bottom</td>
			<td>Fisherbrand</td>
			<td>FB012931</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide Solution</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>XmaI</td>
			<td>New England BioLabs</td>
			<td>R0180S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

a scalable, cell-based method for the functional assessment of ube3a variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

Large-scale functional screening of Ube3a missense variants identifies a broad landscape of loss- and gain-of-function mutations 
Previous work with Ube3a mutants suggested that the Wnt response can serve as a reporter of cellular UBE3A protein activity. These observations were expanded, and additional validation experiments were performed to investigate whether the BAR assay is suitable to report a range of UBE3A activities in the cell. First, HEK293T cells were transfected with v...

Watch this Scientific Journal Video about A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants at JoVE.com

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

a-scalable-cell-based-method-for-functional-assessment-ube3a

Research

JoVE Journal

Neuroscience

1.8K Views.  Washington University School of Medicine. A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

Video: A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...