Name: modeling neuronal death and degeneration in mouse primary cerebellar granule neurons
Uploaded: 2017-11-06T16:30:00
Description: Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

This work is supported by Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research grants to A.J.-A.

This protocol is based on modifications of procedures that have been described previously 18,24,25,26,27. This protocol is approved by the Animal Care Committee at McGill University.
1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.
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Here we provide a simple method for the culturing of primary mouse cerebellar granule neurons (CGNs), loss and gain of function studies, and modeling different mechanisms of cell death. Several factors affect the reproducibility of the results using this procedure which require close monitoring. These include the purity of the culture including the elimination of glial cells in the culture, the confluence of the culture, and maintaining healthy cells. Introducing variability in these factors can bias the results and chal...

Cerebellar granule neurons (CGNs) are well characterized in culture and have served as an effective model to study neuronal death and development 1,2,3,4,5,6. The early expression of N-methyl-D-aspartate (NMDA) receptors in CGN cultures in vitro makes them an attractive model to study NMDA-induced signalling. Activation of these receptors with NMDA in conjunction with membrane depolarization is used to model physiological neuronal activity stimulation, and has allowed for research into the mechanisms of synaptic plasticity 7,8. On the contrary, over-stimulation of these receptors by NMDA ligand can be used to model excitotoxicity, a major mechanism of neuronal loss in acute brain damage and neurodegenerative diseases 9. One mechanism for the induction of excitotoxicity is through ATP starvation with reduced oxygen, as seen with acute neuronal injury. This results in membrane depolarization and elevated levels of glutamate release at the synapse. The subsequent overstimulation of the NMDA receptor by elevated glutamate results in excessive Ca2+ influx via these receptors, which in turn activates several pathways including Ca2+-activated proteases, phospholipases, and endonucleases, resulting in the uncontrolled degradation of critical cellular components and cell death. Additionally, high intracellular Ca2+ leads to the generation of oxygen free radicals and mitochondrial damage 10,11.
While the majority of neuronal loss following NMDA-induced neuronal excitotoxicity is due to calcium influx and is Bax/Bak independent, other mechanisms of cell death cannot be excluded from this model. The appearance of both necrotic and apoptotic like cell death due to excitotoxicity is partially due to the generation of reactive oxygen species (ROS) and DNA damage caused by high intracellular Ca2+ levels 12. DNA damage results in neuronal death through apoptotic mechanisms, being correlated with hallmarks of apoptotic cell death, such as the appearance of chromatin masses and apoptotic bodies. Induction of apoptosis is mediated through the release of cytochrome c from the mitochondria, and has been shown to be dependent on Bax/Bak oligomerization 13. Bax/Bak oligomerization promotes pore formation in the outer mitochondrial membrane, resulting in cytochrome c release and the activation of pro-apoptotic regulators as seen with mild ischemic injury 14.
Generation of ROS is a significant issue in the brain due to the low endogenous levels of antioxidants, coupled with the large oxygen requirement for neuronal functioning 15. When exposed to an ischemic event, nitric oxide synthase is upregulated , producing nitric oxide and increasing reactive oxygen species 14. The increased concentration of oxygen radicals can result in DNA damage and indirectly cause energy starvation. High levels of DNA double-stranded breaks are remedied by the activation of poly ADP-ribose polymerase-1 (PARP-1), an eukaryotic chromatin-bound protein responsible for catalyzing the transfer of ADP-ribose units from NAD+, a process integral to DNA repair 16. However, with excessive damage due to oxidative stress, PARP-1 activation can cause energy starvation due to the increased drain on NAD+, a necessary substrate for ATP production through oxidative phosphorylation. Ultimately, oxidative stress will trigger apoptosis in a Bax/Bak dependant manner leading to mitochondrial cytochrome c release, and has been shown to induce mitochondrial remodelling in CGNs 17.
Finally, changes in concentration of potassium chloride (KCl) in CGN cultures can be used to model low potassium/depolarization mediated apoptosis 18,19,20. When exposed to low levels of K+, CGNs undergo distinct physiological changes, resulting in reductions of both mitochondrial respiration and glycolysis, attributed to decreased cellular demand 21, as well as reduction in levels of nuclear factor-&#954;B (NF&#954;B) which regulates activities including inflammation and synaptic transmission 22. This model is of particular interest for the study of cell death during neuronal development. The low K+ environment more closely resembles physiological conditions, and causes hallmarks of cell death seen during neuronal development 23.
In summary, CGNs provide a longstanding model to investigate the underlying molecular mechanisms of neuronal death and degeneration. The following protocol will allow isolation and culturing of CGNs, expression or repression of a particular genetic pathway using viruses and the induction of neuronal death via different mechanisms representing neuronal injury and degeneration.

