This protocol follows any and all animal care and use guidelines and regulations set by IACUC at the University of Cincinnati. This work is funded by NIH/NINDS NS07722 and the Gardner Family Center for Parkinson&#8217;s Disease and Movement Disorders.

All steps of the protocol follow the animal care and use guidelines and regulations set by the IACUC of the University of Cincinnati.
1. General Considerations
<ol>
	<li>If possible, test mice during their active phase in the dark cycle. Typically, perform testing under low light and at least 1 hr into the dark cycle. However, if testing in the dark cycle is not feasible, perform all three tests during the light cycle. Keep in mind that sniffing times may decrease when mice are tested during their sleep cycle. NOTE: While the tests can be performed during the sleep cycle the mice may show less interest in the buried pellet, blocks, or scented cartridge.</li>
	<li>In order to reduce any effect of outside olfactory cues, wear a base pair of gloves at all times throughout each test and then place a clean pair of gloves over the base pair for each mouse and each trial. As a general rule, when in doubt, put on a clean set of gloves. Have a box of gloves readily available since they will be changed frequently. NOTE: We use nitrile gloves for all animal handling.</li>
	<li>Cleaning test materials and cages between subjects with a disinfectant/sterilizing agent is also an important step in this battery of tests in order to reduce the potential for spreading disease as well as remove undesired olfactory cues.</li>
</ol>
2. Buried Pellet Test
<ol>
	<li>At least two days before testing, record the weight of each mouse and then food restrict to 90% of body weight. Prior to testing and during food restriction, give each mouse 1-2 pieces of the pellets to be used during the test (e.g., a piece of sweetened cereal). Do not skip this step because mice are neophobic with food and may not search for the pellet during the test if they have not eaten the pellets previously.</li>
	<li>On all test days, habituate the mice for 1 hr prior to testing by placing their cage in the testing room with no water bottle or feeder bin. House them in their home cage with just a filter top lid.</li>
	<li>During habituation, fill a clean mouse cage ~3 cm high with clean bedding making certain the bedding is evenly distributed throughout the cage. Set a timer for 5 min.</li>
	<li>Following 1 hr of habituation, bury 1 sweetened cereal pellet 0.5 cm below the bedding so that it is not visible.</li>
	<li>Remove the test mouse from its home cage, place it in the center of the test cage, place the filter top lid on the cage and start the timer.</li>
	<li>Stop the timer when the mouse uncovers the pellet and begins eating it. Note the time to uncover the buried pellet.</li>
	<li>If the mouse does not find the pellet within 5 min, end the trial and note a score of 300 sec for that mouse. Move the pellet to the top of the bedding so the mouse has access to it, and allow the mouse to eat the pellet.</li>
	<li>Following the trial, return the mouse to its home cage.</li>
	<li>Empty the bedding from the test cage and clean the cage with animal room cleaning solution.</li>
	<li>Repeat steps 2.4 to 2.9 for each mouse, and change gloves before and after each mouse.</li>
	<li>After all mice are tested, give them just enough food to maintain them at 90% body weight.</li>
	<li>Perform testing on days 2-5 the same way as test day 1 with one exception; bury the pellet in a different spot in the cage for each trial.</li>
	<li>On testing day 6, perform the surface pellet trial.</li>
	<li>For the surface pellet trial, follow the same procedure for test day 1 but instead of burying the pellet under the bedding place it on top of the bedding. Record the time for the mouse to find and start eating the pellet.</li>
</ol>
3. Block Test
<ol>
	<li>Individually house mice in clean cages and place 5 blocks in the cage (labeled A-E), changing gloves before setting up each cage.</li>
	<li>Perform the test 24 hr later. Move test mice to the testing area, remove the water bottle and feeder bin, and place all 5 blocks from the cage along with a handful of bedding into a plastic bag labeled with the animal&#8217;s identification. Place the sealed plastic bag on top of the mouse&#8217;s home cage.</li>
	<li>Habituate the animals in their home cage without the water bottle, feeder bin, and blocks for 1 hr prior to testing.</li>
	<li>Change gloves between each cage so that the scents are not exchanged between animals/blocks.</li>
	<li>Line up the mouse cages beside each other on a table with at least 10 cm between each cage. Position a video camera so that the front of the cage (not the long side of the cage) is in clear view.</li>
	<li>Set the timer for 30 sec and label a card with the important experiment details (date, mouse ID, trial, etc.). Videotape the label for 2-3 sec.</li>
	<li>Remove blocks A-D from the plastic bag with that mouse&#8217;s identification and place them in the middle of the cage so that they are clearly visible on the video camera. Make sure there is ~1 cm of space between each of the blocks. Place the filter top back on the cage. Start the timer and videotape for 30 sec.</li>
	<li>After 30 sec, stop recording and remove the blocks from the cage and place them back into the plastic bag.</li>
	<li>Change gloves and then move on to the next mouse, repeating the same procedure used for the first trial. Do this for a total of 6 trials for each mouse making sure there is an inter-trial interval of ~5 min.</li>
	<li>On the 7th trial, follow the same procedure for the trials 1-6 except instead of placing block D from the mouse&#8217;s own cage add block E from another mouse&#8217;s cage so that the mouse is exposed to blocks A, B, and C from its own home cage and block E from another mouse&#8217;s cage. Note in the lab book whose block E each mouse is exposed to and alternate where block E is placed in each mouse&#8217;s cage (e.g., ABCE or AEBC). Take block E from a mouse of the same sex but not from an original cagemate. Videotape for 30 sec.</li>
	<li>Following the 7 trials, return animals to a new clean cage with their original cagemates. Clean blocks used in the test with mild detergent and water and allow to air dry for use in later tests.</li>
</ol>
4. Habituation/Dishabituation
<ol>
	<li>Perform the habituation/dishabituation test in a similar manner to the block test but instead of using blocks that have the odor of a mouse, use tissue cartridges holding cotton scented with different pairs of extracts.</li>
	<li>Follow steps 3.1-3.4 using a single, unscented tissue cartridge instead of the blocks.</li>
	<li>Habituate mice in the test room.</li>
	<li>Prepare the scented tissue cartridges in a separate room. Place a small cotton ball into a clean cartridge and then add 5 &#956;l&#160;of extract (e.g., almond extract) into the cotton. Snap the cartridge top over the cotton and place in a dedicated box or bag labeled with that scent.</li>
	<li>Do the same procedure for a second scent (e.g., anise extract). Change gloves between handling different scents. Make sure the cotton ball is completely encased in the tissue cartridge. NOTE: Exposed fibers from the cotton ball may cause the animal to have interest in the cotton ball for nesting purposes rather than the scent itself.</li>
	<li>Then, follow steps 3.6-3.10 using one scented tissue cartridge for the first six trials, and the second scented tissue cartridge for the seventh trial.</li>
</ol>
5. Analysis of Videotapes
<ol>
	<li>For the block and habituation/dishabituation tests, have a rater blind to experimental condition measure the time spent sniffing each block, with sniffing defined as nasal contact with that block. If desired, measure other behaviors such as time to approach the novel block (E) during Trial 7 and total activity (the number of movements made along the length and width of the home cage) during each trial. Score&#160;the time spent sniffing and time to approach from the videotape using a stopwatch.</li>
	<li>Similarly, for the habituation/dishabituation test, have a rater blind to experimental condition measure the time spent sniffing the cartridge, time to approach the cartridge, and locomotor activity.</li>
</ol>
6. Statistics
<ol>
	<li>Analyze the buried pellet test using nonparametric statistics because the latency to find the pellet scores tend to show heterogeneity of variance. Average the latency to locate the pellet across days 3&#8211;5 and then compare between groups of mice using a Mann&#8211;Whitney U-test. Similarly, for the surface pellet part of the test, analyze latency to find the pellet using a Mann&#8211;Whitney U-test to compare genotypes.</li>
	<li>For the block test, analyze the data from Trial 7 (introduction of the novel-scented block). Typically, employ nonparametric analyses to account for heterogeneity of variance. In order to analyze differences in time spent sniffing the novel block as well as time to approach the blocks for different experimental groups, use the Mann&#8211;Whitney U-test. Use the Wilcoxon Signed Rank test to compare time spent sniffing the novel block to time spent sniffing the familiar blocks.</li>
	<li>For the habituation/dishabituation test, conduct parametric analyses on Trials 1, 6 and 7, similar to previous studies20. If mice exhibit little to no sniffing, use nonparametric analyses.</li>
</ol>

