This work was supported by NIH grants R01DA021194 and R01NS058850.

1. Brain slice preparation
<ol>
	<li>Young (15-40 days old) male and female Sprague-Dawley rats are used. (We also use this same protocol in mice.) Under deep urethane anesthesia, animals are decapitated and the brain is quickly dissected out. Then 300 &mu;m-thick coronal brain slices containing the midrostral part of substantia nigra are cut on a Leica vibratome (VT-1200S). The slice cutting procedure is performed in an ice-cold, oxygenated high sucrose cutting solution containing (in mM): 220 sucrose, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 10 D-glucose. It is bubbled with a mixture of 95% O2/5% CO2 to keep oxygenated and maintain pH at 7.4. Keep the cutting solution ice-cold throughout the procedure.</li>
	<li>The brain slices are incubated in an incubation chamber filled with an oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, 10 D-glucose. It is bubbled with a mixture of 95% O2/5% CO2 to keep oxygenated and maintain pH at 7.4. The incubation temperature is 34 &deg;C for 45 min and then at room temperature until the brain slice is transferred to the recording chamber.</li>
</ol>
2. Electrophysiological fingerprinting of nigral neurons
<ol>
	<li>One brain slice is transferred to a patch clamp recording chamber continuously perfused with the oxygenated aCSF at 32 &deg;C.</li>
	<li>Patch pipettes are pulled from autoclaved borosilicate (KG-33) glass capillary tubing (1.10 mm i.d., 1.65 mm o.d., King Precision Glass, Claremont, CA) using a PC-10 puller (Narishige, Tokyo, Japan) and filled with intracellular solution prepared with DNase-RNase-free water (Fisher Scientific) (containing 135 mM KCl, 0.5 mM EGTA, 10 mM HEPES, 1.5 mM MgCl2, pH adjusted to 7.3). The patch pipettes have resistances of 2-3 M&Omega; in the bath.</li>
	<li>The substantia nigra is identified according to its distinct anatomical location (Fig. 1A1-2). Under visual guidance of a video microscope (Olympus BX51W1) equipped with Nomarski optics and 60X water immersion lens, large cells in the substantia nigra pars compacta (SNc) and substantia nigra pars reticulata (SNr) (possible DA neurons and GABA neurons) are selected for recording. Conventional giga-ohm tight seal whole-cell configuration is obtained (Fig. 1B1). Current clamp and voltage clamp analyses can be performed. To minimize mRNA degradation, electrophysiological recording should be brief (&le;5 min). One option is to record only the membrane and spiking properties to provide electrophysiological fingerprints for the neurons of interests (Fig. B2-3). This brief recording takes only about 1 min.</li>
</ol>
3. Cytoplasm aspiration
<ol>
	<li>After electrophysiological fingerprinting and characterization of SNr GABA and DA neurons, gentle mouth suction is applied to aspirate the cytoplasm. The cell nucleus may also be aspirated, resulting in genomic DNA contamination. The tight seal should not be broken; otherwise extracellular debris including mRNA may be sucked into the patch pipette and contaminate the cell content. The integrity of the tight seal is good as long as the increase in the holding current at -70 mV does not exceed 100 pA.</li>
	<li>Then the aspirated cell content is expelled into a 0.2 mL PCR tube by breaking the pipette tip and a small positive pressure. One cell's content is collected into one PCR tube. After accepting the cell content, the collecting PCR tube, pre-cooled on ice, is immediately placed in a -20 &deg;C freezer.</li>
	<li>To rule out contamination of extracellular debris that may contain mRNAs, the patch pipette is lowered into the tissue without actually aspirating cytoplasm and then the pipette content is subjected to RT-PCR.</li>
</ol>
4. Reverse transcription (RT)
<ol>
	<li>After collecting cell content from a reasonable number of neurons, usually 10-20, RT should start immediately to minimize the degradation of mRNAs.</li>
	<li>Since the quantity of the cell content from a single neuron is too small, RNA isolation procedure is not performed. To remove the potential contaminating genomic DNA, the aspirated cell content is first digested by DNase I (5 &mu;L DNase I and 1.6 &mu;L DNase I Buffer, 5 min at 25 &deg;C). Then DNase I is inactivated by adding 1.2 &mu;L 25 mM EDTA and incubating at 75 &deg;C for 5 min.</li>
	<li>cDNA is synthesized using the SuperScript III reverse transcriptase-based Cells-Direct cDNA Synthesis kit (Invitrogen, Table 1). First strand cDNA is synthesized at 50 &deg;C for 50 min, then the reaction is inactivated by incubation at 85 &deg;C for 5 min and 1 &mu;L RNase H is added to digest mRNA (37 &deg;C for 20 min). The single-stranded cDNA is stored at -20 &deg;C for later PCR amplification.</li>
	<li>Complete removal of genomic DNA is verified by RT-minus control in which the reverse transcriptase is omitted while all other reaction components were exactly the same.</li>
</ol>
5. Two stage PCR amplification
<ol>
	<li>Primers were designed according to the sequences in GenBank. Whenever possible, intron-spanning primer pairs were used that help detect genomic DNA contamination. Primer pair sequences are listed in Table 2. The effectiveness of the primer pairs should be first verified by whole brain mRNAs that yield positive products. All primers are synthesized by Integrated DNA Technologies (Coralville, Iowa, USA).</li>
	<li>In the first stage, 5 &mu;L of the 30 &mu;L cDNAs is amplified for 35 cycles in the presence of primer pair (Table 2). The thermal cycling protocol of the thermal cycler (MasterCycler, Eppendorf, Table 1) is 2 min at 94 &deg;C for the initial denaturation, then 35 cycles of 15 s at 94 &deg;C to denature, 30 s at 55&deg;C to anneal, and 45 s at 72&deg;C to extend, followed by 7 min for a final extension. Taq DNA polymerase, deoxynucleotide triphosphates (dNTPs), and all other components necessary for PCR amplification are provided by a PCR super mix (Invitrogen, Table 1)</li>
