We dedicate this article to Dr. Ermelinda Lomazzo. During the finalization of this manuscript, Dr. Ermelinda Lomazzo passed away. She is the embodiment of passion for science and selfless engagement in team work to fulfill a meaningful research purpose. She always dreamed of contributing meaningfully to the greater well-being of humans. Her goodhearted nature was never compromised by the strenuous roads of science and life. She will remain invaluable, and forever, in our hearts.
Julia M. Post was funded by Focus Program for Translational Neuroscience (FTN) at University Medical Center of the Johannes Gutenberg University Mainz and is currently funded by the SPP-2225 EXIT project to LB. Raissa Lerner was partially funded by DZHK project 81X2600250 to LB and Lipidomics Core Facility. Partial funding for these studies was provided by the Lipidomics Core Facility, Institute of Physiological Chemistry, and Intramural funds (to LB) from the University Medical Center of the Johannes Gutenberg University Mainz.

<ol>
	<li>Aronica, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammatory+targets+and+treatments+for+epilepsy+validated+in+experimental+models.">Neuroinflammatory targets and treatments for epilepsy validated in experimental models.</a> Epilepsia. 58, 27-38 (2017).</li><li>Lerner, R., Post, J., Loch, S., Lutz, B., Bindila, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+brain+and+peripheral+plasticity+of+the+lipidome+in+acute+kainic+acid-induced+epileptic+seizures+in+mice+via+quantitative+mass+spectrometry.">Targeting brain and peripheral plasticity of the lipidome in acute kainic acid-induced epileptic seizures in mice via quantitative mass spectrometry.</a> Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 1862 (2), 255-267 (2017).</li><li>Schuhmann, K., Almeida, R., Baumert, M., Herzog, R., Bornstein, S. R., Shevchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+lipidomics+on+a+LTQ+Orbitrap+mass+spectrometer+by+successive+switching+between+acquisition+polarity+modes.">Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes.</a> Journal of Mass Spectrometry. 47 (1), 96-104 (2012).</li><li>Puppolo, M., Varma, D., Jansen, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+analytical+methods+for+eicosanoids+in+brain+tissue.">A review of analytical methods for eicosanoids in brain tissue.</a> Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 964, 50-64 (2014).</li><li>Blewett, A. J., Varma, D., Gilles, T., Libonati, J. R., Jansen, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+high-performance+liquid+chromatography-electrospray+mass+spectrometry+method+for+the+simultaneous+determination+of+23+eicosanoids.">Development and validation of a high-performance liquid chromatography-electrospray mass spectrometry method for the simultaneous determination of 23 eicosanoids.</a> Journal of Pharmaceutical and Biomedical Analysis. 46 (4), 653-662 (2008).</li><li>Lerner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+lipidomic+and+transcriptomic+profiling+in+mouse+brain+punches+of+acute+epileptic+seizure+model+compared+to+controls.">Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls.</a> Journal of Lipid Research. 59, 283-297 (2017).</li><li>Lerner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+lipidomic+and+transcriptomic+profiling+in+mouse+brain+punches+of+acute+epileptic+seizure+model+compared+to+controls.">Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls.</a> Journal of Lipid Research. , 1-48 (2018).</li><li>L&#233;vesque, M., Avoli, M., Bernard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+temporal+lobe+epilepsy+following+systemic+chemoconvulsant+administration.">Animal models of temporal lobe epilepsy following systemic chemoconvulsant administration.</a> Journal of Neuroscience Methods. 260, (2016).</li><li>Eyo, U. B., Murugan, M., Wu, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-Neuron+Communication+in+Epilepsy.">Microglia-Neuron Communication in Epilepsy.</a> Glia. 65 (1), 5-18 (2017).</li><li>Zhu, J., Zheng, X. Y., Zhang, H. L., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kainic+acid-induced+neurodegenerative+model:+Potentials+and+limitations.">Kainic acid-induced neurodegenerative model: Potentials and limitations.