This work was supported by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme [600-IRMI/FRGS 5/3 (033/2019)].

The present study has been approved by the Committee on Animal Research and Ethics (CARE), Universiti Technologi MARA (UiTM) [Reference No: UiTM CARE 346/2021, dated 7 May 2021].
NOTE: The published protocols22,25,26 for standard husbandry and maintenance of the 6-OHDA-lesioned adult zebrafish PD model were utilized. Experiments were conducted with adult male zebrafish (Danio rerio) aged more than five months old with a standardized length of 3.2-3.7 cm.
1. Zebrafish maintenance and pre-ICV microinjection preparations
<ol>
	<li>Maintain the fish in an aerated water tank under a controlled temperature of 28 &#177; 1.0 &#176;C. For zebrafish husbandry and maintenance, use distilled water mineralized with commercial sea salt (1 g/L) throughout the experiment27.</li>
	<li>House a maximum of 25 fish per 45 L tank or one fish per 1.8 L water and expose them to a schedule of 14 h light and 10 h dark photoperiod. Feed the fish at least twice per day with food pellets supplemented with freeze-dried worms.</li>
	<li>Prepare a concentrated stock solution of tricaine methanesulfonate (MS-222) by dissolving 2.5 g of MS-222 and 5 g of sodium bicarbonate in 250 mL of distilled water. Dilute 2 mL of stock solution to produce 200 mL of working anesthesia solution.</li>
	<li>Prepare 99.96&#160;mM of 6-OHDA by first dissolving 0.2 mg of ascorbic acid in 1 mL of 0.9% w/v sterile-filtered NaCl. Filter the solution with 0.2-micron filter before adding 25 mg of 6-OHDA in powder form into the solution. Prepare the solution fresh before each injection and store it in dark at 4 &#176;C. 
	CAUTION: Wear appropriate personal protective equipment (i.e., gloves, laboratory coat, and face mask) and practice good laboratory practices when handling the chemicals. All handlings of the chemicals should be done within a biosafety cabinet.</li>
</ol>
2. Anaesthetisation and ICV injection of zebrafish
<ol>
	<li>Fast the fish for 24 h to avoid regurgitation during anesthesia. Anaesthetise the fish by immersing it into a container containing 0.01% w/v of MS-222 solution for approximately 1 min or until all visible muscular movement ceases.</li>
	<li>Position the anesthetized fish on a water-soaked sponge placed under a stereomicroscope and wet the fish regularly.</li>
	<li>Identify the position for injection based on the intersection between the metopic suture (MS), coronal suture (CS), and sagittal suture (SS) that connects the frontal and parietal skull of the zebrafish brain.</li>
	<li>Make a small hole of 1.0 mm2 area using a sharp 27 G needle in the skull guided by the specific anatomical position on the zebrafish skull (Figure 1A,B).</li>
	<li>Lower the microcapillary injector at a 60&#176; angle until it reaches a depth of 1,200 &#181;m from the cranial roof of the zebrafish skull (Figure 1C). Press the Z limit to fix the position.</li>
	<li>Set the initial injection pressure to 4000 hPa and compensation pressure to 10 hPa. Set the duration of injection to 0.3 s. Lower the intensity of injection with each subsequent injection.</li>
	<li>Inject 0.5 &#181;L of 99.96&#160;mM neurotoxin 6-OHDA (or 0.9% w/v saline for sham control group) and let the microcapillary rest for 20 s. Continue to wet the fish with distilled water throughout the injection process to prevent drying out.</li>
	<li>Slowly remove the microcapillary and resuscitate the fish under running distilled water. Place the fish in an isolated recovery tank and remove any distractions that can potentially disturb the recovery process.</li>
	<li>Flush the microcapillary before the next injection to clear the blockage and ensure that the intensity of injection is sufficient to yield the desired volume of 0.5 &#181;L of 6-OHDA.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63355/63355fig01v2.jpg" /> 