	<li>In the second stage PCR, the product of 1 &mu;L from the 1st stage PCR amplification is used as template and the same primer pair used in the first stage is used (Table 2). 40 cycles are run with the extension time shortened to 30 s. The same PCR super mix is used.</li>
	<li>PCR products from the 2nd stage amplification are separated by 2.5% agarose gel electrophoresis, visualized by ethidium bromide (0.05 mg/100 mL gel) or gelgreen under UV light, and photographed (Fig. B4). The positive bands are then cut out, extracted using a Qiagen extraction kit (Table 1) and sequenced, and compared with the published sequences of the target genes.</li>
</ol>
6. Representative results:
The dopamine (DA) neurons in the substantia nigra pars compacta and pars reticulata and the neighboring GABA projection neurons in the substantia nigra pars reticulata have distinct electrophysiological characteristics (Zhou et al. 2006; Ding et al. 2011). As shown in Fig. 1B2, SNr GABA neurons exhibit high frequency spontaneous spiking around 10 Hz. The spikes have a base duration around 1 ms. Upon hyperpolarizing current injection, SNr GABA neurons display a weak depolarizing sag in response to hyperpolarizing current injection, indicating a weak expression of Ih current in these neurons (Fig. 1B2). In contrast, the nigral DA neurons exhibit low frequency (around 2 Hz) spontaneous spikes that have a base duration around 3 ms. DA neurons also show a pronounced sag in response to hyperpolarizing current injection, indicating a strong expression of Ih current (Fig. 1B3) (Zhou et al. 2006; Ding et al. 2011).
scRT-PCR detected mRNA for glutamic acid decarboxylase 1 (GAD1, the key enzyme for GABA synthesis and a marker for GABA neurons) in electrophysiologically identified SNr GABA neurons, but not in DA neurons (Fig. 1B4,5). In contrast, scRT-PCR detected mRNA for tyrosine hydroxylase (TH, key enzyme for dopamine synthesis and a marker for DA neurons) in electrophysiologically identified SNc and SNr DA neurons, but not in GABA neurons (Fig. 1B4,5). So scRT-PCR results confirm the electrophysiological neuron identification. Next, scRT-PCR was used to profile the expression of voltage-activated Kv3 channel subunits, Kv3.1, Kv3.2, Kv3.3 and Kv3.4. These subunits contribute to form voltage-gated K+ channels of diverse properties depending on subunit composition (Ding et al. 2011). In the example SNr GABA neuron shown in Fig. 1B4, mRNAs for Kv3.1, Kv3.2, Kv3.3 and Kv3.4 were detected. In the example SNr DA neuron shown in Fig.1B5, only Kv3.2, Kv3.3 and Kv3.4 were detected. In our pooled data, Kv3.1 was more frequently detected in SNr GABA neurons than in nigral DA neurons, indicating a higher expression level of Kv3.1 in the fast-spiking SNr GABA neurons (Ding et al. 2011).
<img alt="Figure 1" src="/files/ftp_upload/3136/3136fig1.jpg" /> 
Figure 1. Electrophysiological identification of SNr GABA neurons and nigral DA neurons. A: nigral areas can be identified by their distinct anatomical location. A1 shows the location of SNc and SNr in a coronal Nissl-stained section captured with a 1X objective. The boxed area was captured with a 10X objective and displayed in the middle as indicated by the arrow. The cell-rich SNc and cell-poor SNr are clearly visible. A2 shows the location of SNc and SNr in a live, unstained coronal section captured with a 4X objective. The cell-rich SNc and cell-poor SNr are also clearly identifiable. VTA, ventral tegmental area. B1 shows an SNr GABA neuron being patch clamped in whole-cell mode. B2 shows the typical electrophysiological properties of SNr GABA neurons in current clamp recording mode. These neurons fire spontaneous high frequency action potential of short duration and have a weak Ih-mediated sag response to hyperpolarizing current injection. B3 shows typical electrophysiological properties of nigral DA neurons in current clamp recording mode. They fire spontaneous low frequency action potentials of long duration and have a prominent Ih-mediated sag (arrow head) response to hyperpolarizing current injection. Thus, SNr GABA neurons and nigral dopamine neurons are clearly identified electrophysiologically. B4 shows a gel picture of scRT-PCR products from an electrophysiological identified SNr GABA neuron. Glutamate decarboxylase 1(GAD1) mRNA but no Tyrosine hydroxylase (TH) mRNA was detected. mRNAs for Kv3.1, Kv3.2, Kv3.3 and Kv3.4 were detected in this SNr GABA neuron. B5 shows scRT-PCR detection of neuronal Kv3 channel mRNAs in a SNr DA neuron. TH mRNA but no GAD1 mRNA was detected in an electrophysiologically identified SNr DA neuron. mRNAs for Kv3.2, Kv3.3 and Kv3.4 were detected in this SNr DA neuron. B6 shows pooled data.
7. Potential false negative and false positive results
Based on our experience, several factors can lead to false negative scRT-PCR results. These factors include failure in aspirating sufficient amount of the cytoplasm due to patch pipette tip clogging, RNase contamination, and inefficient PCR primers. Patch pipette tip clogging usually can be seen on the video monitor and indicated by increased access resistance. RNase contamination can be avoided by thorough deactivation and wearing gloves. PCR primer effectiveness should be tested by using total RNA from a brain tissue punch (Zhou et al. 2008; Ding et al. 2011).
Since we always use DNase to first treat our samples before RT, we have not encountered false positive results. Theoretically, without DNase digestion, genomic DNA contamination and thus false positive detection may occur when the gene is intronless or the primer pair does not span the intron region.

