This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.

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The authors have nothing to disclose.

Here we isolate single cells from a heterogeneous population using a fluorescent tag, then study each cell with single-cell RNA-seq. We report that 100% of the live TH-eGFP cells that we harvested and sequenced were indeed dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, DDC and&#160;slc6a3. All of the TH-eGFP positive cells expressed these three genes as assessed by RNA-Seq. This was concordant with electrophysiological studies that showed that each TH-eGFP cell also displayed a repertoire of ion channels associated with dopaminergic neurons8, 9. In this protocol we utilized the GENSAT tyrosine hydroxylase-eGFP mouse strain10 as a reliable marker for live dopaminergic neurons. In WT cultures there is presently no reliable way to identify live dopaminergic neurons based on their morphology. Despite our extensive experience in identifying and recording from dopaminergic neurons, in the wildtype cultures only three out of ten of the suspected dopaminergic cells harvested were dopaminergic neurons. Additionally, we observed that not all of the cells that were negative for TH were GABAergic. We defined cells as GABAergic based on the presence of the Gad1 transcript and the absence of the DA-related transcripts as assessed by RNA-seq.
Because dopaminergic neurons represent a small minority of the neurons expressed in primary VM cultures, the ability to reliably identify these neurons in living cultures will enhance the range of available single-cell studies. This protocol can be used for neuroprotection, neurodegeneration, and pharmacological assays to study the effects of various treatments on the dopaminergic transcriptome. Additionally, one can use this protocol for immunohistochemical, electrophysiological, biophotonic11, and in principle, proteomic assays. These assays are especially useful for Parkinson&#39;s disease and addiction research. But of course analogous protocols, on GENSAT mice or similarly labeled mice, could be used for other disease states or for cells that are rare. Additionally, this protocol gives an emerging view of the heterogeneity within a given neuronal subtype.
In previous studies, after harvesting the single cell we placed it directly into PBS; however now we place the harvested single cell directly into the reaction buffer. This results in better quality RNA-Seq libraries as assessed by numbers of genes detected in each library. One of the main limitations of the technique is that fluorescently tagged cells are required. As stated earlier, there is no truly reliable way to identify a specific cell type in a mixed culture based on morphology. The large range of cell-specific fluorescent or Cre-expressing mouse lines now provides appropriately marked cells in many cases. However, care should be taken when selecting a suitable fluorescent mouse line, as an extensive study of transgenic mouse lines found that TH-Cre knock-in mouse lines exhibit pronounced transgene expression in nondopaminergic cells12.
Additionally, another step that may need to be optimized in future experiments is the desired tip size of the micropipette. If future experiments include the use of cell types other than dopaminergic neurons, the micropipette tip size may need to be optimized for the cell of interest.
RNA deep sequencing is a more powerful technique than microarrays. RNA-Seq gives good run-to-run reproducibility. The dynamic range detected is comparable to the actual transcript abundance within cells. One can detect alternative splice forms and novel isoforms. De novo analysis of samples without a reference gene is possible; and if a new reference gene is discovered the data can be re-analyzed for the specific gene(s) without having to run the wet-work experiment again13.
To summarize, we report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. This technique extends other reports that identify acutely dissociated or long-term cultured dopaminergic neurons from GENSAT mice,14,15 and the technique will have widespread applications in neuroscience and molecular biology.

Parkinson&#39;s Disease (PD) is an incurable, age-related neurodegenerative disorder. The cause of this relatively common disorder remains poorly understood. There is widespread neuronal loss throughout the brain, with pronounced neuronal degeneration of dopaminergic neurons in the Substantia Nigra (SNc), leading to diagnostic clinical features of bradykinesia, rigidity and tremor.
Primary mixed cultures containing SNc dopaminergic neurons are especially relevant for Parkinson&#39;s disease. Ventral Tegmental Area (VTA) dopaminergic neurons have been implicated in reward and addiction. Ventral Mesencephalic (VM) primary mixed embryonic cultures contain both SNc and VTA dopaminergic (DA) neurons and GABAergic neurons. VM primary cultures can be useful for neuroprotection assays and to elucidate the selective vulnerability of dopaminergic neurons. There is no reliable way to identify dopaminergic cells in culture based on morphology. Here we develop techniques to identify and harvest single dopaminergic neurons, and construct single-cell, high-yield RNA sequencing libraries.
We report representative RNA transcriptome data for single dopaminergic and GABAergic neurons isolated from midbrain cultures. This protocol can be used for neuroprotection, neurodegeneration, and pharmacological assays to study the effects of various treatments on the DA/GABA transcriptome. Because dopaminergic neurons represent a small minority of the neurons expressed in primary VM cultures, the ability to reliably identify these neurons in living cultures will enable an enhanced range of single-cell studies. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology.

