We sincerely thank the Imaging core-facility at Midwestern University (MWU) and the group of students [Chanmoly Seng, Christopher Dipollina, Darryl Giambalvo, and Casey Sigerson] for their contributions in cell culture and imaging work. This research work was supported by the MWU&#39;s Intramural grant funding to M.F. and research start-up funds to V.T.

All the procedures including the use of the animals have been approved by the institutional review board-approved protocols (IACUC- Midwestern University).
1. Harvesting DRG from Mouse Embryos
<ol>
	<li>Euthanize the adult mice by asphyxiation method (CO2) followed by decapitation. Immediately proceed to surgically remove the vertebral column.
	<ol>
		<li>Expose the vertebral column by cutting down the skin layer dorsally using fine scissors. Isolate the vertebral column by cutting through the ribs on either side of the column and through the sacrum separating the vertebral column from the rest of the animal.</li>
		<li>Mount the vertebral column (ventral side up) onto a surgical mat using needles/pins.</li>
	</ol>
	</li>
	<li>Using fine scissors make a double cut on both sides of the vertebral bodies to expose the ventral side of the vertebral canal.</li>
	<li>Under a surgical microscope (magnification set to 4X), use scissors to gently move the spinal cord on the side to expose the contralateral dorsal spinal roots and to locate the DRG, along the dorsal roots of the peripheral nerves. Each ganglion is partially hidden inside the intervertebral foramina.</li>
	<li>To harvest the DRG, pinch the dorsal root (between the spinal cord and the DRG) with one forceps and gently pull out the DRG from the intervertebral foramen.</li>
	<li>Place the second forceps on the spinal nerve peripheral to the DRG and pull the DRG together with the spinal nerve and spinal root.</li>
	<li>Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum free media (SFM)34.</li>
	<li>Transfer each individual DRG in a dry glass Petri dish and, under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade. The DRG is easily identifiable as a bulgy transparent structure along the white spinal nerve/root. Blood vessels often are found surrounding the DRG.</li>
	<li>Place the cleaned DRG in a new Petri dish containing ice-cold SFM media.</li>
	<li>Dilute the gelatinous protein mixture (see Table of Materials) in ice-cold SFM (1:1).</li>
	<li>Plate the DRG ex vivo in 12-well plates pre-coated with 10/20 &#181;L of gelatinous protein mixture and set them inside the incubator at 37 &#176;C and 5% CO2 for 30-60 min.</li>
	<li>Gently add 1.5-2 mL of SFM to the culture system to cover the entire explant and maintain the explants at culturing conditions (37 &#176;C and 5% CO2). 
	NOTE: This is a critical step because the DRG is anchored to the glass dishes only by the polymerized gelatinous protein mixture. Time of polymerization and pipetting skills are critical to avoid floating.</li>
	<li>Change the medium of growing DRG every 72 h and let the DRG grow for as long as needed.</li>
</ol>
2. Isolating Single Cell Neurons from DRG
<ol>
	<li>Place all the DRG collected in a 1.5 mL sterile tube with 1.2 mL of F12 media containing 1.25 mg/mL of collagenase IV and incubate it at 37 &#176;C and 5% CO2 for 45 min. Repeat this step for another 45 min after the first incubation.</li>
	<li>Treat explants with 2 mL of F12 media containing trypsin (0.025%) for 30 min immediately after the collagenase IV treatment at 37 &#176;C and 5% CO2.</li>
	<li>Incubate with 2 mL of F12 media containing fetal bovine serum (FBS; 33%) at 37 &#176;C and 5% CO2 for 15 min.</li>
