Name: an improved method for collection of cerebrospinal fluid from anesthetized mice
Uploaded: 2018-03-19T13:00:00
Description: Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

This work was supported by the National Natural Science Foundation of China (81650110527, 81371400) and National Key Basic Research Program of China (2013CB530900).

All animal experiments were performed in accordance with the policies of the Society for Neuroscience (USA) and Fudan University (Shanghai, China) ethical committees. This procedure is for a non-survival surgery.
1. Setup of CSF Collection Apparatus
<ol>
	<li>Pull the glass capillary (inner diameter 0.75 mm, outer diameter 1.0 mm) using a micropipette puller (as shown in Liu et al.10; sharpened capillary is shown in Figure 1).</l...

<ol>
	<li>Anoop, A., Singh, P. K., Jacob, R. S., Maji, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+Biomarkers+for+Alzheimer's+Disease+Diagnosis.">CSF Biomarkers for Alzheimer's Disease Diagnosis.</a> Int. J. Alzheimers. Dis. 2010, 1-12 (2010).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat. Rev. Neurol. 6 (3), 131-144 (2010).</li><li>Schoonenboom, N. S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+biomarkers+of+Alzheimer's+disease:+impact+on+disease+concept,+diagnosis,+and+clinical+trial+design.">CSF biomarkers of Alzheimer's disease: impact on disease concept, diagnosis, and clinical trial design.</a> Adv. Geriatr. 2014, 1-14 (2014).</li><li>Ramautar, 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=Metabolic+profiling+of+mouse+cerebrospinal+fluid+by+sheathless+CE-MS.">Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.</a> Anal. Bioanal. Chem. 404 (10), 2895-2900 (2012).</li><li>Liu, L., Herukka, S., Minkeviciene, R., Vangreon, T., Tanila, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+A&#946;42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF A&#946;42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol. Dis. 17 (3), 516-523 (2004).</li><li>Schelle, J., 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=Prevention+of+tau+increase+in+cerebrospinal+fluid+of+APP+transgenic+mice+suggests+downstream+effect+of+BACE1+inhibition.">Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.</a> Alzheimer's Dement. , (2016).</li><li>You, J. -. S., Gelfanova, V., Knierman, M. D., Witzmann, F. A., Wang, M., Hale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+blood+contamination+on+the+proteome+of+cerebrospinal+fluid.">The impact of blood contamination on the proteome of cerebrospinal fluid.</a> Proteomics. 5 (1), 290-296 (2005).</li><li>Liu, L., Duff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Technique+for+Serial+Collection+of+Cerebrospinal+Fluid+from+the+Cisterna+Magna+in+Mouse.">A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse.</a> J. Vis. Exp. (21), (2008).</li><li>Maia, L. F., 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=Changes+in+amyloid-&#946;+and+Tau+in+the+cerebrospinal+fluid+of+transgenic+mice+overexpressing+amyloid+precursor+protein.">Changes in amyloid-&#946; and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein.</a> Sci. Transl. Med. 5 (194), 194re2 (2013).</li><li>Oshio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cerebrospinal+fluid+production+and+intracranial+pressure+in+mice+lacking+choroid+plexus+water+channel+Aquaporin-1.">Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1.</a> FASEB J. , (2004).</li></ol>

This protocol describes in detail a technique which improves on current methods10 of CSF collection to minimize contamination from blood and allow for the abundant collection of CSF (on average ~ 10-15 &#181;L can be obtained) from mice. When breaking the capillary tip, the tip&#160;of the capillary should not be too small (as then the CSF will be extracted very slowly) or too big (will not be fine enough to collect the CSF and tissue can become lodged in the capillary). Care should be taken when ...