</a> Journal of Biomedicine and Biotechnology. 2011, 457079 (2011).</li><li>Park, S. H., Sim, Y. B., Kim, C. H., Lee, J. K., Lee, J. H., Suh, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+&#945;-CGRP+in+the+regulation+of+neurotoxic+responses+induced+by+kainic+acid+in+mice.">Role of &#945;-CGRP in the regulation of neurotoxic responses induced by kainic acid in mice.</a> Peptides. 44, 158-162 (2013).</li><li>Lerner, R., Cuadrado, D. P., Post, J. M., Lutz, B., Bindila, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+lipidomic+and+transcriptional+changes+of+prophylactic+PEA+administration+in+control+mice.">Broad lipidomic and transcriptional changes of prophylactic PEA administration in control mice.</a> Frontiers in Neuroscience. 13, 527 (2019).</li><li>Post, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiepileptogenic+Effect+of+Subchronic+Palmitoylethanolamide+Treatment+in+a+Mouse+Model+of+Acute+Epilepsy.">Antiepileptogenic Effect of Subchronic Palmitoylethanolamide Treatment in a Mouse Model of Acute Epilepsy.</a> Frontiers in Molecular Neuroscience. 11, (2018).</li><li>Schauwecker, P. E., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+of+susceptibility+to+excitotoxic+cell+death:+Implications+for+gene+targeting+approaches.">Genetic determinants of susceptibility to excitotoxic cell death: Implications for gene targeting approaches.</a> Proceedings of the National Academy of Sciences. 94 (8), 4103-4108 (2002).</li><li>Monory, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endocannabinoid+System+Controls+Key+Epileptogenic+Circuits+in+the+Hippocampus.">The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus.</a> Neuron. 51 (4), 455-466 (2006).</li><li>Konsman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Brain+in+Stereotaxic+Coordinates.">The mouse Brain in Stereotaxic Coordinates.</a> Psychoneuroendocrinology. 28 (6), (2003).</li><li>Spijker, S., Li, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rondent+Brain+Regions.">Dissection of Rondent Brain Regions.</a> Neuroproteomics. 57, 13-27 (2011).</li><li>Gross, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+lipidomics+through+space+and+time.">The evolution of lipidomics through space and time.</a> Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 1862 (8), 731-739 (2017).</li><li>Wang, M., Wang, C., Han, R. H., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+advances+in+shotgun+lipidomics+for+biology+and+medicine.">Novel advances in shotgun lipidomics for biology and medicine.</a> Progress in Lipid Research. 61, 83-108 (2016).</li><li>Abbott, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+high-throughput+lipid+extraction+method+for+the+analysis+of+human+brain+lipids.">An improved high-throughput lipid extraction method for the analysis of human brain lipids.</a> Lipids. 48 (3), 307-318 (2013).</li><li>Matyash, V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A., Schwudke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+extraction+by+methyl-tert-butyl+ether+for+high-throughput+lipidomics.">Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.</a> Journal of Lipid Research. 49 (5), 1137-1146 (2008).</li></ol>

The authors declare no conflict of interest.

The neurolipidomic and transcriptomic methodology described here is a viable mean to investigate any disease or healthy development at high and low spatial resolution in the brain and peripheral organs. Due to the optimized plasma sampling and handling procedures, plasma lipidomic analysis can also be carried out from the same animals sacrificed for tissue lipidomics and transcriptomics, thus improving the reliability of tissue blood molecular correlates and biomarker discovery. The provision of a broad set of data by application of either of the three protocols or combinations thereof, is of value to investigate not only a neurological disease within a context (animal model experiment) but also across and between experimental model contexts. Moreover, a high level of standardization of sampling, processing, and molecular analysis facilitates high reproducibility of molecular data, hence reliably referencing molecular changes between and within studies and laboratories.