<table><tbody><tr><td>Papain</td><td>Worthington Biochemical Corporation&amp;nbsp;</td><td>LS003126</td><td></td></tr><tr><td>Hanks-balanced Salt Solution (HBSS), 1x</td><td>Gibco&amp;nbsp;</td><td>14175-095</td><td></td></tr><tr><td>Donor Equine Serum</td><td>Thermo Scientific&amp;nbsp;</td><td>SH30074.03</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>A7030-50G</td><td></td></tr><tr><td>Medium: Neurobasal medium</td><td>Gibco&amp;nbsp;</td><td>21103-049</td><td></td></tr><tr><td>Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX</td><td>Gibco&amp;nbsp;</td><td>35050-061</td><td>0.5 mM final concentration.</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma&amp;nbsp;</td><td>A7506</td><td></td></tr><tr><td>B27</td><td>Gibco&amp;nbsp;</td><td>17504-044</td><td>50x stock solution, 1x final concentration.</td></tr><tr><td>35 mm glass bottom dishes</td><td>MatTek&amp;nbsp;&amp;nbsp;</td><td>P35GC-1.5-10-C</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma&amp;nbsp;</td><td>P4957</td><td></td></tr><tr><td>Laminin</td><td>Sigma&amp;nbsp;</td><td>L2020</td><td>Stored at -20 &amp;deg;C in 20 &amp;micro;L aliquots</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Sigma&amp;nbsp;</td><td>DN25</td><td></td></tr><tr><td>Blue pipette tips P1000</td><td>Sorenson Bioscience</td><td>&amp;nbsp;Inc. 10130</td><td></td></tr><tr><td>10 mm shallow Petri dish</td><td>VWR&amp;nbsp;</td><td>25384-324</td><td></td></tr><tr><td>37 &amp;deg;C Water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C, 5% humidity incubator</td><td></td><td></td><td></td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>&amp;nbsp;X100-500ML</td><td></td></tr><tr><td>Donkey serum</td><td>Equitech&amp;nbsp;</td><td>SD-0100HI</td><td>100% stock solution, 1% final concentration.</td></tr><tr><td>Standard Pattern surgical scissors</td><td>Fine Science Tools</td><td>14000-14</td><td></td></tr><tr><td>Forceps - 2x3 teeth</td><td>Fine Science Tools</td><td>11022-14</td><td></td></tr><tr><td>Forceps - Dumont #55</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Forceps - Dumont #5</td><td>Fine Science Tools</td><td>11252-23</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Gibco&amp;nbsp;</td><td>70011-044</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-buffered solution (DPBS) 1x</td><td>Gibco</td><td>14190-144</td><td>500 mL</td></tr><tr><td>21 G needle</td><td>BD PrecisionGlide Needle</td><td>305167</td><td>100 needles</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>model P-87</td><td></td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Sutter Instrument</td><td>&amp;nbsp;MP-285</td><td></td></tr><tr><td>Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing</td><td>Clontech</td><td>634936</td><td>100 rnxs,incl Advantage2PCR Kit</td></tr><tr><td>oligonucleotide (12 &amp;micro;M): SMARTer IIA oligonucleotide (12 &amp;micro;M)</td><td></td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Reverse transcriptase (100 units)</td><td>SMARTcribe reverse transcriptase</td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Primer 1</td><td>3&amp;rsquo; CDSIIa primer&amp;nbsp;</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>Primer 2</td><td>IS PCR primer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>50x 2 polymerase mix</td><td>50x Advantage 2 polymerase mix</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>10x PCR buffer</td><td>10x Advantage 2 PCR buffer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>RNA Spikes</td><td>ThermoFisher</td><td>4456740</td><td></td></tr><tr><td>PCR cooler rack</td><td>IsoFreeze PCR cooler rack</td><td></td><td></td></tr><tr><td>DNA Sample Preparation Kit&amp;nbsp;</td><td>(Illumina/Nextera) Nexter</td><td>FC-121-1030</td><td>24 Samples</td></tr><tr><td>PCR master mix</td><td>Nextera PCR master mix (NPM)</td><td></td><td>From the Nextera Kit&amp;nbsp;</td></tr><tr><td>PCR primer cocktail (PPC)&amp;nbsp;</td><td>Nextera PCR primer cocktail (PPC)&amp;nbsp;</td><td></td><td>From the Nextera Kit</td></tr><tr><td>Fluorometer</td><td>The Qubit ThermoFisher</td><td>Q33216</td><td></td></tr><tr><td>HS DNA BioAnalyzer kit</td><td>Agilent</td><td>5067-4626</td><td>110 rxns</td></tr><tr><td>Electrophoresis system&amp;nbsp;</td><td>Agilent 2100 Bioanalyzer.</td><td>G2938C</td><td></td></tr><tr><td>Magnetic beads: Agencourt AMPure&amp;nbsp;</td><td>Beckman Coulter</td><td>A63880</td><td>5 mL</td></tr><tr><td>RNAse wipe: RNAse Zap wipes</td><td>Ambion</td><td>AM9786</td><td>Size 100 Sheets</td></tr><tr><td>Qubit ds HS Assay Kit</td><td>Molecular probes&amp;nbsp;</td><td>Q32854</td><td>500 assays</td></tr><tr><td>Gel extraction kit: Qiaquick Gel Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>28704</td><td></td></tr><tr><td>Glass capillary tubing</td><td>(Kimax-51, 1.5 - 1.8 mm o.d.)</td><td></td><td></td></tr><tr><td>Microforge</td><td>Narishige (or equivalent)&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sequencer</td><td>Illumina HiSeq instrument</td><td></td><td></td></tr><tr><td>GENSAT tyrosine hydroxylase-eGFP mouse strain&amp;nbsp;</td><td></td><td>MMRRC stock number: 000292-UNC</td><td>Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center</td></tr></tbody></table>

reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a tyrosine hydroxylase promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their RNA transcriptome using RNA-seq (Figure 1). The data showed that 100% of the TH-eGFP-positive cells that were harvested and sequenced were dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, dopa decarboxylase (DDC), and the dopamine transporter DAT (slc6a3). All of the TH-eGFP positive cells expressed these three genes as assessed by RNA-Seq. In each single-cell RNA-Seq library 6,000 - 8,000 protein coding genes were detected (Figure 2). The dopaminergic neurons robustly express dopamine-related transcripts in addition to several nicotinic acetylcholine receptor (nAChR) subunits (Table 1). We observed that not all of the cells that were negative for TH were GABAergic; therefore we defined cells as GABAergic based on the presence of the Gad1 transcript as assessed by RNA-seq. The cells that we defined as GABAergic neurons do not express dopamine-related transcripts but as mentioned do express a major gene for GABA synthesis, Gad1. The single-cell libraries also revealed long non-coding RNA, as well as transcripts encoding channels, receptors, nuclear proteins, histones, organellar proteins, and transcription factors.
<img alt="Figure 1" src="/files/ftp_upload/54981/54981fig1.jpg" /> 
Figure 1. RNA-Seq in Individual Neurons, as Performed in Our Experiments on Primary TH-eGFP Mouse Midbrain Cultures. (1) Culture TH-eGFP mouse ventral neurons for 3 weeks. (2) Identify fluorescent single neurons by exciting GFP using an Hg light source and FITC/GFP filter. (3) With the same 40X objective under phase optics, visualize single neurons and correctly position the pipette. (4 - 5) Aspirate the single cell into the glass micropipette. Images show a cell after harvesting: (4) Phase 40X; (5) eGFP fluorescence. (6) Lyse cells with lysis buffer with oligo-dT primer at 72 &#176;C. (7) cDNA synthesis; 26 cycles amplification. (8) Transposon-mediated &#34;tagmentation&#34; of cDNA. (9) cDNA quality control check and multiplexed sequencing. (10) Subsequent computational analysis using Tophat, Cufflinks and Cuffdiff7. Relative abundance of expressed genes was measured in units of fragments per kilobase of transcript per million mapped reads (FPKM). The goal of RNA-seq is to provide a list of genes expressed in a cell and their relative abundance. Images (2 - 4) are from the current work, (6 - 10) are modified from a previous report1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54981/54981fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54981/54981fig2.jpg" /> 
Figure 2. The Number of Protein Coding Genes Detected in the Single Dopaminergic RNA-Seq Libraries Generated from TH-eGFP-positive Cells. 6,000 - 8,000 protein genes were detected in the single cell libraries. FPKM: Fragments per kilobase of transcript per million mapped reads (FPKM). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54981/54981fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genes</td>
			<td>DA single cells n = 10</td>
