Name: in vivo microdialysis method to collect large extracellular proteins from brain interstitial fluid with high-molecular weight cut-off probes
Uploaded: 2018-09-26T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes at JoVE.com

This work was supported by &#39;&#39;Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control)(15H01552) from MEXT and Grant-in-Aid for Young Scientists (B) (16K20969). The author thanks Dr. David M. Holtzman and Dr. John R. Cirrito for the technical advices during the development of this method.

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Graduate School of Medicine at the University of Tokyo.
1. Pre-surgical Procedure
<ol>
	<li>Before starting surgery, wipe everything with 70% ethanol to maintain sterile conditions.&#160;Thermal support using a heating pad is recommended.</li>
	<li>Anesthetize the mice by intraperitoneal injection of chloral hydrate (400 mg/kg). Confirm anesthetization by performing a toe pinch.&#160;Use o...

<ol>
	<li>Lei, Y., Han, H., Yuan, F., Javeed, A., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+interstitial+system:+Anatomy,+modeling,+in+vivo+measurement,+and+applications.">The brain interstitial system: Anatomy, modeling, in vivo measurement, and applications.</a> Progress in Neurobiology. 157, 230-246 (2017).</li><li>Kushikata, T., Hirota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+microdialysis+in+free-moving+animals.">Neuropeptide microdialysis in free-moving animals.</a> Methods in Molecular Biology. 789, 261-269 (2011).</li><li>Cirrito, J. 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=In+vivo+assessment+of+brain+interstitial+fluid+with+microdialysis+reveals+plaque-associated+changes+in+amyloid-beta+metabolism+and+half-life.">In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.</a> The Journal of Neuroscience. 23 (26), 8844-8853 (2003).</li><li>Emmanouilidou, 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=Assessment+of+&#945;-synuclein+secretion+in+mouse+and+human+brain+parenchyma.">Assessment of &#945;-synuclein secretion in mouse and human brain parenchyma.</a> PLoS One. 6 (7), 1-9 (2011).</li><li>Yamada, 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=In+vivo+microdialysis+reveals+age-dependent+decrease+of+brain+interstitial+fluid+tau+levels+in+P301S+human+tau+transgenic+mice.">In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice.</a> The Journal of Neuroscience. 31 (37), 13110-13117 (2011).</li><li>Ulrich, J. D., 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=In+vivo+measurement+of+apolipoprotein+E+from+the+brain+interstitial+fluid+using+microdialysis.">In vivo measurement of apolipoprotein E from the brain interstitial fluid using microdialysis.</a> Molecular Neurodegeneration. 8 (1), 13 (2013).</li><li>Emmanouilidou, 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=GABA+transmission+via+ATP-dependent+K++channels+regulates+&#945;-synuclein+secretion+in+mouse+striatum.">GABA transmission via ATP-dependent K+ channels regulates &#945;-synuclein secretion in mouse striatum.</a> Brain. 139 (3), 871-890 (2016).</li><li>Takeda, S., 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=Seed-competent+high-molecular-weight+tau+species+accumulates+in+the+cerebrospinal+fluid+of+Alzheimer's+disease+mouse+model+and+human+patients.">Seed-competent high-molecular-weight tau species accumulates in the cerebrospinal fluid of Alzheimer's disease mouse model and human patients.</a> Annals of Neurology. 80 (3), 355-367 (2016).</li><li>Yamada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Microdialysis+of+brain+interstitial+fluid+for+the+determination+of+extracellular+tau+levels.">In vivo Microdialysis of brain interstitial fluid for the determination of extracellular tau levels.</a> Methods in Molecular Biology. 1523, 285-296 (2017).</li><li>Kang, J. -. 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=Amyloid-&#946;+dynamics+are+regulated+by+orexin+and+the+sleep-wake+cycle.">Amyloid-&#946; dynamics are regulated by orexin and the sleep-wake cycle.</a> Science. 326 (November), 1005-1008 (2009).</li><li>Cirrito, J. 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=Synaptic+activity+regulates+interstitial+fluid+amyloid-beta+levels+in+vivo.">Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo.</a> Neuron. 48 (6), 913-922 (2005).</li><li>Bero, A. W., 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=Neuronal+activity+regulates+the+regional+vulnerability+to+amyloid-&#946;+deposition.">Neuronal activity regulates the regional vulnerability to amyloid-&#946; deposition.</a> Nature Neuroscience. 14 (6), 750-756 (2011).</li><li>Yamada, 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=Neuronal+activity+regulates+extracellular+tau+in+vivo.">Neuronal activity regulates extracellular tau in vivo.</a> Journal of Experimental Medicine. 211 (3), 387-393 (2014).</li><li>Pooler, A. M., Phillips, E. C., Lau, D. H. W., Noble, W., Hanger, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+release+of+endogenous+tau+is+stimulated+by+neuronal+activity.">Physiological release of endogenous tau is stimulated by neuronal activity.</a> EMBO Reports. 14 (4), 389-394 (2013).</li><li>Castellano, 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=Human+apoE+isoforms+differentially+regulate+brain+amyloid-&#946;+peptide+clearance.">Human apoE isoforms differentially regulate brain amyloid-&#946; peptide clearance.</a> Science Translational Medicine. 3 (89), 89ra57 (2011).</li><li>Yamada, 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=Analysis+of+in+vivo+turnover+of+tau+in+a+mouse+model+of+tauopathy.">Analysis of in vivo turnover of tau in a mouse model of tauopathy.</a> Molecular Neurodegeneration. 10 (1), 55 (2015).</li><li>Taylor, H., 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=Investigating+local+and+long-range+neuronal+network+dynamics+by+simultaneous+optogenetics,+reverse+microdialysis+and+silicon+probe+recordings+in+vivo.">Investigating local and long-range neuronal network dynamics by simultaneous optogenetics, reverse microdialysis and silicon probe recordings in vivo.</a> Journal of Neuroscience Methods. 235, 83-91 (2014).</li></ol>