However, to attain this, the setup of an experimental design that offers the maximum readout potential for the defined study aim is critical. To attain a reliable comparison of the molecular changes between experimental groups, it is recommended to use a minimum of ten animals to compensate for animal variability and the biological range of lipid levels. When procurement of animals and/or logistics of animal handling are restrictive, use of a minimum of six animals per group is imperative to afford confident statistical analysis. Group size calculations need to compensate for model-related mortality rates (i.e., a minimum of six, ideally ten, animals per group are required for the study despite the possible mortality rate of the model). A critical requisite is to ensure age-, gender-, and strain-matched animals per experimental group. For the discovery phase, it is essential to use the same provider for experimental animals for all studies and the same animal batch if possible, in order to avoid bias of the findings due to possible behavioral and molecular phenotype differences between animal batches. To ensure reliability and reproducibility of the molecular and behavioral phenotype determined in the studies, it is critical to carry out a biological replicate analysis whenever possible.
Another crucial step is to set up a standardized, scheduled experimental work with animal groups. It is imperative to treat the animals within the same time-window of the day to circumvent circadian molecular variability. The time of the day should be set according to the known impact of circadian rhythm on synthesis and degradation of the target molecules or kept consistent for all experimental groups when no information on circadian rhythm effects is available. Similarly, the housing and feeding conditions prior to animal sacrifice must be maintained consistent and strictly controlled across experimental models. This is particularly relevant for lipidomic profiling due to influence of nutrition on lipid plasma and tissue metabolism. Administration of therapeutic or disease-inducing drugs should invariably be carried out in parallel with vehicle administration in control groups, whereby the vehicle must be the same as the one used for the drug administration. In order to choose the most suitable rodent strain and/or substrains for the purpose of the study, the drug-based treatment strategies should be carried out according to drug specificity in terms of time and frequency of administration, doses and route of administration, and the specific susceptibilities to drugs of different strains inferred from literature and/or prior experience. A time course investigation of a disease and response to therapy involving large cohort groups or multiple animal groups is impossible to carry out in one day in terms of treatment, sacrificing, and sampling. In such cases, animal group processing must be scheduled and carried out in consecutive days, but maintaining the same conditions in terms of time of the day, experimental design, processing time, researchers, etc. The preparation of chemical injections is another critical step. Drug- or disease-inducing compounds must be freshly prepared prior to administration and according to drug specifications. The use of the same production batch of the drug is recommended for all cohorts to be compared. This is especially important in the case of natural compound formulations such as kainic acid (KA) used in this study for epilepsy induction.
To enable reliable lipidomic and or transcriptomic profiling, the animal sacrificing procedure must be performed consistently across the animal groups within a timeframe of 5 minutes. If blood is collected after decapitation, it is important to maintain a constant amount of isoflurane in the glass chamber. For this purpose, soak frequently (after five uses) with isoflurane and do not exceed 10 s for the duration of anesthesia using isoflurane, in order to avoid onset of arrhythmia and palpitations and ensure proper blood pressure for plasma sampling.
Conditions for biological material sampling and handling (e.g., the time window and the order of biological material sampling and handling) must be strictly followed and maintained identical for all groups. To avoid variable degrees of tissue thawing and hence inconsistent ex vivo tissue changes of lipid and/or mRNA levels, it is essential to maintain strictly controlled time and temperature conditions for post-sampling 
storage. tissue dissection or punching, and subsequent sample processing and analysis (see sections 3 and 4). Freshly sampled whole brains can be immediately dissected on a precooled metal plate (4 &#176;C) without prior snapfreezing, if the size of the experimental animal groups allows for animal sacrifice, removal, and dissection without altering the timeframe indicated here for each of these procedures. If the size of experimental groups is not practical for these procedures, snap-freezing of the brains and subsequent dissection is recommended to allow comparable and controlled time for processing. When strictly following the protocols and timeline guidelines indicated here, no discrepancies were observed between molecular levels obtained by extraction of freshly dissected brain regions and brain regions dissected from frozen brains.