The CSF is a valuable body fluid for analysis in neuroscience research. The CSF is primarily made from blood plasma, containing few cells (no red blood cells and only a few white blood cells) and is almost protein-free. It is one of the fluids in close contact with the central nervous system (CNS) and it can pass many electrolytes out from the brain and spinal cord to the peripheral system. In humans, CSF samples can be collected to aid in diagnosing disease or for research purposes in clinical trials, as a spinal tap (or lumbar puncture) is a minor, invasive procedure: the CSF fluid can reflect changes in the CNS without having to directly access these tissues. Thus, in recent years, for research purposes in the clinic, CSF samples have been obtained from patients of neurodegenerative diseases such as Alzheimer&#39;s disease and other dementias1,2,3. There are many biomarker assays which have been developed using CSF samples to potentially aid in diagnosing diseases in the clinic2,3. However, there is much debate on the reliability of these assays to produce consistent, sensitive results to specifically diagnose disease4,5. So, there is a great need for the development of better assays and targets, which can be found in the CSF, to aid in producing a standard technique to diagnose neurodegenerative diseases with greater sensitivity and specificity. Due to the potential importance of human CSF samples in disease, the collection of CSF from rodents in neuroscience research is also of interest.
Mice are important animals in biological and medical research and allow for the testing of potential therapeutic compounds and proof-of-concept studies before human clinical trials. However, in mice it is difficult to obtain CSF samples due to its closeness to the brain in a small animal, as the usual method of CSF collection in mice is to obtain it via the cisterna magna, an opening between the cerebellum and dorsal surface of the medulla oblongata. This causes difficulty in collecting CSF samples as this area is difficult to dissect to and in close proximity to blood vessels, increasing the risk of contamination from blood cells. Due to these difficulties, most researchers can only obtain a small amount of CSF for analysis (usually stated as 5-7 &#181;L) and the contamination of CSF samples by blood cells is a primary concern for analyses6,7,8,9. Blood contamination can obscure results and not truly reflect the state of the CNS. Furthermore, limited sample collected can impact research as the usual amount collected from mice is enough for only one measurement (in duplicate or triplicate) using enzyme-linked immunosorbent assay (ELISA). Thus, CSF samples are usually pooled from multiple mice in order to have enough sample to run multiple assays. Developing a protocol for the abundant, uncontaminated collection of CSF from mice is greatly desired and will be beneficial in improving neuroscience research using rodents.
In this protocol, a technique for the abundant (an average of 10-15 &#181;L) collection of CSF from anesthetized mice is described in detail and improves on a currently known method of CSF collection to minimize contamination from blood10. A robust protocol for CSF collection will aid in the development of CSF-based biomarker assays, which could be used to aid in diagnosing disease, as well as improve research into the mechanisms that underlie diseases affecting the CNS.

<table>
	<tbody>
		<tr>
			<td>Chloral hydrate (used as anesthetic)</td>
			<td>Sinopharm Chemicals Reagen Co. Ltd.</td>
			<td>30037517</td>
			<td>CAS number 302-17-0.</td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>66 vision technology</td>
			<td>54002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting curved forceps</td>
			<td>66 vision technology</td>
			<td>53072</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting straight forceps</td>
			<td>66 vision technology</td>
			<td>53070</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse adapter (with ear bars)</td>
			<td>Made in-house.</td>
			<td>N/A</td>
			<td>Similar equipment available from World Precision Instruments.</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Meiji Labax</td>
			<td>Model 15381</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic base for micromanipulator</td>
			<td>Kanetec</td>
			<td>MB-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1ml)</td>
			<td>Tansoole</td>
			<td>02024692</td>
			<td>For 1ml.</td>
		</tr>
		<tr>
			<td>Microtubes (1.5ml)</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor Cocktail Set III EDTA-free</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>Human A&#946;42 ELISA kit</td>
			<td>Invitrogen</td>
			<td>KHB3441</td>
			<td></td>
		</tr>
		<tr>
			<td>Piping (teflon tubing)</td>
			<td>World Precision Instruments</td>
			<td>MMP-KIT</td>
			<td>Obtained from a microinjection kit and attached to the capillary holder and syringe.</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Tiangen Biotech</td>
			<td>OSE-MC8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Obtained from any household store/pharmacy.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved method for collection of cerebrospinal fluid from anesthetized mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

Using the procedure outlined here (Figure 1 and Figure 2), the CSF immediately collected in the capillary should be clear (Figure 2E), not pink or red. If there is a pink to red tinge to the fluid collected in the capillary, then there was contamination with blood.
As an example of the application of the CSF sample collected with this metho...

Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

The overall goal of this procedure is to improve collection of cerebrospinal fluid from mice without contamination from blood. This method provides an easy and reliable way to collect CSF for monitoring the biomarkers of neural diseases. The main advantage of this technique is that the addition with the micromanipulator to aid in CSF collection from mice decreases the contamination of blood during collection.Begin by placing a pulled glass capillary into a capillary holder which is firmly mounted on a micromanipulator. Next, while viewing through a dissecting microscope, use straight forceps to break the tip of the sharpened capillary so that the inner diameter of the broken tip is about 10 to 20 microns. Then attach a thin tube and a syringe to the other end of the capillary holder, connect the thin tube and the syringe with a three-way valve and shift the three-way valve to open.Then plunge the syringe to expel any contaminants and create positive pressure in the capillary tube. Repeat this a few times by disconnecting and reconnecting the syringe to the three-way valve. Then draw up air to 100 to 200 microliters before the final reconnection of the syringe with the three-way valve.Move the micromanipulator with the glass capillary to the side to prevent damage during the surgical procedure. Before beginning the surgery, ensure that the mouse has been fully anesthetized by pinching all four limbs and observing the absence of a reaction. Then use dissecting scissors to cut and trim the hair at the back of the head of the mouse from above the eyes between the ears to the bottom of the neck.Secure the head of the mouse using a stereotaxic frame mouse adapter. Make sure the head cannot move and is secured tightly in the adapter by gently pushing down onto the head of the mouse. Use scissors and curved forceps to cut through the mouse skin across the back of the neck and then down the middle of the skull between the ears and eyes of the mouse.Pull the skin and tissue apart and out of the area over the cisterna magna. While viewing through the dissecting microscope, carefully dissect away the last thin layers of muscle over the base of the skull using forceps and move these layers of muscle to the side and out of the area of interest. Once the dura over the cisterna magna is exposed, use a wet cotton bud to wipe the membrane clean and then use a dry cotton bud to dry the area.The cisterna magna appears triangular with one to two large blood vessels running through the area. Either side of or between the blood vessels is optimal for capillary insertion and CSF collection. Once the cisterna magna is exposed, use the micromanipulator to align the glass capillary to the back of the mouse head so that the sharpened point is just behind the membrane at a 30 to 45 degree angle.Next, plunge the syringe once or a few times and then move the plunger back 100 to 200 microliters to make sure there are no contaminants and there is negative pressure in the glass capillary. Then move the capillary tip closer to the membrane until resistance can be seen, but without puncturing the membrane. Knock on the micromanipulator controls to gently tap the capillary tube through the membrane.CSF should automatically be drawn into the capillary tube once the opening has been punctured. Leave the capillary in place to slowly collect CSF. If the CSF has stopped flowing, slowly draw the syringe up approximately 50 microliters to create more negative pressure in the capillary.Ten to 15 microliters of CSF is collected in 10 to 30 minutes. Once the desired volume of CSF has been collected, close the tubing by shifting the three-way valve to close and remove the syringe connected to the tubing. Open and then close the tubing to create equalized pressure in the capillary, the tubing, and the area outside the capillary.Then reconnect the syringe before gently removing the glass capillary from the mouse using micromanipulator. Next, place the collection tube containing one microliter of 20X protease inhibitor under the sharpened point of the glass capillary. Open the tubing and gently plunge the syringe.The CSF should flow out of the capillary and into the collection tube. After collection, pulse centrifuge the CSF to mix the sample with the protease inhibitor at the bottom of the collection tube. CSF samples can be aliquoted and then stored for further analysis at minus 80 degrees Celsius.CSF collected from APP/PS1 mice demonstrated a significant increase in levels of human A beta 42 at 12 months of age. In wild type mice, no human A beta 42 was detected. Once mastered, this procedure can be done in one hour if performed properly.While attempting this procedure, it is important to remember to avoid blood vessels when inserting the capillary and to only make minor adjustments with the micromanipulator during CSF collection to avoid contamination with blood.

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Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&amp;#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, difficulty in obtaining HECF in real time from conscious rats has long restricted the evaluation of CNS disease progression and the effectiveness of ethnomedicine therapy. Encouragingly, a brain microdialysis technique can be used for continuous sampling with the advantages of dynamic observation, quantitative analysis, and a small sampling size. This allows the monitoring of changes in the extracellular fluid content for compounds from traditional herbs and their metabolites in the brain of living animals. The aim of this study was thus to accurately implant a cerebrospinal fluid microdialysis probe into the hippocampal region of Sprague Dawley (SD) rats with a three-dimensional brain stereotaxic apparatus, cutting off molecular weights greater than 20 kDa. The high-quality HECF was then obtained from conscious rats using a microdialysis sampling control system with an adjustable sampling rate from 2.87 nL/min - 2.98 mL/min. In conclusion, our protocol provides an efficient, rapid, and dynamic method to obtain HECF in awake rats with the help of microdialysis technology, which provides us with unlimited possibilities to further explore the pathogenesis of CNS-related diseases and evaluate drug efficacy.

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer&#039;s Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...