<ol>
	<li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+pacemaker+frequency+of+individual+dopaminergic+neurons+by+Kv4.3L+and+Kchip3.1+transcription.">Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and Kchip3.1 transcription.</a> EMBO J. 20, 5715-5724 (2001).</li><li>Surmeier, D. J., Song, W. J., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+expression+of+dopamine+receptors+in+neostriatal+medium+spiny+neurons.">Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.</a> J. Neurosci. 16, 6579-6591 (1996).</li><li>Zhou, F. W., Matta, S. G., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutively+active+TRPC3+channels+regulate+basal+ganglia+output+neurons.">Constitutively active TRPC3 channels regulate basal ganglia output neurons.</a> J. Neurosci. 28, 473-482 (2008).</li><li>Zhou, F. W., Jin, Y., Matta, S. G., Xu, M., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-short+dopamine+pathway+regulates+basal+ganglia+output+neurons.">An ultra-short dopamine pathway regulates basal ganglia output neurons.</a> J. Neurosci. 29, 10424-10435 (2009).</li><li>Ding, S., Matta, S. G., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv3-like+potassium+channels+are+required+for+sustained+high-frequency+firing+in+Basal+Ganglia+output+neurons.">Kv3-like potassium channels are required for sustained high-frequency firing in Basal Ganglia output neurons.</a> J. Neurophysiol. 105, 554-570 (2011).</li><li>Zhou, F. W., Xu, J. J., Zhao, Y., Ledoux, M. S., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+functions+of+histamine+H1+and+H2+receptors+and+H3+receptors+in+substantia+nigra+pars+reticulata.">Opposite functions of histamine H1 and H2 receptors and H3 receptors in substantia nigra pars reticulata.</a> J. Neurophysiol. 96, 1581-1591 (2006).</li></ol>

No conflicts of interest declared.

The combination of patch clamp recording in brain slice with scRT-PCR we demonstrated here provide an excellent method to investigate the mRNA expression profiles for ion channels, receptors, and key enzymes for neurotransmitter synthesis in individually characterized neurons. This is particularly useful when the protein in question can not be detected and localized using other methods such as immunohistochemistry due to low expression level and/or lack of suitable antibody (Surmeier et al. 1996; Zhou et al. 2009). The h...

<img src="/files/ftp_upload/3136/3136table1.jpg" alt="Table 1" /> 
Table 1. Key reagents and equipment
<img src="/files/ftp_upload/3136/3136table2.jpg" alt="Table 2" /> 
Table 2. Primer pairs for rat neuronal Kv3 channel, tyrosine hydroxylase (TH) and glutamic acid decarboxylase 1 (GAD1) mRNAs for scRT-PCR

profiling voltage-gated potassium channel mrna expression in nigral neurons using single-cell rt-pcr techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Watch this Scientific Journal Video about Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques at JoVE.com

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

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15.2K Views.  University of Tennessee College of Medicine. Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

Video: Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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.

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, ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...