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We have no conflicts of interest.

Each of the tests described in this study measure different aspects of olfactory function in mice. The buried pellet test measures the food motivation aspect of olfaction, testing the ability of hungry (food restricted, not food deprived) mice to detect a palatable piece of sweetened cereal buried under bedding. The block test measures more of the social aspect of olfactory function, testing the ability of mice to discriminate between their own scent and that of a conspecific. The habituation/dishabituation test assesses the ability of the mouse to discriminate between familiar and novel, innocuous scents. Using multiple tests is important when characterizing a new model because anomalies in food motivation or fear could contribute to differences observed in mutant mice compared to controls. For example, if only the block test is performed and decreased sniffing of the block that has the odor of another mouse is observed, it is unclear whether there is an olfactory deficit or an enhanced fear response to the conspecific that may lead to avoidance of that block. When multiple tests are performed and deficits are observed in all tests, it strongly supports an interpretation of olfactory impairment. However, if differences are observed in only one test but not the others then there may be a more subtle olfactory impairment but it will be essential to rule out other explanations (i.e., enhanced fear or reduced food motivation). Additional important factors to keep in mind when testing olfactory function mice include the background strain and sex of the mice. Different genetic background strains (i.e., C57BL/6, DBA, etc.) can have profound effects on mouse behavior therefore, it is recommended to adapt each protocol in wildtype mice of the same background before performing the entire experiment. It is also recommended that male and female mice be tested separately from each other because the male olfactory system is highly sensitive to females in estrous and olfactory detection of a female in estrous could interfere with performance in all three tests.
Some steps are absolutely critical to follow when testing olfaction in mice. It is important to be cognizant of the odors the experimenter is introducing to the animals. Wearing gloves for the procedures is essential and frequent changing of gloves between animals is required. It is always a good practice for the experimenter not to wear cologne or perfume on olfaction testing days. While some parts of the procedure are inflexible, there are other parts that can be modified and adapted without reducing the validity or sensitivity of the test. For example, the number of habituation trials can be increased or decreased depending on the strain of mice being tested and how quickly they habituate to the stimuli. The major limitation of these tests is that they are all driven by some form of motivation, be it food or social, making it difficult to completely rule out anomalies in motivation as an explanation when an altered response observed. This can be minimized by analyzing additional parameters, such as activity during testing and time to approach stimuli.
Once mastered, the tests can be easily used in examining the phenotype of novel mouse models of neurodegenerative disease. In addition, the tests that have the highest power can be included in preclinical studies testing potential therapeutics (for an example see ref. 25).

