Name: a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels
Uploaded: 2018-05-07T15:00:00
Description: Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

This work was supported by grants from the National Institutes of Health (R01-NS084941) and the Arizona Biomedical Research Commission (ADHS16-162406) to PTR. WA has received past support from a pre-doctoral appointment to a National Institutes of Health Training Grant (T32-HL007249).

All procedures outlined below have been approved by an Institutional Animal Care and Use Committee (IACUC) and conform to National Institutes of Health (NIH) and Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. The procedural flow for the protocol is depicted in Figure 1.
1. Set-up for the Procedure
<ol>
	<li>Prepare the brain microvessel buffer (BMB). Start by weighing 54.66 g D-mannitol, 1.90 g EGTA, and 1.46 g 2-amino-2-(hydroxymet...

<ol>
	<li>Rolfe, D. F., Brown, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+energy+utilization+and+molecular+origin+of+standard+metabolic+rate+in+mammals.">Cellular energy utilization and molecular origin of standard metabolic rate in mammals.</a> Physiol Rev. 77 (3), 731-758 (1997).</li><li>Brzica, H., Abdullahi, W., Ibbotson, K., Ronaldson, P. T. <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+Transporters+in+Central+Nervous+System+Drug+Delivery+and+Blood-Brain+Barrier+Protection:+Relevance+to+Treatment+of+Stroke.">Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke.</a> J Cent Nerv Syst Dis. 9, 1179573517693802 (2017).</li><li>Ronaldson, P. T., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+transporters:+promoting+blood-brain+barrier+repair+in+response+to+oxidative+stress+injury.">Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury.</a> Brain Res. 1623, 39-52 (2015).</li><li>Witt, K. A., Mark, K. S., Hom, S., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia-reoxygenation+on+rat+blood-brain+barrier+permeability+and+tight+junctional+protein+expression.">Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression.</a> Am J Physiol Heart Circ Physiol. 285 (6), H2820-H2831 (2003).</li><li>McCaffrey, G., 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=Occludin+oligomeric+assemblies+at+tight+junctions+of+the+blood-brain+barrier+are+altered+by+hypoxia+and+reoxygenation+stress.">Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.</a> J Neurochem. 110 (1), 58-71 (2009).</li><li>Lochhead, J. 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=Oxidative+stress+increases+blood-brain+barrier+permeability+and+induces+alterations+in+occludin+during+hypoxia-reoxygenation.">Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation.</a> J Cereb Blood Flow Metab. 30 (9), 1625-1636 (2010).</li><li>Lucke-Wold, B. P., 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=Bryostatin-1+Restores+Blood+Brain+Barrier+Integrity+following+Blast-Induced+Traumatic+Brain+Injury.">Bryostatin-1 Restores Blood Brain Barrier Integrity following Blast-Induced Traumatic Brain Injury.</a> Mol Neurobiol. 52 (3), 1119-1134 (2015).</li><li>Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+inhibition+prevents+inflammatory+pain+induced+changes+in+the+blood-brain+barrier.">Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier.</a> Brain Res. , 6-13 (2008).</li><li>Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+signaling+alters+substrate+permeability+and+tight+junction+protein+expression+at+the+blood-brain+barrier+during+inflammatory+pain.">Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain.</a> J Cereb Blood Flow Metab. 29 (6), 1084-1098 (2009).</li><li>Seelbach, M. J., Brooks, T. A., Egleton, R. D., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+inflammatory+hyperalgesia+modulates+morphine+delivery+to+the+brain:+a+role+for+P-glycoprotein.">Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein.</a> J Neurochem. 102 (5), 1677-1690 (2007).</li><li>Ronaldson, P. T., Finch, J. D., Demarco, K. M., Quigley, C. E., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+pain+signals+an+increase+in+functional+expression+of+organic+anion+transporting+polypeptide+1a4+at+the+blood-brain+barrier.">Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier.</a> J Pharmacol Exp Ther. 336 (3), 827-839 (2011).</li><li>Pop, V., 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=Early+brain+injury+alters+the+blood-brain+barrier+phenotype+in+parallel+with+beta-amyloid+and+cognitive+changes+in+adulthood.">Early brain injury alters the blood-brain barrier phenotype in parallel with beta-amyloid and cognitive changes in adulthood.</a> J Cereb Blood Flow Metab. 33 (2), 205-214 (2013).</li><li>Thompson, B. 