Figure 1: Injection site of neurotoxin, 6-OHDA. (A) The point of microcapillary entry is guided by the intersection between the metopic suture (MS), coronal suture (CS), and sagittal suture (SS) that connects the frontal and parietal skull of the zebrafish brain (plan view). (B) A schematic drawing (plan view) of the zebrafish skull and brain shows the microcapillary, which is lowered directly above the habenula (Hab), and its point of entry at the intersection between hemispheres. (C) A schematic drawing (sagittal section) of the zebrafish brain shows the angle of injection and depth of penetration. The black dot represents the lesioned site that is situated above the targeted area, the ventral diencephalon. Abbreviations: 6-OHDA: 6-hydroxydopamine, CS: coronal suture, Dn: diencephalon, Hab: habenula, Hyp: hypothalamus, MS: metopic suture, OB: olfactory bulb, POA: preoptic area, PT: posterior tuberculum, SS: sagittal suture, Tec: tectum, and Tel: telencephalon. <a href="https://www.jove.com/files/ftp_upload/63355/63355fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Locomotor assessment
NOTE: Locomotor assessment of zebrafish (n = six / group; sham vs lesioned) was assessed individually via the open tank test using established protocols28,29 at day three and day 30 post-6-OHDA lesion.
<ol>
	<li>Video recording
	<ol>
		<li>Place the experimental tank (length 20&#160;cm, width 11.5&#160;cm, height 13&#160;cm) with its walls covered with white paper on a raised platform (Figure 2A).</li>
		<li>Illuminate the tank from the bottom using a light source. Fill the tank with distilled water (80%-90% full) and maintain the temperature at 28 &#177; 1.0 &#176;C. Measure the temperature using a thermometer and regulate it using a commercial aquarium heater.</li>
		<li>After a minimum of 2 min of acclimatization, record the fish swimming behavior from a plan view on the 2-dimensional (2D) plane of the experimental arena using a video camera for 5 min (Figure 2B). To avoid inconsistency in the swimming behavior of the earlier and the last batch of recordings, do not exceed the acclimatization by 10 min30.</li>
		<li>Analyze the videos using video tracking software with the open-tank protocol for the acquisition of distance traveled (cm) and mean speed (cm/s) of each subject.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63355/63355fig02.jpg" /> 
Figure 2: Experimental setup of an open tank test for assessment of zebrafish locomotor behavior. (A) The experimental tank (front view) is placed on a raised platform that is illuminated from below. The four walls of the tank are covered with white paper and the recordings are captured axially. The temperature is measured using a thermometer and regulated at 28 &#177; 1.0 &#176;C using a commercial aquarium heater. (B) Screenshot (plan view) of video recording that is captured using the setup. <a href="https://www.jove.com/files/ftp_upload/63355/63355fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Data analysis
	<ol>
		<li>Double click on the icon to open the video tracking software. Click on the File tab and select Create New Empty Experiment. This will allow the user to customize the experiment parameters according to the aims of the investigation.</li>
		<li>Click on the Protocol tab, select Video Sources, and click on Add New Video Source. Click on the available drop-down list and select the Video File option. This will prompt the file browsing pop-up from which the video recordings of interest can be selected.</li>
		<li>Click on the Apparatus subtab and select the Rectangular icon to set up the apparatus. Drag the rectangular icon to cover the whole experimental arena. Set the scale bar accordingly and input the numerical value of the scale measurement used in the length of the ruler section. The present experiment used a 10 mm scale for the open tank test.</li>
		<li>Set the animal color by selecting The Animals are Darker than The Apparatus Background. Leave the other available options in Tracking to the preset default settings.</li>
		<li>In the Zones subtab, click on the previously drawn apparatus. This zone is set as the standard zone of which the position is the same for all tests.</li>
		<li>Select the following options under test scheduling and test data report: Test Duration, Total Distance Traveled, and Average Speed. Other available tests on the list are optional and dependent on the researcher&#39;s investigative interest.</li>
		<li>In the Experiment tab, assign the animals according to their test group by typing in the group name under the Name section and the number of animals per group in the Number of Animals section.</li>
		<li>Switch to Tests tab to run the experiment. Click on the Start Test icon and wait until all the videos are analyzed.</li>
		<li>In the Results tab, click on the View the Report icon to view the locomotor data in text report form.</li>
	</ol>
	</li>
</ol>