A critical aspect for attaining reproducible and minimal variability of the lipid levels within and between groups, apart from the sample processing under strictly controlled temperature conditions, is the provision of antioxidants (see sections 2 and 3). Avoiding any stress factors (e.g., the smell of blood) of the animals prior to sacrificing is of paramount importance, since many lipids involved in neuronal activity such as eCBs can rapidly change in response to stress.
For lipid extraction and analysis, it is essential to freshly prepare the internal standards, calibration solutions, and extraction solvents on the day of the extraction. The same source of internal standards must be used for both calibration curve preparation and for sample extractions. Also, following strictly controlled temperature conditions for sample extraction, storage, and analysis is paramount to minimize and control ex vivo enzymatic or chemical alterations of molecules. For LC/MRM analysis, the set of targeted lipids can be tailored to the study aim by adding or removing targets and correspondingly internal standards and calibrants, provided that the separation, detection, and MRM transitions for a new set of lipids are optimized. The presented extraction protocols allow the provision of two LC/MRM replicates for eCBs and eiCs, which is instrumental for cases of technical failure or when replicate analysis is of significance to the study. PL extraction protocols render sample/extract amounts suitable for at least 10 analyses per extract (e.g. multiple scan experiments based on precursor ion and neutral loss scanning2,&#160;respectively; additional LC/MRM analyses; or LC/MRM replicates to compensate for technical failure during a run). The lipid analysis is not restricted to LC/MRM; in fact the lipid extracts obtained with any of these protocols are amenable for untargteted, high-end mass spectrometriy analysis. Except for brain punches or minute amount of tissues obtained from discrete regions (less than 3 mg), frozen pulverized tissues of regions larger than 2&#8722;3 mg can be aliquoted and used for multiple extraction modules as described here for replicate analysis and/or for other investigations amenable in tissue powder.
A general advantage of the protocols described here compared to commonly used ones is the increased overall time-effectiveness and sensitivity for multicompound extraction and analysis at decreased expenditure of animal resources, consumables, and analysis costs. Importantly, the dual lipid/mRNA extraction protocol also affords a higher efficiency of mRNA extraction20,21 and integrity of the mRNA compared to corresponding available standard protocols, and simultaneously increased efficiency of the lipid extraction7. This is likely also due to the decreased matrix effect for each of the lipid and mRNA fractions when dually extracted. Due to this, the method is readily applicable for high spatial resolution profiling such as in brain punches.
However, a current limitation of the protocol is that the inflammatory lipids are not amenable for analysis and quantification using the dual lipid/mRNA extraction. Thus, the protocol is subject for further refinement. To this end, tissue and plasma inflammatory lipids and endocannabinoids can be co-extracted and co-analyzed, which is an optimized tool to concurrently investigate neuroinflammatory processes and endocannabinoids-modulated neuronal activity (see coextraction of eiCs and eCBs). Inclusion of phospholipids in this latter assay is expected to be feasible.
In view of prospective multi-omic approaches for neurological diseases, the proteomic analysis of protein fractions obtained after the lipid extraction protocol (i.e., co-extraction of eCBs and eiCs, as well as co-extraction of PLs and eCBs) is expected to be feasible. However, this is not yet possible when using the dual lipid/mRNA protocol. For the latter, the chemical environment of the extraction precludes even protein amount determination using standard protein assays such as the bicinchoninic acid assay (BCA). Further developments to overcome this limitation and expedite the inclusion of proteomic profiling in these protocols are planned.
Using the modular protocol described here, it was possible to attain a brain regional map of eiCs, eCBs, and PLs in an animal model of acute epileptic seizures (Figure 4). The protocol showed the hippocampal modulation of inflammatory processes by eiCs and of neuronal activity modulation by eCBs in treated and untreated mice with KA-induced acute seizures13. Subregional brain localization of phospholipid, endocannabinoid, and mRNA changes at acute epileptic seizure states, respectively, were also observed (Figure 5). These results highlight the value and applicability of the methods described here in advancing the knowledge on a broad spectrum of lipids involved in modulation of a complex neurological diseases such as epilepsy in brain regions and subregions. These protocols are of general applicability in neurological disease investigation and beyond while further development of the protocols and applications for cell populations continues.