	<li>Wash explants three times with 2 mL of F12 media and proceed to mechanically dissociate them with a glass pipette until the media turns cloudy. 
	NOTE: This procedure involves collection of clean/trimmed DRG from the animal as explained in the prior section. When performing mechanical dissociation of explants, be gentle and do not use excessive force since it can lead to spillage and loss of working material.</li>
	<li>Filter the dissociated cell culture through a 0.22 &#181;m filter to remove any impurities and excess connective tissue. Centrifuge the filtered cell lysate at (2,500 x g) for 2 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 500 &#181;L of neurobasal media containing supplement for neuronal culture (1x), antibiotic mixture (1x), L-glutamate (0.5 mM), and nerve growth factor (5 &#181;g/mL) (see Table of Materials).</li>
	<li>Plate the dissociated cells onto laminin-coated cover slides (50 &#181;g/mL; see Table of Materials) at a preferred cell density. Determine the cell density using a cell counter. 
	NOTE: We plated cells at 25,000 cells/cover slide. This technique allows dissociation of both neurons and glial satellite cells. The glial component co-cultured with the pseudounipolar neurons plays a critical role for neuronal survival.</li>
</ol>
3. HSV-1 Infection of DRG Explants and DRG-derived Dissociated Cells
NOTE: This work was done by strictly following the biosafety level-2 (BSL-2) requirements to which we have a fully equipped lab that is approved by the Midwestern University biosafety committee. A KOS strain of HSV-1 was used in this study. Please take appropriate measurements and safety precautions as per local institutions guidelines if working with virus strains.
<ol>
	<li>Determine and prepare the virus in the correct dilutions in SFM media to infect the model. The virus used in this study was KOS strain of HSV-1. When working with DRG-derived dissociated cells, use 1 unit of multiplicity of infection (MOI) for infection, which means the number of virus equal the number of cells.</li>
	<li>If infecting DRG explants, use the number of virions (e.g., 10,000 virions) because an exact number of cells in an explant cannot be determined.</li>
	<li>Place the explant/cells to be infected with virus in a sterile cell plate or tube containing a mixture of SFM media and virus. 
	NOTE: We used 25,000 virions for a cover slide containing 25,000 cells (1 MOI). To infect an explant, we use 10,000 virions.</li>
	<li>Place the cell plate or tube at 37 &#176;C for infection to take place; time of viral exposure may vary depending on viral infectivity. 
	NOTE: Viral entry was confirmed by using ortho-nitrophenyl-&#946;-galactoside (ONPG) and 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) assays26.</li>
</ol>
4. Immunofluorescence
<ol>
	<li>Fix the explants and single cell samples in 4% formalin prepared in phosphate buffered saline (PBS). Wash samples 3 times for 10 min each in PBS.</li>
	<li>Incubate the samples with desired primary antibodies (anti-&#946;-tubulin, anti-peripherin, anti-heparan sulfate (HS), and/or anti-glycoprotein D (gD) antibody; see Table of Materials for dilutions) diluted in PBS buffer with 0.3% Triton-X (PBST) and 10% normal goat serum. Store samples at 4 &#176;C overnight.</li>
	<li>Wash the samples 3 times for 10 min each with PBS. Incubate samples at room temperature for 1 h in the appropriate secondary antibody (488 and/or Cy3; see Table of Materials for dilutions) diluted in PBS.</li>
	<li>Wash the samples 3 times for 10 min each with PBS. Incubate the samples with Hoechst dye (1.5 &#181;M) in PBS for 20 min prior to mounting step.</li>
	<li>Mount the samples on glass slides using a fluorescence mounting medium and coverslip.</li>
</ol>