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=Hypoxia/reoxygenation+stress+signals+an+increase+in+organic+anion+transporting+polypeptide+1a4+(Oatp1a4)+at+the+blood-brain+barrier:+relevance+to+CNS+drug+delivery.">Hypoxia/reoxygenation stress signals an increase in organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: relevance to CNS drug delivery.</a> J Cereb Blood Flow Metab. 34 (4), 699-707 (2014).</li><li>Tome, M. 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=P-glycoprotein+traffics+from+the+nucleus+to+the+plasma+membrane+in+rat+brain+endothelium+during+inflammatory+pain.">P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain.</a> J Cereb Blood Flow Metab. 36 (11), 1913-1928 (2016).</li><li>Slosky, L. 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=Acetaminophen+modulates+P-glycoprotein+functional+expression+at+the+blood-brain+barrier+by+a+constitutive+androstane+receptor-dependent+mechanism.">Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism.</a> Mol Pharmacol. 84 (5), 774-786 (2013).</li><li>Artus, C., 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+Wnt/planar+cell+polarity+signaling+pathway+contributes+to+the+integrity+of+tight+junctions+in+brain+endothelial+cells.">The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells.</a> J Cereb Blood Flow Metab. 34 (3), 433-440 (2014).</li><li>Yu, 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=Long-term+exposure+to+ethanol+downregulates+tight+junction+proteins+through+the+protein+kinase+Calpha+signaling+pathway+in+human+cerebral+microvascular+endothelial+cells.">Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Calpha signaling pathway in human cerebral microvascular endothelial cells.</a> Exp Ther Med. 14 (5), 4789-4796 (2017).</li><li>Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-9+increases+the+functional+expression+of+organic+anion+transporting+polypeptide+1a4+at+the+blood-brain+barrier+via+the+activin+receptor-like+kinase-1+receptor.">Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor.</a> J Cereb Blood Flow Metab. 37 (7), 2340-2345 (2017).</li><li>Mesev, E. V., Miller, D. S., Cannon, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceramide+1-Phosphate+Increases+P-Glycoprotein+Transport+Activity+at+the+Blood-Brain+Barrier+via+Prostaglandin+E2+Signaling.">Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling.</a> Mol Pharmacol. 91 (4), 373-382 (2017).</li><li>Betz, A. L., Csejtey, J., Goldstein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hexose+transport+and+phosphorylation+by+capillaries+isolated+from+rat+brain.">Hexose transport and phosphorylation by capillaries isolated from rat brain.</a> Am J Physiol. 236 (1), C96-C102 (1979).</li><li>Yousif, S., Marie-Claire, C., Roux, F., Scherrmann, J. M., Decleves, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+drug+transporters+at+the+blood-brain+barrier+using+an+optimized+isolated+rat+brain+microvessel+strategy.">Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy.</a> Brain Res. 1134 (1), 1-11 (2007).</li><li>McCaffrey, G., 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=Tight+junctions+contain+oligomeric+protein+assembly+critical+for+maintaining+blood-brain+barrier+integrity+in+vivo.">Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.</a> J Neurochem. 103 (6), 2540-2555 (2007).</li><li>Brzica, 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=The+liver+and+kidney+expression+of+sulfate+anion+transporter+sat-1+in+rats+exhibits+male-dominant+gender+differences.">The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences.</a> Pflugers Arch. 457 (6), 1381-1392 (2009).</li><li>Ronaldson, P. T., Bendayan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+viral+envelope+glycoprotein+gp120+produces+oxidative+stress+and+regulates+the+functional+expression+of+multidrug+resistance+protein-1+(Mrp1)+in+glial+cells.">HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells.</a> J Neurochem. 106 (3), 1298-1313 (2008).</li><li>Pustylnikov, S., Sagar, D., Jain, P., Khan, Z. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+C-type+lectins-mediated+host-pathogen+interactions+with+dextran.">Targeting the C-type lectins-mediated host-pathogen interactions with dextran.</a> J Pharm Pharm Sci. 17 (3), 371-392 (2014).</li><li>Obermeier, B., Daneman, R., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+maintenance+and+disruption+of+the+blood-brain+barrier.">Development, maintenance and disruption of the blood-brain barrier.</a> Nat Med. 19 (12), 1584-1596 (2013).</li><li>Abdullahi, W., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Expression+of+P-glycoprotein+and+Organic+Anion+Transporting+Polypeptides+at+the+Blood-Brain+Barrier:+Understanding+Transport+Mechanisms+for+Improved+CNS+Drug+Delivery?.">Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?.</a> AAPS J. 19 (4), 931-939 (2017).</li></ol>

