This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

NOTE: All research volunteers in this study were administered informed consent documents approved by the Institutional Review Board of Howard University and Johns Hopkins University. Only participants who consented by signing the informed consent documents were enrolled into the study. The study base for this analysis consisted of: 20 subjects (12 BD and 10 controls; 30% males) and mean age (s.d.) of 38.2 (14.1) years. 
NOTE: Spray all surfaces with RNase Zap to remove RNase contamination from glass and plastic surfaces.
1. Biopsy Procedure
<ol>
	<li>Spray topical lidocaine (xylocaine) liquid 4% and oxymetazoline HCl 0.05% in the nasal cavity of the study participant and allow 15 mins of numbing time.</li>
	<li>Soak &#189; x 3 inch surgical cottonoids in a mixture of lidocaine and oxymetazoline and insert in the nasal cavity along superior portion of the nasal septum and allow to sit for 15 min.</li>
	<li>Using a 30&#176; rigid nasal endoscope to locate the upper section of the nasal cavity, remove nasal mucosa using up-biting nasal forceps from the superior portion of the nasal septum, taking several small specimens.</li>
</ol>
2. Preparation of Tissue Blocks
<ol>
	<li>Immediately prior to biopsy, fill standard sized cryomolds with tissue freezing medium.</li>
	<li>Using autoclaved tweezers, mount fresh tissue in the freezing medium and freeze on dry ice. Fill remainder of cryoblock with the freezing medium, freeze cryoblock on dry ice, and store blocks at -80&#186;C.</li>
</ol>
3. Cryosectioning
<ol>
	<li>Prepare polyethylene naphthalate (PEN) membrane glass slides immediately prior to cryosectioning. Spray slides with RNase Zap solution for 5 min. Then rinse slides in diethylpyrocarbonate (DEPC) water and let air dry for 30 min. Activate dried slides under UV light for 30 mins.</li>
	<li>Cryosection each block entirely at 30 &#181;m. Keep slides on dry ice, and sections frozen as much as possible. Store slides at -80 &#186;C upon completion.</li>
</ol>
4. Laser-Capture Microdissection
<ol>
	<li>Begin by dehydrating slides immediately prior to microdissection. Let frozen slides thaw for 10 sec, and then apply the following ethanol series to each slide: 60% (2 ml, 15 sec), 80% (2 ml, 15 sec), 95% (3 ml, 25 sec), 100% (7 ml, 60 sec).</li>
	<li>Turn photoactivated localization microscopy (PALM) microscope on and set to the following conditions: For 10X magnification set the energy to 82, focus to 74, and speed to 25 - 30. For 20X magnification, set the energy to 63, focus to 67, and speed to 5 - 6. Adjust as necessary to obtain optimal cutting results.</li>
	<li>Visually identify neurons using 20X or 10X magnification. Distinguish the thin surface olfactory neuron layer from underlying sub-mucosa under a microscope without the use of staining procedure. Observe neurons as a dense columnar appearance18.</li>
	<li>Using the Pencil function in the afore-mentioned microscope in step 4.2, trace around the neuronal layer. Trace slightly away from neurons to prevent burning of neurons from laser. This is essential for obtaining high quality RNA. Then initiate laser guided cutting.</li>
	<li>Pick up dissected sections using fine tip microdissection forceps and put tissue in a microtube containing 100 &#181;l lysis buffer on ice. Repeat for all neuronal sections obtained from a single sample. Total dissection time per sample should not exceed 50 min.</li>