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modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

With careful dissection, the intact brain should be removed with minimal damage as seen in Figure 1A-B. Effort should be taken to minimize damage to the brain during removal, particularly damage to the cerebellum. Damaging the cerebellum makes for more difficult identification and complete removal of the meninges, and increases the likelihood of contamination of the neuronal culture. Once the meninges have been removed, the cerebellum can be ...

Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

The overall goal of this procedure is to produce healthy pure populations of cerebellar granule neurons and to genetically manipulate them and model different mechanisms of neuronal injury in the primary culture in vitro. This method can help answer key questions in the field of neuronal injury. We use this protocol to investigate the underlying molecular mechanisms of neural injuries following acute brain damage and neuro-generative diseases.The main advantage of this protocol is that we can model different mechanisms of cell death such as excitotoxicty, oxidative stress, DNA damage, and developmental event using the culture system. Although this method can provide insights into neuronal injury it can also be used with rat's cerebellar neurons to study neuronal activity and morphogenesis in response to growth factors and side events. To extract the brain of a six to seven day old mouse, use a pair of forceps to grasp a head, and with micro-dissection scissors, cut the skin anteriorly towards the head.Push back the skin to reveal the skull. Next, penetrate the skull with the tip of the scissors and cut anteriorly and laterally. Taking care not to damage the cerebellum, and facilitate identification and removal of meninges, then use forceps to peel back the skull exposing the brain.With a pair of forceps or spatula, gently tease out the brain into cool dissection solution. To isolate the cerebellum, place the brain in the dissection solution supplemented with magnesium sulfate and keep the solution and the brain on ice. Under the dissection microscope, remove the meninges by using fine forceps, then dissect out the cerebellum from the brain in magnesium sulfate supplemented dissection solution.This helps in peeling off the remaining meninges and allows one to go in between layers to clean the cerebellar folds. The presence of meninges is neuronal culture results in unhealthy cells and eventual cell death. As such, it's important to ensure complete removal of meninges before proceeding with culture.After that, turn the cerebellum to its ventral side and ensure the removal of the choroid plexus. Next, pull the cerebella into a 35 millimeter dish containing one milliliter of magnesium sulfate supplemented dissection solution. Chop the tissues into small pieces and transfer them into a 50 milliliter tube containing 30 milliliters of magnesium sulfate buffered dissection solution.In this step, centrifuge the 15 milliliter tube containing the chopped cerebral tissue for five minutes at 644 times g, and four degrees Celsius. After that remove the supernatant, and add 10 milliliters of trypsin dissection solution. Then shake the table at high speed for 15 minutes at 37 degrees Celsius.Add two milliliters of trypsin inhibitor solution one to the tube and rock gently for two minutes. Subsequently, centrifuge the tube for five minutes at 644 times g and four degrees Celsius. After five minutes, remove the supernatant, and add two milliliters of trypsin inhibitors solution two before transferring it to a 15 milliliter tube.Then triturate the tissue in the 15 milliliter tube until the solution becomes murky. Let it settle for five minutes. Then remove the clear supernatant and transfer it to a new tube containing one milliliter of calcium chloride supplemented dissection solution.Add another two milliliters of trypsin inhibitor solution two to the bottom of the tube containing the pellet. Triturate it again and let it settle for five minutes. Remove the supernatant and add it to the tube containing supernatant from the previous step.Repeat this process until most of the tissue is mechanically dissociated. Add 0.3 milliliters