In this article, a simple and effective method of preparing membrane protein samples from microvessels freshly isolated from rat brain tissue is described. Several approaches for isolation of rat brain microvessels and/or generation of membrane preparations from isolated microvasculature have been reported in the literature13,20,21,22,24. Although the microves...

The blood-brain barrier (BBB) exists at the interface between the central nervous system (CNS) and the systemic circulation and plays an essential role in the maintenance of brain homeostasis. Specifically, the BBB functions to precisely control solute concentrations in brain extracellular fluid and to efficiently supply those nutrients that are required by brain tissue to fulfill the considerable metabolic demands of the CNS1. These roles imply that the BBB, which exists primarily at the level of the microvascular endothelial cell, must possess discrete mechanisms that enable some substances to access brain parenchyma while ensuring that potentially harmful xenobiotics cannot accumulate. Indeed, brain microvascular endothelial cells are not fenestrated and exhibit limited pinocytosis, which ensures a lack of non-selective permeability2. Additionally, brain microvessel endothelial cells express tight junction and adherens junction proteins that act to form a physical &#34;seal&#34; between adjacent endothelial cells and greatly restrict paracellular diffusion of blood-borne substances into brain parenchyma. Indeed, selective permeability of endogenous and exogenous substances requires functional expression of uptake and efflux transporters3. Overall, tight junctions, adherens junctions, and transporters work in concert to maintain the unique barrier properties of the BBB.
The BBB is a dynamic barrier that responds to physiological, pathophysiological, and pharmacological stimuli. For example, hypoxia/reoxygenation stress has been shown to modulate expression of critical tight junction proteins (i.e., occludin, zonulae occluden-1 (ZO-1)), which is associated with increased paracellular permeability to vascular markers such as sucrose4,5,6. Similar observations have been made at the BBB in the setting of traumatic brain injury7 and peripheral inflammatory pain8,9. These same diseases can also modulate transport mechanisms at the BBB10,11,12,13,14. Indeed, hypoxia/reoxygenation injury enhances functional expression of organic anion transporting polypeptide 1a4 (Oatp1a4) at the BBB, which can lead to significant increases in the blood-to-brain transport of specific Oatp transport substrates such as taurocholate and atorvastatin13. BBB properties can also be altered by pharmacotherapy itself, a mechanism that can form a basis for both profound changes in the drug effectiveness in the brain and for drug-drug interactions. For example, acetaminophen targets nuclear receptor signaling mechanisms in the brain microvascular endothelial cells, increases functional expression of the critical efflux transporter P-glycoprotein (P-gp), and modifies time-dependent analgesia conferred by morphine, an opioid analgesic drug and established P-gp transport substrate15. A thorough understanding of BBB changes, that can be induced by diseases or by drugs, also requires identification and characterization of specific regulatory mechanisms that control these modifications. Indeed, discrete signaling pathways have been identified in brain microvascular endothelial cells that control the molecular expression of tight junction proteins16,17 and transporters15,18,19. Taken together, these observations indicate that complex molecular pathways are involved in the regulation of BBB tight junctions and transporters in both health and disease.
A significant challenge in the study of the BBB is the absolute requirement of a simple and effective method for isolation of microvessels from brain tissue derived from experimental animals and subsequent preparation of membrane samples. These samples must be prepared so that they are both enriched in brain microvascular endothelial cells and limited in presence of other cell types. Over the past several years, multiple methodologies for isolation of microvasculature from rodent brain have been reported in the scientific literature13,20,21,22. This article describes a simple, robust, and reproducible method for isolation of microvessels from rat brain and for preparation of endothelial membrane-enriched samples that can be used for the analysis of protein expression. An advantage of this microvessel isolation protocol is the ability to obtain sample preparations of high quality and with sufficient protein yield from an individual experimental animal. This enables the consideration of inter-animal variability in protein expression. Such an advance in this protocol has greatly improved the robustness of BBB studies because over-estimation (or under-estimation) of the true magnitude of protein changes at the BBB can now be avoided. Additionally, the inclusion of multiple centrifugation steps with dextran enables improved enrichment of microvessels in experimental samples while facilitating removal of unwanted cellular constituents such as neurons.