	<li>Once dissection is complete, immediately vortex sample lysates and keep on dry ice. Store samples at -80&#186;C for total RNA isolation.</li>
</ol>
5. Isolation of Total RNA and Quality Control Analysis
<ol>
	<li>Thaw sample lysates on ice. Carry out total RNA isolation&#160;with RNAqueous Micro Kit with DNase-I inactivation by following manufacturer&#39;s protocol with no modifications (see Figure 1). The total elution volume is approximately 20 &#181;l.</li>
	<li>Measure RNA concentration and evaluate RNA quality with RNA Integrity Number (RIN) using Bioanalyzer. Samples that meet the quality control criteria in Figure 2 can be used for cDNA synthesis.</li>
</ol>
6. cDNA Synthesis
<ol>
	<li>Synthesize cDNA using a validated commercial kit (see Table of Reagents). Perform the annealing step by combining 0.1 - 1.0 ng of RNA (depending on availability) and RNase free water to a total volume of 8 &#956;l per tube. Then add 1 &#956;l of oligo d(T)20 primer and 1 &#956;l of 10 mM dNTP mix per tube, for a total volume of 10 &#956;l.</li>
	<li>Gently vortex and centrifuge samples and anneal samples at 65 &#176;C for 5 min. After the reaction is completed, immediately cool samples on ice.</li>
	<li>Prepare master mix solution according to the specifications in Table 1. Then add 9.5 &#181;l master mix and 0.5 &#181;l of commercially prepared reverse transcriptase (RT) to each tube. Add 0.5 &#181;l RNase-free water to control tube instead of RT.</li>
	<li>Run a RT reaction with the first incubation at 50 &#186;C for 50 min, then 85 &#186;C for 5 min and a final 4 &#186;C hold, with no cycling.</li>
	<li>Dilute all samples by 5x with water and aliquot. Store samples at -80 &#186;C.</li>
</ol>
7. Real Time PCR &#8211; Confirm Neuronal Enrichment
<ol>
	<li>Prepare the master mix solution according to specifications in Table 2. Use an internal control such as GAPDH. Compare olfactory marker protein (OMP) expression in microdissected tissue with OMP expression in undissected tissue to determine neuronal enrichment. 
	NOTE: OMP is a marker for olfactory neurons. 
	OMP sequence: forward, 5&#8217;-CTGTCGGACCTGGCCAAG-3&#8217; 
	reverse, 5&#8217;-CACCCTCGCCAAAGGTGA-3&#8217; 
	GAPDH sequence: forward, 5&#8217;-CATGAGAAGTATGACAACAGCCT-3&#8217; 
	reverse, 5&#8217;-AGTCCTTCCACGATACCAAAGT-3&#8217;.</li>
	<li>Set up PCR plate by adding 7 &#181;l of master mix in each well and 3 &#181;l of cDNA.</li>
	<li>Centrifuge plate for 5 min at 300 x g (1,300 rpm). Run Real Time-PCR reaction using the default thermal cycling conditions specified for commercial supermix such as SYBR GreenER qPCR.</li>
	<li>Analyze data (see Figure 2 for specifications).</li>
</ol>
8. Real Time PCR &#8211; Expression of Genes of Interest
<ol>
	<li>Perform on samples that show neuronal enrichment of 2 fold or more.</li>
	<li>Prepare master mix on ice for each probe (FAM-labeled probe of interest and VIC-labeled GAPDH) in separate tubes according to the specifications in Table 3.</li>
	<li>Set up a PCR plate with 8 &#181;l master mix in each well and 2 &#181;l of cDNA.</li>
	<li>Centrifuge the plate for 5 min at 300 x g (1,300 rpm). Run Real Time-PCR reaction using the default thermal cycling conditions as per the gene expression master mix protocol provided by the manufacturer. Analyze expression results (see Figure 2 for specifications).</li>
</ol>