The authors declare no conflicts of interest.

The present work successfully demonstrated the locomotor assessment of the established 6-OHDA-induced, adult zebrafish-based PD model. The entire experiment involved three major steps: pre-ICV microinjection preparations, ICV microinjection of zebrafish, and locomotor assessment. To ensure the healthy recovery of adult zebrafish following the ICV microinjection procedure and good experimental outcome, some good practices for each step have been recommended in the present study.
Pre-ICV microin...

Parkinson&#39;s disease (PD), a disease distinctively characterized by muscle rigidity, resting tremor, and bradykinesia, is the fastest growing neurological disease in the world1,2. The risk and prevalence of PD increase rapidly with age especially in individuals aged 50 years and above3. The etiology and pathogenesis of PD hitherto remain poorly understood. This has often left the early-onset of PD undiagnosed. At present, the lack of dopamine and the loss of dopaminergic neurons (DpN) in PD patients are strongly linked to the manifestation of motor symptoms4. Capitalizing on this relationship, several treatments have been designed either to act directly as dopamine replacement (i.e., levodopa) or to compensate for the loss of DpN (i.e., deep brain stimulation). Although these treatments bring about symptomatic benefits, they do not modify the deteriorating course of the disease5. In view of this significant weakness, cell replacement therapy has been proposed. The efficacy of this approach is, however, inconsistent given the challenges of graft preparation, cell growth control, and phenotype instability. Cell replacement therapy, which had raised ethical concerns, also poses the risk of inducing brain tumors and unwanted immune reactions6,7.
The limitations of current therapeutic strategies have led to a greater emphasis on the regeneration of DpN as a potential approach in treating PD. Regeneration of DpN or neuroregeneration has emerged as one of the promising breakthroughs in the management of PD, not only due to its potential as a new therapeutic method but also as means to understand the mechanism of the disease8,9. This approach focuses on the restoration of neuronal function through differentiation, migration, and integration of existing progenitor cells into the lesioned circuitry10. In order to further explore neuroregeneration, various in vivo studies have been undertaken. It was found that vertebrates such as mammals, amphibians, and reptiles generate new brain cells following injury11,12. Among the vertebrates, mammalian animals are more sought after given their genetic resemblance to human beings. Mammals, however, exhibit limited and poor reparative capacity in the central nervous system (CNS) that can last through adulthood following a brain lesion13. In general, mammals are unsuited as animal models for understanding neuroregeneration given that the low number of neurons produced will not be sufficient to restore damaged neural circuits observed in PD. As such, the teleost-based model, specifically in zebrafish, is greatly favored for its high proliferative rate, capability to continuously self-renew, and close brain homology with humans14,15.
Zebrafish is most commonly used to study disordered movement in PD16. The zebrafish-based PD model is usually induced by neurotoxins, which include 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA)17. Although effective in inducing specific loss of DpN and decrease of dopamine levels, MPTP-based models do not closely mimic the conditions of PD as the DpN loss is not restricted solely to the CNS18. The inability of 6-OHDA to cross the blood-brain barrier restricted its effects on cellular and functional changes within the brain when it is administered intracranially as opposed to intramuscularly19. Peripheral administration of 6-OHDA caused a global reduction of dopamine levels throughout the nervous system20. While administration of 6-OHDA into the cerebrospinal fluid caused ablation of DpN throughout the CNS21, which does not mimic the condition as seen in PD whereby the loss of DpN occurs specifically at the substantia nigra of the human brain. ICV administration of 6-OHDA, on the contrary, specifically induced significant ablation of DpN in the area of ventral Dn in the zebrafish brain, which closely resembled substantia nigra22. Interestingly, recovery of DpN was reported 30 days post 6-OHDA-induced lesion and these neurons survived over the course of life23,24. The functional recovery of DpN was demonstrated through a locomotor assessment of distance traveled (cm) and mean speed (cm/s) using the 6-OHDA-induced adult zebrafish-based PD model22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-Hydroxydopamine (6-OHDA)</td>
			<td>Sigma-Aldrich, Missouri, USA</td>
			<td>162957</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Thermo Fisher Scientific, California, USA</td>
			<td>FKC#A/8882/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable pasteur pipette, 3 mL</td>
			<td>Thermo Fisher Scientific, California, USA</td>
			<td>FB55348</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube, 0.2 mL</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30124332</td>
			<td></td>
		</tr>
		<tr>
			<td>Nice conical flask, 100 mL</td>
			<td>Evergreen Engineering &#38; Resources, Semenyih, Malaysia</td>
			<td>SumYau0200</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Missouri, USA</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, Missouri, USA</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (MS-222)</td>
			<td>Sigma-Aldrich, Missouri, USA</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ANY-maze software</td>
			<td>Stoelting Co., Illinois, USA</td>
			<td>-</td>
			<td>version 7.0; video tracking software</td>
		</tr>
		<tr>
			<td>Cubis II Micro Lab Balance</td>
			<td>Sartorius, G&#246;ttingen, Germany</td>
			<td>SE 2</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet IV microinjector</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5192000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Femtotip II, sterile injection capillary</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5242957000</td>
			<td></td>
		</tr>
		<tr>
			<td>InjectMan 4 micromanipulator</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5192000027</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Portable Lamp</td>
			<td>MR. DIY, Selangor, Malaysia</td>
			<td>9023251</td>
			<td>20 mAh</td>
		</tr>
		<tr>
			<td>PELCO Pro Superalloy, offset, fine tips</td>
			<td>Ted Pella, California, USA</td>
			<td>5367-12NM</td>
			<td></td>
		</tr>
		<tr>
			<td>Shanda aquarium heater</td>
			<td>Yek Fong Aquarium, Selangor, Malaysia</td>
			<td>SDH-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Sera Precision, Heinsberg, Germany</td>
			<td>52525</td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>D3100</td>
			<td></td>
		</tr>
	</tbody>
</table>

locomotor assessment of 6-hydroxydopamine-induced adult zebrafish-based parkinson's disease model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present experiment assessed the changes in adult zebrafish swimming behavior following ICV microinjection with 6-OHDA. The reason for using 6-OHDA as the neurotoxin of choice was due to its inability to cross the blood-brain barrier, which produced specific and targeted ablation of DpN in the area of interest-ventral diencephalon (Dn)16. The DpN subpopulation here holds anatomical resemblance to the DpN subpopulation in the human&#39;s substantia nigra pars compacta31.<...

Watch this Scientific Journal Video about Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model at JoVE.com

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

locomotor-assessment-6-hydroxydopamine-induced-adult-zebrafish-based

Research

JoVE Journal

Neuroscience

2.9K Views.  Universiti Teknologi MARA (UiTM), Cawangan Selangor, Kampus Puncak Alam. The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

Video: Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...