<table>
	<tbody>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>#M4125</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>#E3889</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>#T1503</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Dextran (MW 75,000)</td>
			<td>Spectrum Chemical Mftg Corp</td>
			<td>#DE125</td>
			<td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td>
		</tr>
		<tr>
			<td>Zetamine</td>
			<td>MWI Animal Health</td>
			<td>#501072</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Western Medical Supply</td>
			<td>#5530</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Western Medical Supply</td>
			<td>N/A</td>
			<td>General anesthetic diluent</td>
		</tr>
		<tr>
			<td>Filter Paper (12.5 cm diameter)</td>
			<td>VWR</td>
			<td>#28320-100</td>
			<td>Used for removal of meninges from brain tissue</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td>#60.540.386</td>
			<td>Disposable tubes used for dextran centrifugation steps</td>
		</tr>
		<tr>
			<td>Pierce&#8482; Coomassie Plus (Bradford) Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>#23236</td>
			<td>Measurement of protein concentration in membrane preparations</td>
		</tr>
		<tr>
			<td>Wheaton Overhead Power Homogenizer</td>
			<td>DWK Life Sciences</td>
			<td>#903475</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>10.0ml glass mortar and pestle tissue grinder</td>
			<td>DWK Life Sciences</td>
			<td>#358039</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>#H1758</td>
			<td>Required for pH adjustment of buffers</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>#23210</td>
			<td>Protein standard for Bradford Assay</td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>#91100-12</td>
			<td>Used for dissection of brain tissue</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>#16020-14</td>
			<td>Used for opening skull to isolate brain</td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>#352070</td>
			<td>Used for collection of brain tissue following isolation</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>ThermoFisher Scientific</td>
			<td>#13-678-20C</td>
			<td>Used for aspiration of cellular debris following dextran spins</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>#459836</td>
			<td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>#41121703</td>
			<td>Used for ultracentrifugation of samples</td>
		</tr>
	</tbody>
</table>

a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

The experimental flow for isolation of rat brain microvessels and for the preparation of microvessel membrane samples is shown in Figure 1. Using the procedure presented here, successful isolation of intact microvessels from rat brain is demonstrated (Figure 2A). These vessels were obtained following completion of centrifugation with dextran and immediately prior to commencing ultracentrifugation to prepare membrane samples (i.e....