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The authors report no financial relationships with commercial interests.

A new platform for obtaining enriched olfactory neuronal layers by combining nasal biopsy and LCM is presented and has been validated in this study. This technique can have widespread implications. It can be applied towards biomarker studies, including those for treatment response, and drug discovery efforts for other neuropsychiatric conditions, making a broader impact in the field.
To obtain high quality neurons amenable to downstream applications, a few critical and troubleshooting steps mu...

Bipolar disorder (BD) is a severe neuropsychiatric disorder, characterized by pathological changes in mood, drive and cognition1. Lithium used for treatment of BD has been shown to alter steady state mRNA levels of a large number of genes in animal studies2, but it remains unknown if any of these molecules is associated with clinical response in humans2. Understanding the mechanism of lithium response would require investigating lithium-induced molecular changes in neuronal tissues. Unfortunately, it is not practical to obtain brain biopsies of BD patients pre- and post-lithium therapy to identify molecular signatures of lithium response. Post-mortem brain tissues have been used to study biomarkers in BD, however, they cannot be used to assess molecular markers associated with dynamic changes in emotions, cognition and drive; and the validity of retrospectively ascertained lithium treatment response can be problematic4. Lymphocytes and other blood cells could be useful, but molecular changes in blood cells may not reflect neuronal alterations3-5. Cerebrospinal fluid may be insufficient to obtain information on intracellular molecules that may reflect disease-associated and medication-reversible intrinsic changes.
The olfactory epithelium (OE) is a unique part of the central nervous system (CNS), embryologically-related to limbic structures6; and it is easily accessible through nasal biopsies. It consists of glial-like supporting (i.e., sustentacular) cells, basal proliferating cells, and olfactory receptor neurons at different stages of development7-9. Therefore, OE provides a unique opportunity to accessibly study dynamic changes in the CNS of patients with neuropsychiatric diseases7. Studies are demonstrating the utility of the OE as a surrogate tissue for investigating disease associated events that reflect those occurring in brain neurons8,9. For example, studies have utilized OE to investigate molecular profiles associated with psychiatric conditions10-14. Olfactory system also serves to identify clinical endophenoytpes such as smell deficits that are associated with negative symptoms of schizophrenia15. Additionally, neurodevelopmental processes continue in the OE throughout life, providing a useful avenue to model the underlying pathophysiology of psychiatric conditions8,9.
However, a drawback to the use of this tissue is the substantial contamination of olfactory biopsies with non-neuronal cells16. For instance, total RNA used for gene expression studies in previous OE studies contained RNA extracted from the entire nasal biopsied tissue including RNA from non-neuronal cells17. Therefore, previous approaches have been limited by the quality of cells. To overcome this issue, a novel approach to obtain enriched neuronal cell populations by combining nasal biopsies with laser-capture microdissection (LCM) has been developed18.
LCM is a technique that allows for the selective isolation of cells using UV laser cutting combined with infra-red laser19-21. Combining LCM with OE approach minimizes substantial contamination of OE by non-neuronal cells, thereby enhancing enrichment of neuronal cells18. Moreover, the neuronal layer can be distinguished from the submucosa layer under a microscope, thereby eliminating the need for staining. Neuronal cell types can further be distinguished from other cell populations using primary antibodies which are expressed by the cell type of interest7. Therefore, this procedure establishes an easier method for the enrichment of almost purely neuronal cell population that can be used for gene expression studies, immunohistochemistry and other morphologic investigations.
This study aims to establish an experimental platform to investigate molecular changes in olfactory neurons associated with disease states and treatment response. To address this, a small set of non-smoking patients who met the DSM-IV diagnostic criteria for BD based on the Diagnostic Interview for Genetic Studies (DIG)22 was recruited to undergo two nasal biopsies: one biopsy pre-treatment with lithium and the second biopsy, after 6 weeks of daily oral lithium therapy. Furthermore, eligible BD patients must be: symptomatic for depression, based on scoring &#8805;10 out of 60 in the clinician administered Montgomery-Asberg Rating Scale (MADRS)23; symptomatic for hypomania or mania, based on a score &#8805;10 out of 56 on the clinician administered Young-Mania Rating Scale (YMRS)24; or &#8805;10 on both MADRS and YMRS. Rater-Inter-Rater coefficient of agreement between clinicians for both scales is &#62;0.96. After biopsies, neurons were enriched from OE by LCM. Following additional quality control measures, to ensure extraction of high quality RNA from the tissue and neuronal enrichment, Real Time RT PCR was conducted to investigate pre- and post-treatment expression levels of genes of interest. The ensuing sections contain a description of the validation of this approach, highlighting the optimization of the protocol and the strategies that were applied for trouble-shooting the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Reagent</td>
			<td style="width:143px;">Manufacturuer&#160;</td>
			<td style="width:175px;">Manufacturer Catalog #</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold Molds</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryosectioning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Membrane Slide 1.0 PEN (D)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>415190-9041-000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase Zap</td>
			<td>Ambion</td>
			<td>AM9780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC Treated Water</td>
			<td>Quality Biological</td>
			<td>351-068-131</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software: Robo v3.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No.5 Dumont Microdissction Forceps</td>
			<td>Roboz</td>
			<td>RS-49085</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous Micro Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cDNA Synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III First Strand Synthesis Kit</td>
			<td>Invitrogen&#160;</td>
			<td>18080-051</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OMP qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR GreenER qPCR SuperMix</td>
			<td>Invitrogen&#160;</td>
			<td>11760-500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Expression Assay Probes</td>
			<td>Applied Biosystems</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
		</tr>
	</tbody>
</table>

olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

The protocol and troubleshooting strategy in the study of molecular signatures were successfully optimized. RNA quality and neuronal enrichment criteria for samples to be used in further downstream Real Time-PCR analysis were standardized. RIN and RNA concentration were examined as confounding factors to detected expression levels. Based on the analysis of several samples with RINs ranging from 1 - 10, it was determined that a minimum RIN of ~3.0 and above is sufficient for downstream analysis in this study. As a result,...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

Research

JoVE Journal

Neuroscience

9.7K visualizações.  Johns Hopkins University.  Neste estudo, um romance plataforma para investigar assinaturas moleculares intraneuronal de resposta ao tratamento do transtorno bipolar (TB) foi desenvolvido e validado. O epitélio olfatório de pacientes com TB foi obtido através de biópsias nasais. Em seguida, captura a laser microdissection foi combinado com Tempo real RT PCR para investigar a assinatura molecular de resposta de lítio em BD. 

Vídeo: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.