			<td>GABAergic single cells n = 7&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">FPKM (Mean &#177;SEM)</td>
		</tr>
		<tr>
			<td>TH</td>
			<td>4714 &#177;764</td>
			<td>1.2 &#177;0.14</td>
		</tr>
		<tr>
			<td>DDC</td>
			<td>779 &#177;167</td>
			<td>0.8 &#177;0.1</td>
		</tr>
		<tr>
			<td>slc6a3/DAT</td>
			<td>506 &#177;144</td>
			<td>0.5 &#177;0.04</td>
		</tr>
		<tr>
			<td>Drd2</td>
			<td>14 &#177;8</td>
			<td>0.1 &#177;0.004</td>
		</tr>
		<tr>
			<td>chrna6 (&#945;6)</td>
			<td>174 &#177;57</td>
			<td>0.0</td>
		</tr>
		<tr>
			<td>chrna4 (&#945;4)</td>
			<td>17 &#177;5</td>
			<td>26.4 &#177;2.3</td>
		</tr>
		<tr>
			<td>chrnb2 (&#946;2)</td>
			<td>9 &#177;3</td>
			<td>17.3 &#177;1.2</td>
		</tr>
		<tr>
			<td>chrnb3 (&#946;3)</td>
			<td>38 &#177;13</td>
			<td>0.0</td>
		</tr>
		<tr>
			<td>Gad1</td>
			<td>0.0</td>
			<td>959.0 &#177;70.0</td>
		</tr>
		<tr>
			<td>Htr3a</td>
			<td>0.0</td>
			<td>0.6 &#177;0.01</td>
		</tr>
		<tr>
			<td>Htr3b</td>
			<td>0.0</td>
			<td>0.0</td>
		</tr>
	</tbody>
</table>
Table 1. Dopamine-related and nAChR Gene Transcripts in Dopaminergic&#160;and GABAergic Neurons at Day 21 in VentrMidbrain Cultures. RNA-Seq data for TH-eGFP positive and negative neurons are shown. Single cells were harvested on culture day 21. RNA-Seq data show that TH-eGFP positive neurons had high levels of DA-related gene transcripts, including tyrosine hydroxylase (TH), dopa decarboxylase (DDC), the dopamine transporter DAT (slc6a3), and nicotinic acetylcholine receptor (nAChR) subunits including &#945;6, &#945;4, &#946;2 and &#946;3. However, these cells had low levels of the GABAergic marker gene glutamic acid decarboxylase 1 (Gad1) and the serotonin 5-HT3 receptor A and B subunits (Htr3A and Htr3B). GABAergic neurons have low to no levels of DA-related gene transcripts, chrna6, chrnb3, and Htr3A/B, but have high levels of Gad1. RNA-Seq (Cuffdiff) data are shown for dopamine-related, GABAergic, nicotinic receptor, and serotonin transcripts. FPKM: Fragments per kilobase of transcript per million mapped reads (FPKM).

Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

reliable-identification-living-dopaminergic-neurons-midbrain-cultures

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.3K Views.  California Institute of Technology (Caltech).  Bij de ziekte van Parkinson (PD), substantia nigra (SNC) dopaminerge neuronen degenereren, waardoor motorische stoornissen. Hier melden wij een protocol voor het kweken van ventrale middenhersenen neuronen van een muis tot expressie eGFP gedreven door een tyrosine hydroxylase (TH) promotor sequentie, het oogsten van individuele fluorescerende neuronen uit de culturen, en het meten van hun transcriptoom met behulp van RNA-seq. 

Video: Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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

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

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

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

Developmental Biology

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

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