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The authors have nothing to disclosure.

The ex vivo DRG model is extremely useful to investigate a wide spectrum of events such as neuron-glia interaction as well as the effect of the microenvironment on both neuronal and glial metabolism37. Further, the DRG-model could be used as a cost-effective tool to address relevant questions regarding pathogenic mechanism and associated markers by developing ex vivo systems for acute chronic and latent phase of infection or in a given disease. In addition, a screening library of...

In this manuscript, we report a method to obtain an organotypic ex vivo model of a mouse derived DRG model system as a preserved tissue-like microenvironment to investigate a wide range of biological responses to PNS insults ranging from neuron-glial interaction, neuroplasticity, inflammatory markers, to viral infection. In addition, we further developed a protocol to create a primary co-culture of DRG-derived single sensory neurons and satellite cells.
The DRG are satellite gray-matter-units located outside the central nervous system (CNS) along the dorsal spinal roots of spinal nerves. The DRG, located in proximity of intervertebral foramina, house pseudounipolar sensory neurons and satellite glial cells. The pseudounipolar neurons feature a single neurite that splits into a peripheral process carrying somatic and visceral inputs from peripheral targets to the cell body, and a central process that submits sensory information from the cell body into the CNS. A connective capsule defines and isolates this peripheral cluster of neurons and glial cells from the CNS. No postnatal cell migration to or from the DRG has ever been described and a local stem cell niche is responsible for neurogenic events occurring throughout life1. Therefore, this model is particularly suitable to study adult neurogenesis, axonogenesis, response to traumatic lesion, and cell death2,3,4,5,6,7,8,9 .
In the field of neuroregeneration, the DRG harvested from in vivo and explanted in vitro reproduces axonotmesis, an injury condition in which axons are fully severed and the neuronal cell body is disconnected from the innervated target10,11. It is well known that peripheral nerve injury can cause decreased and increased gene expression in the DRG and many of these changes are a result of regenerative processes but many may also be a result of immune response or another response from non-neuronal cells. By using an ex vivo system of isolated DRG, some of this complexity is removed and mechanistic pathways can be more easily investigated.
Besides its central role in conveying sensory inputs to the CNS, the abundance of receptors for many neurotransmitters including GABA12,13,14,15 at the level of the neuronal soma as well as evidence of interneuronal cross-excitation may suggest that DRG are sophisticated preliminary integrators of sensory inputs16,17. These new findings confer to the DRG explant the characteristics of a mini-neuronal network system similar to other &#34;mini-brain&#34; models, which are nervous-tissue-specific organoids used for broader experimental fields of investigation and therapeutic approach to neurological diseases18,19. These evidences together with the fact that the DRG is a discrete and well-defined cluster of neuronal tissue surrounded by a connective capsule, make it a suitable organ for ex vivo transplantation.
Culturing mouse DRG presents an attractive multicellular option to model human pathophysiologies due to structural and genetic similarities between the species. Additionally, a large repository of transgenic mouse strains is highly conducive to future mechanistic studies. Neurite extension both during development and after injury requires mechanical interactions between growth cone and substrate20,21. Nano- and micro-patterned substrates have been used as tools to direct neurite outgrowth and demonstrate their capacity to respond to topographical features in their microenvironments. Neurons have been shown to survive, adhere, migrate, and orient their axons to navigate surface features such as grooves in substrates22,23. However, these studies have typically utilized cultured cell lines and it is difficult to predict how primary neuronal cells will respond to well-defined, physical cues in vivo or ex vivo.
The ex vivo explant model of mouse DRG used for this proposal mimics the real cell-cell interaction and biochemical cues surrounding growing axons. Among many different experimental paradigms ranging from axonal regeneration, neurosphere production, to neuroinflammation, the DRG explant model continues to serve as a valuable tool to investigate the viral infection and latency aspect within sensory ganglia24,25,26,27.
The nervous system (NS) in general is target for viral infections28,29,30. Most viruses infect epithelial and endothelial cell surfaces and make their way from the surface tissue to the NS via peripheral nerve sensory and motor fibers. In particular, the herpes simplex virus type 1 (HSV-1) after an initial infection in epithelial cells establishes a life-long latency in the sensory ganglia preferably, the DRG of the PNS31,32. HSV-1 neuroptropic capability of infecting the PNS ultimately leads to neurological diseases33.

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			<td>Adult Mice NIH/Swiss</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Cell Treat</td>
			<td>229635</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel ECM</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
			<td>gelatinous protein mixture</td>
		</tr>
		<tr>
			<td>F12 Media</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F6178</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22um filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Supplement for neuronal culture</td>
		</tr>
		<tr>
			<td>PSN antibiotics</td>
			<td>Gibco</td>
			<td>15640-055</td>
			<td>*Part of SFM media 
			Antibiotic mixture</td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Alomone Labs</td>
			<td>N-100</td>
			<td>Nerve growth factor</td>
		</tr>
		<tr>
			<td>Laminin coated coverslide</td>
			<td>Neuvitro</td>
			<td>GG-14-Laminin</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG subtrate</td>
			<td>Pierce</td>
			<td>34055</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Invitrogen</td>
			<td>15520034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-B-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T8328</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Antibody anti-peripherin</td>
			<td>Millipore</td>
			<td>AB1530</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Hoechst dye</td>
			<td>Thermo Fisher</td>
			<td>62249</td>
			<td>1.5 &#181;M final concentration</td>
		</tr>
		<tr>
			<td>Anti-heparan sulfate</td>
			<td>US Biological</td>
			<td>H1890-10</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>Anti gD antibody</td>
			<td>Virostat</td>
			<td>196</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-100G</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>Gibco</td>
			<td>21010-046</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>KIT (Insulin-transferrin-Selenium-A)</td>
			<td>Gibco</td>
			<td>51300-044</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Vitamin-C</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>488 (goat anti-mouse)</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 (goat anti-rabbit)</td>
			<td>Jackson Immunoresearch laboratories</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat serum&#160;</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT5014-120ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield</td>
			<td>Vector</td>
			<td>H-1500</td>
			<td>Flurescence mount</td>
		</tr>
		<tr>
			<td>Diamond White Glass Coverslides</td>
			<td>Globe Scientific</td>
			<td>1380-20</td>
			<td></td>
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adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

Multiple aspects of neuroplasticity and neuron-environment interaction can be investigated using DRG and a single dissociated cell culture model. We began the studies by isolating a DRG explant and DRG-derived dissociated cells as schematically represented in Figure 1. Both tissue and single cells models can be analyzed by using a variety of molecular techniques such as immunofluorescence, Western blot, genomic assays, and other analytical techniques depending on the nature of experimental d...

Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

adult-mouse-drg-explant-dissociated-cell-models-to-investigate

Research

JoVE Journal

Neuroscience

20.9K Views.  Midwestern University. In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

Video: Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...