This method paper was funded by Eranet Neuron Neuvasc (Badaut), Agence Nationale de la Recherche Nanospace (Badaut), CNRS IRP IINOVAtion (Badaut) and NIH NINDS Grant No. 1R01NS119605 (Obenaus, Badaut) and 1RF1NS138032 (Obenaus, Territo).&#160;Figure 1 is created with BioRender.com.

Experiments were reproduced on 2 sites, the University of Bordeaux (France) and the University of California Riverside (CA, USA).
In Bordeaux, all experiments were performed on male and female P17 C57Bl6J mice bred in the University animal facility. Initial breeders were obtained from a commercial source and raised under standard conditions with a 12/12-h dark/light cycle, a temperature of 21 &#176;C &#177; 1 &#176;C, and a humidity of 55% &#177; 10%, and food and water provided ad libitum. Experimental procedures are in accordance with the University of Bordeaux Animal Care Committee regulations, French laws governing laboratory animal use (authorization #29324-2021012118549817 v3), the European Council directives (86/609/EEC), and the ARRIVE guidelines. This protocol, being a mild model of traumatic brain injury, does not produce pain or distress. As such, it does not necessitate any specific pain management outside of regular pain management. Weaning of the pups (TBI, Shams) was effective at P25.
At Riverside, E13 pregnant C57Bl6J female mice were commercially obtained, and all experiments were performed on the resultant litters in both male and female pups aged P17. Animals were raised with an automated 12/12-h dark/light cycle and a temperature of 21 &#176;C. Dams and pups had ad libitum access to food and water. All experimental procedures were approved by the University of California, Riverside Institutional Animal Care and Use Committee and complied with the Animal Welfare Act and Public Health Service policy. All pups (TBI, Sham) in that facility were weaned on P21.
The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.
1. Preliminary steps
<ol>
	<li>To prevent hypothermia, warm up the chamber for anesthesia induction and the recovery cages in advance with warming pads.</li>
	<li>Set the stereotaxic apparatus with the impactor mounted at a 90&#176; angle.</li>
	<li>Verify that all parts are properly tightened and secured and that the piston is firmly screwed to the impactor.</li>
	<li>Cut an aluminum sheet that will fit the stereotaxic apparatus (in this case, 18 cm x 30 cm). The aluminum used here is of standard aluminum quality. Wrap the stereotaxic frame with the foil to form a pad on which the animal will be laid on. Secure the position and tension of the foil with regular lab tape in order to sustain the weight of the animal.</li>
	<li>Turn on the impactor controller and, in the function of the severity of the injury, set the speed of impact to 2 m.s-1 for grade 1 (G1) or 3 m.s-1 for grade 2 (G2) for a more severe impact (modifying this parameter will impact the severity of the concussion).</li>
	<li>Weight out the animals. At P17, the weight of the animals must be over 5.9 g for C57Bl6 mice. Animals that fall under that weight are excluded. 
	NOTE: For some studies, CD1 mice were used, and for P17, the threshold was fixed to be over 6.5 g (personal data).</li>
</ol>
2. Juvenile mild traumatic brain injury procedure
<ol>
	<li>Anesthetize the mouse with a 5 min exposure to isoflurane. Set the gas composition to a 2.5% mixture (in this case, the carrier is a synthetic air mixture with 21% oxygen) with a flow rate of 1.5 L/min. 
	NOTE: From this step, all the actions must be done swiftly, as unimpacted animals will wake up within 5 min.</li>
	<li>Position the mouse head under the impactor. The pup should be spread out at all lengths. The tip is 3 mm in diameter and covered with rubber to minimize the effect of the metal on the skull.</li>
	<li>Lower the impactor by switching the knob to the Extend position.</li>
	<li>Roughly lower the impactor and position the mouse so that the impactor is just between the ears. Then, slide the mouse so that the piston tip is moved forward to the equivalent of one piston tip, and one piston tip to the left of the animal (see Figure 1). 
	NOTE: Sham animals are positioned slightly away from the piston (no part of the animal should be impacted).</li>
	<li>Lower the impactor so that it contacts the head of the animal (Figure 1E2).</li>
	<li>Set the impactor to Retract (Figure 1E3).</li>
	<li>In order to determine the severity of the injury, move the piston 1 mm down for grade 1 (G1). For more severe impact grade 2 (G2), move the piston 3 mm down (Figure 1E4).</li>
	<li>Set the dwell time to 0.1 s regardless of the severity (modifying this parameter will not impact the severity of the concussion).</li>
	<li>Press Impact on the right knob (Figure 1E5).</li>
	<li>Rapidly set the left knob to the off position so that the system doesn&#39;t overheat. See the flow chart for more information (Figure 2).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67667/67667fig01.jpg" /> 