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

The overall goal of this procedure is to isolate cerebral microvessels from rat brain for biochemical studies aimed at understanding molecular characteristics of the mammalian blood-brain barrier. This method can help to answer key questions in blood-brain barrier physiology and neuropharmacology, such as regulation, localization, and expression of tight junction proteins and endogenous transporters in health and disease. The main advantage of this technique is that we can isolate a sufficient number of high-quality microvessels from an individual animal while taking into account the natural variability in protein expression between individual animals.Demonstrating this technique will be Bianca Reilly, an outstanding undergraduate student from my laboratory. After euthanizing and decapitating a Sprague-Dawley rat according to the text protocol, use surgical scissors to resect the skin from the rat skull by making a single transverse cut. Using rongeurs, carefully remove the skull plate, and expose the brain.Then, with a spatula, remove the organ. Detach the cerebrum, and place the isolated brain tissue into a 50-milliliter conical tube with five milliliters of BMB and 5.0 microliters of protease inhibitor cocktail. To process the tissue, transfer it to a clean Petri dish.Then, using forceps, gently roll the brain on a 12.5-centimeter diameter filter paper to remove the outer meninges, which are loosely adhered to the cerebral cortex. During this step, care should be taken to ensure that all meningeal tissue has been removed from the brain tissue specimen. The meninges can be visualized as a clear membranous tissue that can be easily pulled away from the brain tissue.Gently press the brain tissue against the filter paper, and roll the tissue again, turning the filter paper frequently during this step. Then, using forceps, separate the choroid plexus from the cerebral hemispheres. Then, gently flatten the brain tissue again, and use forceps to remove the olfactory bulbs adjacent to white matter.Place the cortical brain tissue into a chilled glass mortar. Then, add five milliliters of BMB with protease inhibitor cocktail. Using an overhead power homogenizer, insert a pestle into the mortar, and homogenize the brain tissue using 15 up and down strokes at 3, 700 rpm.Then, pour the homogenate into labeled centrifuge tubes. Between samples, use 70%ethanol to clean the pestle. To carry out centrifugation, add 8.0 milliliters of 26%dextran solution to each labeled centrifuge tube containing brain homogenate.Invert the tube twice, and then thoroughly vortex the sample. Conduct the vortexing of each sample using multiple angles to ensure a thorough mixing of brain homogenate solution with 26%dextran solution. Centrifuge the samples at 5, 000 g and four degrees Celsius for 15 minutes.Aspirate the supernatant, and resuspend the pellet in 5.0 milliliters of BMB with protease inhibitor cocktail. Then, vortex the pellet to ensure thorough mixing. Next, add 8.0 milliliters of 26%dextran to each centrifuge tube, and vortex as just demonstrated.Then, centrifuge the samples again. Using a vacuum flask and glass pipette, aspirate the supernatant, and ensure that the pellet containing the brain microvessel is not disrupted. Resuspend the pellet in BMB with protease inhibitor and then 26%dextran two more times.After the final centrifugation, add 5.0 milliliters of BMB to each pellet and vortex to resuspend the sample. Using the protocol demonstrated in this video, successful isolation of intact microvessels from rat brain was carried out. In this image, the microvessel is stained using an antibody against Oatp1a4, a transporter that is well-expressed at the plasma membrane of brain microvascular endothelial cells.As demonstrated by western blot, membrane preparations from microvessels harvested from female Sprague-Dawley rats were enriched in PECAM-1, also known as CD31, a marker protein for brain microvasculature. During optimization of this protocol, PECAM enrichment was observed in membrane samples prepared using two and four dextran centrifugation steps. As determined by the Bradford protein assay and listed in this table, membrane preparations typically yield protein concentrations ranging between 5.0 milligrams per milliliter and 10.0 milligrams per milliliter.The protein values were determined based on a standard curve that was generated using BSA as the standard. Shown here, is a western blot for the BBB transport protein Glut1. Clear single bands at 54 kilodaltons were detected for this highly expressed BBB protein in each membrane sample.This graph illustrates a higher expression of Glut1 at the BBB in male versus female rats. No difference in expression of sodium/potassium ATPase was observed between male and female animals. Once mastered, this technique can be done in two to 2 1/2 hours if it is performed properly.While attempting this procedure, it is important to remember to keep all samples on ice throughout the protocol and to take care when aspirating the supernatant from the microvessel pellet after the centrifugation steps. Following this procedure, other methods, like co-immunoprecipitation analysis, can be performed on microvessel samples to answer additional questions, like the association of different proteins within the same protein complex at the blood-brain barrier. After its development, this technique paved the way for researchers in the field of blood-brain barrier physiology and neuropharmacology to explore molecular characteristics of the blood-brain barrier in health and in rodent models of neurological diseases.After watching this video, you should have a good understanding of how to isolate high-quality microvessels from rat brain for your own studies of blood-brain barrier biology in health and disease.

a-simple-reproducible-method-to-prepare-membrane-samples-from-freshly

Research

JoVE Journal

Neuroscience

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...