Microdialysis with high molecular weight cut off membranes has to be operated by a push-pull mode, thus it is critical that the flow rate is accurate and constant. The inaccuracy in the flow rates can be the cause of air bubble generation and inconsistency in the sample concentration. If the flow is inconsistent, check all connections for leakage. If the problem still persists, it may be necessary to re-start with new probes and tubings.
Microdilaysis probes are continuously perfused by the pe...

ISF comprises 15-20% of total brain volume and offers a microenvironment critical for signal transduction, substrate transport and waste clearance1. Therefore, the ability of collecting ISF from living animals will provide greater implications for various biological processes as well as disease mechanism. In vivo microdialysis is one of the few methods that sample and quantify extracellular molecules from ISF from awake, freely moving animals and thereby serves as a useful tool in neuroscience research field2,3. In this method, microdialysis probes with semipermeable membranes are inserted in the brain and perfused with perfusion buffer at the relatively slow flow rate (0.1-5 &#181;L/min). During this perfusion, extracellular molecules in ISF passively diffuse into the probe according to the concentration gradient and collect as a dialysate. Although this article focuses on the method to sample ISF in the brain, both the principle and the method can be applied to other organs by appropriate modification if necessary.
Microdialysis was first employed in the early 1960s, and since then it has been extensively used to collect small molecules including amino acids or neurotransmitters in brain. However, recent commercial availability of microdialysis probes with high-molecular weight cut off membranes (100 kDa-3 MDa) has extended its application to relatively larger proteins in ISF as well4,5,6,7. The studies using these probes has led to the finding that proteins such as tau or &#945;-synuclein that were long thought to be exclusive cytoplasmic are also physiologically present in ISF4,5,8.
One of the difficulties using microdialysis probes with large cut off membranes (typically over 1,000 kDa) is that they are more susceptible to ultrafiltration fluid loss due to the inner pressure accumulated in the probes. Microdialysis probes used here have a unique structure to avoid this issue. The pressure will not be built up due to this structure, thus microdialysis with these probes should be operated in a &#34;push-pull&#34; mode using a syringe pump to perfuse the probes (=push) and a roller/peristaltic pump to collect the dialysate coming from the probe outlet (=pull)9 (Although it needs both push and pull pumps, due to pressure cancelling vent holes present in the probes, the system is technically only driven by the pull pump). This article starts with the stereotaxic surgery of a guide cannula implantation and describes how to set up microdialysis lines in order to collect ISF through microdialysis probes with 1,000 kDa cut-off membranes.