Recent advances in the function of lipids and their role in the onset and progression of neurological diseases open new research and development venues of new therapeutic targets and disease mechanism elucidation1. Documented differences in lipid composition in different brain regions, emphasized by modern molecular imaging techniques such as mass spectrometry imaging and advanced mass spectrometry profiling, shifts the paradigm of lipid investigation from whole brain toward functionally distinct and discrete brain regions. The fact that lipid composition varies in different brain regions prompts new conceptualization of both membrane lipid sensitivity and downstream lipid signaling in response to a brain insult or stimuli across the functionally distinct brain regions. Hence, lipid protocols require new developments to address the challenge of low tissue amounts for higher spatial resolution detection and quantification, and concurrently, analysis of multiple lipid components of cell membranes and signaling pathways. Also, determination of enzymes, lipid ligands, and receptors involved in the regulation of their levels and function is paramount to elucidate the signaling pathways affected in a neurological disease and guide new mechanistic investigations in a pathophysiological context.
In addition to the increased brain spatial resolution, there are two major difficulties challenging the development of new neurolipidomic approaches. First, the lipid signaling molecules are typically of very low abundance compared to membrane constitutive lipids. Second, the lipidome exhibits a high structural heterogeneity, difficult to dissect using a single analytical approach. Hence, extraction and analytical methods are tailored to different lipid categories and commonly performed in distinct tissue samples2. Shotgun lipidomic methods3 are excellent tools to rapidly reveal a broad profile of membrane lipids, while increased sensitivity and selectivity afforded by the targeted discovery and quantification mass spectrometric methods are capitalized upon for investigation of low abundant signaling lipids including: i) inflammatory lipids and ii) lipids involved in the modulation of neuronal activity, such as endocannabinoids (eCBs), amino acid-linked lipids, etc.4,5. To encompass lipid changes at both the cell membrane and&#160;signaling level occurring in brain regions of neurological disease models, typically the lipid extraction and analysis are carried out in distinct tissue samples, obtained from distinct animal batches or from different hemispheres, or by dissecting a larger tissue region into multiple pieces. When mRNA levels of enzyme receptors are also of interest, their investigation typically requires the procurement of a distinct tissue sample. For example, the investigation of membrane lipids, endogenous cannabinoids, and mRNA would require three different tissue samples, (e.g., two samples for the two lipid extraction methods-membrane lipids and signaling lipids- and subsequent two lipid analysis methods- and one sample for mRNA analysis). Investigation of inflammatory lipids and endogenous cannabinoids require two distinct tissue samples, extraction methods, and analysis methods, respectively. Another example is the investigation of mRNA and of any lipid category in a brain punch or laser microdissection sample which consequently requires two distinct animals to procure two samples per brain (sub)region. A substantial extent of variability and/or poor reproducibility of the results frequently occur in such cases, originating from biological variability and/or tissue heterogeneity. Guided by these practical limitations of multimolecular analysis, occurring particularly at high spatial resolution in the brain, a three-module neurolipidomics protocol was designed encompassing: 1) coextraction and co-analysis by LC/MRM of inflammatory lipids (e.g., eicosanoids (eiCs)) and lipids involved in modulation of neuronal activity, such as eCBs2; 2) co-extraction of phospholipids (PLs) and eCBs with subsequent multiscan LC/MRM and precursor/neutral loss scan analysis2; and 3) dual extraction of membrane (phospho)lipids and eCBs as well as mRNA, with subsequent LC/MRM and qPCR or RNA sequencing analysis6. Depending on the biological question to be addressed in a neurological disease and the brain region of interest, a combination of the first and the second protocol, or the first and the third protocol, can be applied on the same tissue specimen for tissues weighing around 4 mg. The first and third protocols can be independently applied for tissues around 2 mg. The second protocol can be applied for tissues weighing as little as 0.5 mg. Irrespective of the neurolipidomic protocol module selected, the tissue sampling and pre-analytical processing, the brain isolation and region dissection, as well as the procedure for sacrificing the animal model are standardized and identical for all three modules of the protocol. In our investigation of neurological diseases, peripheral organs that are relevant for the pathological consequences of the disease are always also collected and analyzed using these modular protocols. Additionally, blood is regularly sampled for plasma lipidomics to serve as a readout tool of neurological diseases with a view on prospective translational applications. The here presented modular lipidomics protocol is very versatile: scaleable to larger tissue amounts and readily applicable for virtually any tissue type and disease. For the application of the modular protocol (Figure 1) in neurological diseases, any standardized rodent model of onset and progression of neurological disorders, such as traumatic brain injury, Parkinson&#39;s disease, Alzheimer&#39;s disease, or epilepsy are amenable.