of calcium chloride supplemented dissection solution to the supernatant collection for every milliliter of supernatant. Mix the contents of the tube and then centrifuged for five minutes at 644 times G at room temperature.Following that, remove the supernatant. Add 10 milliliters of fresh media to the pellet and mix. Then count the live cells and dilute them to a concentration of 1.5 times 10 to the six cells per milliliter.Plate the cells on the previously prepared poly D-lysine plates. For four well plates, plate 0.5 millimiters of the sample giving 7.5 times 10 to the fifth cells per well. For 35 millimeter dishes, plate four milliliters of the sample, giving six times 10 to the sixth cells per plate.For cover slips, plate 0.5 milliliters giving 7.5 times 10 to the fifth cells per well. After 24 hours, add AraC to the plates to reduce glial contamination. If the cells are to be maintained for seven to eight days, repeat this treatment on day three and maintain the cultures in a 5%CO2 incubator at 37 degrees celsius.For the cultures maintained longer than five days feed the culture with glucose every two days starting on day five. To induce neuronal excitotoxicity with NMDA, following seven days in vitro, treat the cerebellar granule neurons with 100 micromolar NMDA and 10 micromolar glycine for one hour. Then, replace the medium with conditioned medium from the parallel cultures with no treatment.This concentration results in 50%cell death at 24 hours post treatment. For ROS induced cell death, treat the neurons with hydrogen peroxide at 75 to 100 micromolar for five minutes. After five minutes, switch it to the conditioned media from parallel cultures.Due to the instability of hydrogen peroxide, the concentration must be optimized to a level that induces between 50 and 70%cell death after 24 hours. This concentration in usually between 75 and 100 micromolar. To induce cell death by DNA damage, treat the cerebellar granule neurons with 10 micromolar camptothecin to induce more than 50%cell death within 24 hours.For low potassium induced neuronal apoptosis in cerebellar granule neurons, change the media containing 25 millimolar potassium to low potassium medium with five millimolar potassium following seven days in vitro. Neurons were transduced with lentivirus for RFP at MOI of three at the time of plating. Fixed and stained at seven days in vitro.The co-localization of RFP signal MAP2 and Hoechst is shown to demonstrate healthy neurons that are fully transduced by lentiviruses. The images shown here represent live-dead assay analyses of neurons infected with different MOI of adenovirus to measure toxicity. Cerebellar granule neurons infected with adenovirus expressing LacZ at an MOI between 25 and 50 allows for maximal efficiency while maintaining minimal toxicity.Less than a 1%difference in cell survival compared to control is seen when infecting at this MOI. These figures show the Hoechst staining of control cerebellar granule neurons and cerebellar granule neurons treated with NMDA to induce cell death. Note the formation of pyknotic nuclei with NMDA treatment.This is an indication of cell death and can be seen in approximately 50%of the culture 24 hours after treatment with 100 micromolar NMDA and 10 micromolar glycine. Once mastered, this technique can be performed in two and a half hours with six mouse pops, if performed properly. When performing this technique, it's important to use aseptic technique and chained surgical instruments as required in order to minimize risk of culture contamination.After its development, this technique paved the way for researchers in the field of neuroscience to study neuronal development and neuronal injury in primary mouse neurons. After watching this video, you should be able to culture cerebellar granular neurons, genetically manipulate them using viruses and induce varying mechanisms of neural injury. Following this procedure, other methods like immunofluorescence or cell viability assays can be performed to answer questions like whether a gene of interest enhances cell survival in response to excitotoxicity, oxidative stress, or DNA damage.

modeling-neuronal-death-degeneration-mouse-primary-cerebellar-granule

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...