<table>
	<tbody>
		<tr>
			<td>The Univentor 820 Microsampler</td>
			<td>Univentor</td>
			<td>8303002</td>
			<td>Refrigerated fraction collector</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>KD scientific</td>
			<td>KDS-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller pump</td>
			<td>Eicom microdialysis</td>
			<td>ERP-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Raturn Stand-Alone System</td>
			<td>BASi</td>
			<td>MD-1409</td>
			<td>Free-moving system</td>
		</tr>
		<tr>
			<td>Dual species cage kit</td>
			<td>BASi</td>
			<td>CX-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>AtmosLM Microdialysis probe (shaft length 8 mm, membrane length 2 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PEP-8-02</td>
			<td>Shaft length for a probe, a guide, a dumy probe and a stereotaxic adaptor should be identical.</td>
		</tr>
		<tr>
			<td>Microdialysis guide (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PEG-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis dummy probe (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PED-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone screw</td>
			<td>BASi</td>
			<td>MD-1310</td>
			<td></td>
		</tr>
		<tr>
			<td>Super bond C&#38;B set</td>
			<td>Sunmedical</td>
			<td></td>
			<td>Dental cement</td>
		</tr>
		<tr>
			<td>Small animal Stereotaxic Instrument with digital display console</td>
			<td>Kopf</td>
			<td>Model 940</td>
			<td>Stereotaxic apparatus</td>
		</tr>
		<tr>
			<td>Mouse and neonatal rat adaptor</td>
			<td>Stoelting</td>
			<td>51625</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Ear Bars and Rubber Tips for Mouse Stereotaxic</td>
			<td>Stoelting</td>
			<td>51648</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin solution from bovine serum</td>
			<td>Sigma</td>
			<td>A7284-50ML</td>
			<td>30% BSA solution</td>
		</tr>
		<tr>
			<td>FEP tubing (70 cm)</td>
			<td>Eicom microdialysis</td>
			<td>JF-10-70</td>
			<td>Internal volume = 0.5 &#181;L/cm</td>
		</tr>
		<tr>
			<td>Teflon tubing (50 cm)</td>
			<td>Eicom microdialysis</td>
			<td>JT-10-50</td>
			<td>Internal volume = 0.08 &#181;L/cm</td>
		</tr>
		<tr>
			<td>Byton tube</td>
			<td>Eicom microdialysis</td>
			<td>JB-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Intramedic luer stab adaptor 23G</td>
			<td>BD</td>
			<td>427565</td>
			<td>Blunt end needle</td>
		</tr>
		<tr>
			<td>Roller tube</td>
			<td>Eicom microdialysis</td>
			<td>RT-5S</td>
			<td>Internal volume = 4 &#181;L</td>
		</tr>
		<tr>
			<td>Cap nut</td>
			<td>Eicom microdialysis</td>
			<td>AC-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mL microcentrifuge tube with cap</td>
			<td>QSP</td>
			<td>503-Q</td>
			<td>Tubes for fraction collector</td>
		</tr>
		<tr>
			<td>Sterotaxic adaptor (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PESG-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Connection needle</td>
			<td>Eicom microdialysis</td>
			<td>RTJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse animal collar</td>
			<td>BASi</td>
			<td>MD-1365</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Rotary Micromotor kit</td>
			<td>FOREDOM</td>
			<td>K.1070</td>
			<td>Drill</td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw driver for bone screws</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clipper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo microdialysis method to collect large extracellular proteins from brain interstitial fluid with high-molecular weight cut-off probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

To stimulate or inhibit neuronal activity in reverse microdialysis11,12,13, picrotoxin, GABAA receptor antagonist or tetrodotoxin, Na+ channel blocker have been used. It has been shown that tau release is stimulated by increase of neuronal activity13,14. Consistent with these previous observations, when 50 &#181;M pi...