Figure 1: Positioning of the impactor tip. (A) The impactor tip is first positioned on the midline between the ears. (B) The tip is then moved 3 mm forward (the equivalent of a tip diameter). (C) Finally, the piston tip is moved 3 mm on the left side of the mouse. (D,E) Possible positions of the impactor tip in regard to the magnetic barrel. (D) The impactor has 3 positions in relation to the piston body (neutral, retracted, or extended/impact) with 5 mm range between them. (E) Positions of the impactor tip in relation to the skull surface at the different stages of the procedure. (1) Position of the impactor in the neutral position; (2) the piston is moved to gently touch the skull of the animal (step 2.5); (3) the impactor is then set to retract (step 3.6); (4) the whole body of the impactor is moved of x mm down (step 2.7). For G1 grade, a speed of impact (y) of 2 m.s-1 with 1 mm depth (x) for G1 or a speed of impact (y) of 3 m.s-1 with 3 mm (x) depth for G2. (5) Once the lever impact is pressed, the impactor tip moves x mm below the skull surface at a y speed during dwell time upon moving back to the neutral position. <a href="https://www.jove.com/files/ftp_upload/67667/67667fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67667/67667fig02.jpg" /> 
Figure 2: CHILD flow chart. Flow chart detailing the steps to induce CHILD in PND17 mice color-coded to denote different phases of concussion induction. Photographs of the setup include (A) the entire CHILD induction setup (anesthesia device is not visible), including the Impact controller, the stereotactic device with the impactor and tip positioned above taut tin foil, and the recovery box located on top of a heating pad. (B) Rubber impact tip (3 mm) with scale. (C) The stereotactic device with tin foil stretched across the stereotactic frame and the impactor solenoid with the impact tip positioned above the foil. (D) Recovery chamber sitting on the heat pad. (E) The impact controller with two control switches, highlighted as in the flow chart (red box = extend/retract/off; purple box= impact toggle). <a href="https://www.jove.com/files/ftp_upload/67667/67667fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Post-trauma follow-up
NOTE: The following steps can be videorecorded for offline analysis, allowing the experimenter to perform CHILD on multiple animals in a row. Nonetheless, regular and repeated assessments of animal well-being should be performed.
<ol>
	<li>Note the duration of apnea, if present (immediately after impact), and if there was a head rotation during impact.</li>
	<li>Position the mouse on its right flank in a recovery cage.</li>
	<li>Note the righting time of the mouse (rearing on 4 legs) and the time of resuming an exploratory behavior. Typically, sham mice awake within 3-4 min and resume an exploratory behavior 3-4 min later (Figure 3). CHILD mice awake within 7-12 min after impact (Figure 3A) and resume exploration within 12-18 min after impact (Figure 3B), according to the sex and impact severity (Figure 3A,B). 
	NOTE: If the impacted animal doesn&#39;t resume its exploratory behavior within 30 min, the animal is excluded.</li>
	<li>Once fully recovered, return the animals to their home cage. At this point, the behavior of sham and CHILD mice (G1 or G2 severity) should be exactly similar, and animal groups shouldn&#39;t be distinguishable within the litter. 
	NOTE: The animals can be maintained up to 18 months post-injury14 in conditions indicated at the beginning of the methods. Behavioral outcomes assessments can be performed using a battery of tests in association with MRI measurements, miniscope imaging, histology, and immunohistochemistry12. Immunohistochemistry for GFAP and NeuN can be used at early time points to validate the model and the presence of tissue changes.</li>
</ol>
4. Immunohistochemistry
NOTE: One day post-injury, animals from the G2 and sham groups (n = 4 mice for G2 and n = 4 for Sham) were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) prepared in phosphate-buffered saline (PBS) 0.1 M, pH 7.4 at 4 &#176;C. Brains were extracted10 from the skull and immersed in the same PFA solution overnight, and then transferred to PBS with sodium azide (0.1%) before cutting. Coronal sections were then prepared at 50 &#181;m thickness using a vibratome. For long storage, sections were placed in cryoprotective medium (30% ethylene glycol and 20% glycerol in PBS) at -20 &#176;C until use. To characterize the impact of the concussion on brain gliosis and neuronal loss, immunohistochemistry targeting GFAP and NeuN was performed as described here below.
<ol>
	<li>Wash the brain sections 4 times for 10 min in PBS.</li>
	<li>Transfer the sections in blocking solution (1% BSA, 0,3% Triton X-100 in PBS) for 10 min at room temperature (RT).</li>
	<li>Incubate sections overnight at 4 &#176;C with the following primary antibodies diluted in blocking solution: chicken anti-GFAP (1:3000) and rabbit anti-NeuN (1:500).</li>
	<li>Wash the slices 4 times for 10 min in PBS under agitation.</li>
	<li>Then, transfer the sections in blocking solution containing the secondary fluorescent antibodies diluted 1:1000 for 1 h at RT, Alexa-Fluor-488-nm goat anti-rabbit, and Alexa-Fluor-568-nm goat anti-chicken.</li>
	<li>Wash then the sections 4 times for 10 min in PBS under agitation.</li>
	<li>Mount the sections on slides using a mounting medium and add a cover slip. Keep the slides at 4 &#176;C until the image acquisition.</li>
	<li>Aquire immunofluorescence using an epifluorescence microscope and micromanager software. Use a stitching plugin with a 10x lens to obtain tiled images of ipsilateral and contralateral hemispheres. 
	NOTE: Negative control staining where the primary antibody or secondary antibody was omitted showed no detectable labeling. For more detailed image analysis, Fiji software15 can be used to quantify the immunostaining parameters in various regions of interest16.</li>
</ol>