These protocols have been extensively applied to study changes in the tissue lipidome and/or transcriptome at the acute phase of epilepsy in the kainic acid (KA)-induced mouse model of epilepsy2,7, a model widely used in preclinical studies due to the resemblance to human temporal lobe epilepsy (TLE)8,9,10,11. Using these protocols, the therapeutic potential of drugs such as Palmitoylethanolamide (PEA)12,13 was assessed in the same mouse model of epilepsy. The study identified lipid and mRNA changes at high and low spatial resolution in the brain and periphery, at the time point of maximal acute seizure intensities (at 60 min postseizure induction), and upon subchronic and acute treatment with PEA at four different timepoints (20, 60, 120, and 180 min) post KA-seizure induction, a time-window covering the acute phase of epilepsy. Plasma, brains, and peripheral organs of untreated KA-injected mice, acute and subchronically PEA-treated mice, as well as vehicle and PEA-vehicle control mice, were collected at each time point12,13, and investigated with this molecular analysis. The molecular data were correlated with behavioral phenotypes obtained by seizure scoring, as well as with immunohistochemistry-derived data on neurodegenerative processes, in order to unravel the progression of the acute epilepsy phase and PEA&#39;s potential to alleviate it.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12(S)-HETE</td>
			<td>Biomol</td>
			<td>Cay10007248-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>12(S)-HETE-d8</td>
			<td>Biomol</td>
			<td>Cay334570-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>1200 series LC System</td>
			<td>Agilent</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>5(S)-HETE-d8</td>
			<td>Biomol</td>
			<td>Cay 334230</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>ABI 7300 Real-Time PCR cycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Acetonitrile LC-MS Chroma Solv</td>
			<td>Honeywell</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>amber eppendorf tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Analyst 1.6.2 Software</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Arachidonic Acid-d8 MS Standard</td>
			<td>Biomol</td>
			<td>Cay-10007277</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Bessmann Tissue Pulverizer</td>
			<td>Spectrum Laboratories, Inc. (Breda, Netherlands)</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Bino</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>cleaved Caspase 3 antibody</td>
			<td>Cellsignaling</td>
			<td>9661S</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Cryostat, Leica CM3050 S</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>CTC HTC PAL autosampler</td>
			<td>CTC Analytics AG</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Dumont Curved Forceps Dumoxel #7</td>
			<td>FST</td>
			<td>11271-30</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Dumont Forceps Super fine tip #5SF (x2)</td>
			<td>FST</td>
			<td>11252-00</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>EDTA 1000 A R&#246;hrchen</td>
			<td>Kabe Labortechnik</td>
			<td>078001</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>EP-1 EconoPump</td>
			<td>BioRAD</td>
			<td>700BR07757</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Fine Forceps Mirror Finish</td>
			<td>FST</td>
			<td>11412-11</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Fine Iris Scissors straight sharp</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Fine Scissor Tungsten Carbide straight</td>
			<td>FST</td>
			<td>14568-09</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Iris Spatulae</td>
			<td>FST</td>
			<td>10094-13</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Kainic acid</td>
			<td>Abcam</td>
			<td>ab120100</td>
			<td>Epileptic drug</td>
		</tr>
		<tr>
			<td>Lipid View software</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>LPC 17:0</td>
			<td>Avanis Polaris</td>
			<td>855676P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>LPC 18:0</td>
			<td>Avanis Polaris</td>
			<td>855775P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Luna 2,5&#181;m C18(2)- HAST 100A LC column</td>
			<td>Phenomenex</td>
			<td>00D-4446-B0</td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Magnifying lamp</td>
			<td>Maul GmbH</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Methanol LC-MS Chroma Solv 99.9%</td>
			<td>Honeywell</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>Motic Camara</td>
			<td>Motic</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>MTBE</td>
			<td>Honeywell</td>
			<td>34875-1L</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>MultiQuant 3.0 quantitation software package</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>PA 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>840857P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PA 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1404</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Palmitoyl Ethanolamide</td>