Olfactory dysfunction is linked to a number of neurodegenerative disorders including Parkinson&#8217;s disease (PD), Alzheimer&#8217;s disease, and Huntington&#8217;s disease1. In PD, olfactory impairments include deficits in odor identification, detection, and discrimination and are found in up to 70&#8211;95% of patients2-5. These deficits can precede the cardinal motor symptoms of PD by up to 4 years, indicating that olfactory dysfunction may signal the early stages of PD6-10. The early occurrence of olfactory deficits in PD has led to a keen interest in olfactory dysfunction and the underlying mechanisms involved. In preclinical studies in rodents, olfactory dysfunction could be a sensitive outcome measure to predict the therapeutic potential of novel therapeutic strategies.
Many tests have been designed and extensively used to characterize sensorimotor impairments in rodent models of PD and to test the therapeutic potential of novel treatments11-15. Even though olfactory deficits are well documented in PD, olfactory function has not been routinely measured in many models. This view is changing though with the discovery of genetic forms of PD and the more accepted notion that PD is a systemic disorder affecting more than just sensorimotor function. Currently, there are numerous studies in genetic mouse models of PD and other neurodegenerative disorders that now include analysis of olfaction in the characterization16-24. Given the growing interest of olfactory dysfunction in neurodegenerative disorders, we sought to assemble a battery of olfactory tests that can be used to characterize novel models of neurodegeneration as well as test potential disease-modifying therapies in preclinical studies. The tests described in the present study have been used in both characterization and preclinical studies18,25.
The tests highlighted in this study have been shown to be sensitive in detecting olfactory dysfunction in a frequently utilized alpha-synuclein overexpressing mouse model of Parkinson&#8217;s disease18. They include the buried pellet test26,27, an adapted version of the block test23,24,28,29, and the habituation/dishabituation test30. It is important to note that there are several adaptations of the tests described in this study that are sensitive measures of olfactory function in mice, the ones highlighted in this study are the tests our laboratory has the most experience with and routinely use.

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			<td align="right" style="width:88px;">2.7243E+10</td>
			<td style="width:88px;">60 minute long play&#160;</td>
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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.

The buried pellet, block, and habituation/dishabituation tests are all highly advantageous evaluations of olfaction in mice. Using these tests, we have found significant changes in olfaction in multiple genetic mouse models of Parkinson&#8217;s disease, including alpha-synuclein overexpressing (Thy1-aSyn)18 and Atp13a2 knockout mice. Both Thy1-aSyn and Atp13a2 knockout mice take longer to find the buried pellet than controls18. Figures 1-3 show data collected from wildtype and Atp13a2 knockout mice. In the buried pellet test, Atp13a2 knockout mice show increased latency to find the buried pellet compared to wildtype control mice (Figure 1). In the block test both wildtype and Atp13a2 knockout mice sniff the novel block from another mouse&#8217;s cage compared to blocks from their own home cage (Figure 2). In the habituation/dishabituation test wildtype mice show habituation to one odor and then increased sniffing when a novel odor is introduced (Figure 3).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51804/51804fig1highres.jpg" width="500" /> 
Figure 1. Buried pellet test. Latency to find the pellet in wildtype (n = 10) and Atp13a2 KO (n = 14) at 20-27 m of age. * p &#60; 0.05, Mann-Whitney U.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51804/51804fig2highres.jpg" width="500" /> 
Figure 2. Block test. Time sniffing the novel block (E) in the block test in wildtype (n = 10) and Atp13a2 KO (n = 12) female mice at 20 - 27m of age. &#916;&#916; represents p &#60; 0.01 compared to blocks A, B, and C from the same genotype. Mann-Whitney U.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51804/51804fig3highres.jpg" width="500" /> 
Figure 3. Habituation/dishabituation test. Mean time sniffing a scented cartridge in wildtype mice (age 9 m, n = 7) across 6 trials and the introduction of a cartridge with a novel scent on trial 7.

Watch this Scientific Journal Video about Olfactory Assays for Mouse Models of Neurodegenerative Disease at JoVE.com

Olfactory Assays for Mouse Models of Neurodegenerative Disease

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 ...

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21.8K Views.  University of Cincinnati. 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.

Video: Olfactory Assays for Mouse Models of Neurodegenerative Disease

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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 ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...