CHILD Model &#8212; Recapitulating Juvenile Concussion and Long-Term Outcomes in Mice

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The authors have nothing to disclose.

Critical steps in the protocol 
The CHILD protocol described here requires the use of an electromagnetic impactor (Figure 2A) that (1) standardizes the impact parameters (speed, depth, duration), (2) ensures reproducibility within and across laboratories, and (3) is readily available commercially. Another critical component is the impactor tip that strikes the rodent&#39;s head. A variety of possibilities can be used, ranging from silicone to metal tips, but this protoc...

Pediatric Traumatic Brain Injury (TBI) is a major health concern as it represents the leading cause of pediatric emergency medical care visits and is associated with debilitating health outcomes later in life. Even mild TBI (mTBI), which makes up 75% of all TBI cases1, leads to long-term abnormalities, including physiological, psychological, neurological, and cognitive dysfunctions2. Despite the high occurrence of pediatric mTBI and its significant associated health risks, the pathological, cellular, and molecular mechanisms that precede the chronic deficits are still underexplored. This gap in knowledge impedes the development of effective treatments and diagnostic methods for pediatric mTBI.
Adult rodent TBI models include weight drop, controlled cortical impact, and fluid percussion injury and can be titrated to elicit moderate to severe injuries (dependent upon outcome measures)3,4. Many of these adult models have been adapted for pediatric TBI5,6. During the last decade, models of mTBI have been developed in rodents with closed head injury as well as with rotational models7. However, implementation of these models in developing rodents is sparse compared to the adult TBI literature8. To address the clinical relevance, encompassing a wide range of severities that model pediatric clinical heterogeneity, this study developed the Closed Head Injury with Long-term Disorder (CHILD) TBI model. CHILD is a non-surgical, unrestrained concussion model using P17 mice, a developmental age that corresponds to the peak of myelination observed in 2-3-year-old children9. A concussive force is delivered using an electromagnetic impactor over the somatosensory cortex of a lightly isoflurane-anesthetized mouse. Impacting the unrestrained head results in rotational forces on the brain10, as reported in many clinical concussion reports. Consistent with clinical mTBI, the CHILD model does not lead to skull fracture or visible abnormality on conventional neuroimaging.
The CHILD model has several advantages. First, both early and long-term pathological features have been observed in CHILD, mimicking the pathological clinical features observed in juvenile concussion patients. It has been observed at late time points gliosis, altered diffusion tensor MRI, and progressive behavioral perturbations with age10,11, modified neuronal activity, and plasticity11,12. Interestingly, unsuspected peripheral consequences were also found with cardiac abnormalities in the long term after a single pediatric injury13. Second, this model is replicable and showed comparable results between researchers and between labs13. Third, the CHILD model is flexible and can be expanded to encapsulate the heterogeneous nature of TBIs. For instance, previous studies have shown that different speeds and depths of impact result in different severities10,11. Finally, an additional strength of this model is that CHILD can be administered repeatedly and can be easily applied to differential locations to probe responses to repeated injury and identify vulnerable brain regions.
Overall, the CHILD model replicates clinical pediatric mTBI and concussion and serves as a research tool to facilitate the understanding of the pathophysiological characteristics of pediatric mTBI. While no model encapsulates every aspect of the features described in real-life TBI cases, this model can address many of the outstanding research questions that remain unanswered.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl6J mice&#160;</td>
			<td>Charles River Laboratory France CRLF 9 All. Moulin Berger, 69130 &#201;cully, France</td>
			<td></td>
			<td>Bordeaux</td>
		</tr>
		<tr>
			<td>Electromagnetic impactor</td>
			<td>Leica Biosystems, Richmond, IL USA</td>
			<td>Impact One stereotaxic impactor</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus, BX41, Center Valley, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylen glycol</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>102466</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP primary antibody</td>
			<td>Millipore, Billerica, MA</td>
			<td>AB5541</td>
			<td>chicken anti-GFAP</td>
		</tr>
		<tr>
			<td>GFAP secondary antibody</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>A11034</td>
			<td>goat anti-rabbit AlexaFluor&#160; 488</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Heavy duty aluminium foil</td>
			<td>Kirkland</td>
			<td>RK611</td>
			<td>obtained at Cosco</td>
		</tr>
		<tr>
			<td>Micromanager software&#160;</td>
			<td>(NIH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN primary antibody</td>
			<td>AbCam, Discovery Drive, Cambridge 
			Biomedical Campus, 
			Cambridge, CB2 0AX, UK</td>
			<td>AB128886</td>
			<td>rabbit anti-NeuN</td>
		</tr>
		<tr>
			<td>NeuN secondary antibody</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>A11041</td>
			<td>goat anti-chicken AlexaFluor 568</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>1.04003</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>26628-22-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame - base</td>
			<td>KOPF instruments 7324 Elmo St, Tujunga, CA 91042, &#201;tats-Unis</td>
			<td>900-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame - electrode manipulator</td>
			<td>KOPF instruments 7324 Elmo St, Tujunga, CA 91042, &#201;tats-Unis</td>
			<td>960</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame - u-frame</td>
			<td>KOPF instruments 7324 Elmo St, Tujunga, CA 91042, &#201;tats-Unis</td>
			<td>900-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma Aldrich Chimie S.a.r.l 
			80 Rue de Luzais 
			L&#39;lsle-d&#39;Abeau Chesnes - BP701 
			38297 Saint-Quentin-Fallavier Cedex 
			France</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector laboratories</td>
			<td>H-1000-10</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems, Richmond, IL USA</td>
			<td>VTS1000</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Jackson laboratory The Jackson Laboratory 600 Main Street Bar Harbor, ME USA 04609</td>
			<td></td>
			<td>UCR</td>
		</tr>
	</tbody>
</table>