Watch this Scientific Journal Video about In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes at JoVE.com

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

This method can help answer key questions in the neuroscience research field about how signal transduction substrate transport and a waste clearance occur in the brain extracellular spaces. The main advantage of this technique is that it allows the sampling and quantification of large extracellular molecules in the Interstitial Fluid or ISF of awake freely-moving animals. After confirming a lack of response to toe pinch, remove the hair from the skull of an anesthetized mouse and use the ear bars and a nose clamp to securely the fix animal onto an adapter.Apply ointment to the animal's eyes and place the adapter onto the stereotaxic apparatus. Use a scalpel to make a skin incision sagittally over the skull and attach a drill onto the manipulator of the stereotaxic frame. Lower the drill until it gently touches lambda and reset the DV coordinate of the drill to zero.Then, move the drill to bregma for resetting of the AP and ML coordinates to zero. Move the drill from bregma to the vertical coordinate. Lower the drill to the skull and set the DV coordinate to zero again.After repeating the procedure for a third coordinate, drill a burr hole carefully at the target coordinate, followed by a second hole on the contralateral side of the parietal bone. Insert a bone screw into the second hole and place the locking piece from a 1.5 milliliter microcentrifuge tube lid onto the skull so that the burr holes are within the circle. Next, place a guide cannula on the shorter arm of the stereotaxic adapter and fasten the cannula with a cap nut.Set the longer arm of the stereotaxic adapter on the electrode clamp and attach the arm on the manipulator of the stereotaxic apparatus. Rotate the DV stereotaxic assembly on the manipulator arm by 12 degrees and move the guide cannula to the burr hole. Then, slowly lower the guide cannula 1.2 millimeters into the brain.Add dental cement to the crown until the metal part of the guide cannula, the bone screw, and any exposed skull are covered and secured and allow the cement to dry. After 12 to 20 minutes, remove the stereotaxic adapter from the electrode clamp, remove the cap nut, and replace the stereotaxic adapter with a dummy probe. Then, refasten the cap nut, release the mouse from the stereotaxic apparatus, and house the mouse alone in an individual cage.Before setting up the microdialysis, fill a disposable one milliliter syringe with distilled water and use a viton tube to connect the syringe to the outlet of a microdialysis probe. Manually cover the vent holes and gently depress the syringe plunger to infuse the probe with water. Confirm that water appears in the probe inlet and that there is leakage onto the surface of the microdialysis membrane.To activate the probe, submerge the probe membranes in 70-100%ethanol for two seconds, followed by a second distilled water infusion. Attach a connection needle to one inlet and one outlet line and load a disposable three milliliter syringe with freshly prepared perfusion buffer. Connect a syringe equipped with a blunt-end needle to the inlet end of the tubing and use a syringe pump to fill the entire tubing with perfusion buffer.When the tube is full, replace the connection needle between the inlet and outlet lines with the activated microdialysis probe and the cap nut and mount a roller tube into the outlet tubing on a roller pump. Start the syringe pump at 10 microliters per minute and the roller pump at 9.5-9.8 microliters per minute. Now place the collar around the neck of the anesthetized guide cannula implanted animal and remove the cap nut and dummy probe.Slowly insert the microdialysis probe through the guide cannula and fasten the cap nut. Then, place the mouse in a cage connected to a free-moving system and tether the mouse with the collar. After at least one hour, sequentially stop the roller pump and the syringe pump, restarting the pumps with the syringe pump set to 20%faster than the roller pump and place the free end of the outlet tubing on a refrigerated fraction collector to collect the brain ISF.When the appropriate experimental volume has been obtained, remove the probe and handle the mouse recovery as just demonstrated. Consistent with previous observations, when 50 micromolar Picrotoxin is administered via reverse microdialysis as demonstrated in awake C57B6J mice, an increase in interstitial endogenous tau levels is observed compared to levels measured in animals treated with a vehicle control. In addition to drug administrations, microdialysis can be combined with other in vivo methods like EEG recording or optogenetics to answer additional questions about how neuronal activity influences substrate concentration in ISF.

in-vivo-microdialysis-method-to-collect-large-extracellular-proteins

Research

JoVE Journal

Neuroscience

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 ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...