			<td>Biomol</td>
			<td>Cay90350-100</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Palmitoyl Ethanolamide-d5</td>
			<td>Biomol</td>
			<td>Cay9000573-5</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1004</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PE 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850757P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PE 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1104</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PG 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>840457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PG 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1204</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PI 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1504</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Precelleys 24</td>
			<td>Peqlab</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Precellys Keramik-K&#252;gelchen</td>
			<td>Peqlab</td>
			<td>91-pcs-ck14p</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Precellys Stahlkugeln 2,8mm</td>
			<td>Peqlab</td>
			<td>91-PCS-MK28P</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Precellys-keramik-kit 1,4 mm</td>
			<td>VWR</td>
			<td>91-PCS-CK14</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Prostaglandin D2</td>
			<td>Biomol</td>
			<td>Cay 12010</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin D2-d4</td>
			<td>Biomol</td>
			<td>Cay 312010</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>Biomol</td>
			<td>Cay10007211-1</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin E2-d9</td>
			<td>Biomol</td>
			<td>Cay10581-50</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PS 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1304</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Q Trap 5500 triple-quadrupole linear ion trap MS</td>
			<td>AB SCIEX</td>
			<td>AU111609004</td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Real Time PCR System</td>
			<td>Appliert Biosystem</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Resolvin D1</td>
			<td>Biomol</td>
			<td>Cay10012554-11</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Rneasy Mini Kit - RNAase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Security Guard precolumn</td>
			<td>Phenomenex</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Shandon coverplates</td>
			<td>Thermo Fisher</td>
			<td>72110017</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Shandon slide rack and lid</td>
			<td>Thermo Fisher</td>
			<td>73310017</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>SM 18:0</td>
			<td>Avanis Polaris</td>
			<td>860586P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>SM d18:1/12:0</td>
			<td>Avanis Polaris</td>
			<td>LM-2312</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Standard Forceps straight Smooth</td>
			<td>FST</td>
			<td>11016-17</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Surgical Scissor ToughCut Standard Pattern</td>
			<td>FST</td>
			<td>14130-17</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>T3000 Thermocycler</td>
			<td>Biometra</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Thromboxane B2</td>
			<td>Biomol</td>
			<td>Cay19030-5</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Thromboxane B2-d4</td>
			<td>Biomol</td>
			<td>Cay319030-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Tissue Lyser II</td>
			<td>Qiagen/ Retsch</td>
			<td>12120240804</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Tissue Tek</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Toluidinblau</td>
			<td>Roth</td>
			<td>0300.2</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Vapotherm</td>
			<td>Barkey</td>
			<td>4004734</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Wasser LC-MS Chroma Solv</td>
			<td>VWR</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
	</tbody>
</table>

lipidomics and transcriptomics in neurological diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

Watch this Scientific Journal Video about Lipidomics and Transcriptomics in Neurological Diseases at JoVE.com

Lipidomics and Transcriptomics in Neurological Diseases

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

lipidomics-and-transcriptomics-in-neurological-diseases

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.3K Views.  University Medical Center of the Johannes Gutenberg University Mainz. This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Video: Lipidomics and Transcriptomics in Neurological Diseases

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...