a pediatric concussion model in mice: closed head injury with long-term disorders (child)

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life exhibiting debilitating physiological, neurological, and psychosocial outcomes. The cellular and molecular pathophysiological mechanisms are relatively unknown, and this significant gap effectively precludes investigations into specific therapeutic strategies to mitigate chronic sequelae. No single animal concussion model recapitulates all the features reported in human subjects, and current models are designed to answer specific research questions. We set out to develop a juvenile concussion that recapitulates clinical early, and long-term symptomology encountered, termed Closed Head Injury with Long-term Disorder (CHILD). In this model, a concussive force via an electromagnetic impactor is delivered to the head of a lightly anesthetized unrestrained post-natal day 17 (P17) mouse, allowing free rotation of the head. Mice are placed on a tightly stretched tin foil across a stereotactic device, and the impactor is carefully aligned with the targeted cortical region. The electromagnetic impactor facilitates the selection of impact depth, dwell, and duration to determine injury severity. A strength of this model is that it allows head rotation, an important clinical feature. After impact, mice are immediately monitored for righting time and time to explore their environment, followed by their return to their dam. Increasing the severity of concussion results in intracranial bleeds in a subset of mice. Mice can be routinely monitored for behavior and neuroimaging over their lifespan, as desired. Various injury severities mimic the heterogeneous nature of juvenile concussions. The current standard CHILD model exhibits no skull fracture, no observable conventional neuroimaging change (similar to clinical), but leads to progressive and persistent neuronal death, altered diffusion MRI, modified neuronal activity and plasticity, increased gliosis, and progressive behavioral perturbations with age. In summary, this CHILD model mimics the early and long-term features observed in many clinical concussion patients.

The CHILD model is a relatively rapid and reproducible model of mTBI in pediatric mice, also allowing for variations in severity and location. The CHILD mice, as expected, exhibited significantly longer recovery times from closed head injury (Figure 3). When cohorts (n = 36 female sham, n = 140 male sham, n = 30 female G1, n = 55 male G1, n = 32 female G2, n = 141 male G2) were aggregated, a delay in righting time was observed (Figure 3A, 3-way ANOVA p ...

A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

Pediatric concussions can result in long-term sequelae with physiological and psychological perturbations. This pediatric concussion mouse model results in long-term neuroinflammation, white matter alterations, and neuronal modifications with behavioral dysfunction. The model can be adapted to differential impact locations or repeated concussions to assess lifetime changes.

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life ...

a-pediatric-concussion-model-mice-closed-head-injury-with-long-term

Research

JoVE Journal

Neuroscience

236 Views.  UMR 7372 CNRS/Université de La Rochelle. Pediatric concussions can result in long-term sequelae with physiological and psychological perturbations. This pediatric concussion mouse model results in long-term neuroinflammation, white matter alterations, and neuronal modifications with behavioral dysfunction. The model can be adapted to differential impact locations or repeated concussions to assess lifetime changes.

Video: A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...