Name: using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models
Uploaded: 2017-05-03T13:00:00
Description: Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

This work was supported by the National Institute of General Medical Sciences (MNR; U54GM104942), NIA (MNR; R15AG045812), Alzheimer's Association (MNR; NIRG-12-242187), WVU Faculty Research Senate Grant (MNR), and WVU PSCOR Grant (MNR).

1. Coating the Microelectrode Array with Enzymes or Matrix Layer
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	<li>Preparing the protein matrix solution
	<ol>
		<li>Weigh out 10 mg of bovine serum albumin (BSA) and transfer to the 1.5 mL microcentrifuge tube.</li>
		<li>Add 985 &#181;L of DI water to the microcentrifuge tube containing BSA. Mix the solution by manual agitation (re-pipetting using 1,000 &#181;L pipette ~ 3 times, until the BSA is dissolved). 
		NOTE: Do not use vortex to mix the solution as doing so may introduce air int...

<ol>
	<li>Bito, L., Davson, H., Levin, E., Murray, M., Snider, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentrations+of+free+amino+acids+and+other+electrolytes+in+cerebrospinal+fluid,+in+vivo+dialysate+of+brain,+and+blood+plasma+of+the+dog.">The concentrations of free amino acids and other electrolytes in cerebrospinal fluid, in vivo dialysate of brain, and blood plasma of the dog.</a> J Neurochem. 13 (11), 1057-1067 (1966).</li><li>Cavus, I., 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=Extracellular+metabolites+in+the+cortex+and+hippocampus+of+epileptic+patients.">Extracellular metabolites in the cortex and hippocampus of epileptic patients.</a> Ann Neurol. 57 (2), 226-235 (2005).</li><li>Montgomery, A. J., Lingford-Hughes, A. R., Egerton, A., Nutt, D. J., Grasby, P. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+effects+on+glutamate+uptake+by+human+astrocytes.">Cytokine effects on glutamate uptake by human astrocytes.</a> Neuroimmunomodulation. 7 (3), 153-159 (2000).</li><li>He, X., 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+association+between+CCL2+polymorphisms+and+drug-resistant+epilepsy+in+Chinese+children.">The association between CCL2 polymorphisms and drug-resistant epilepsy in Chinese children.</a> Epileptic Disord. 15 (3), 272-277 (2013).</li><li>Xie, Y., 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=Resolution+of+High-Frequency+Mesoscale+Intracortical+Maps+Using+the+Genetically+Encoded+Glutamate+Sensor+iGluSnFR.">Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR.</a> J Neurosci. 36 (4), 1261-1272 (2016).</li><li>Marvin, J. 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=An+optimized+fluorescent+probe+for+visualizing+glutamate+neurotransmission.">An optimized fluorescent probe for visualizing glutamate neurotransmission.</a> Nat Methods. 10 (2), 162-170 (2013).</li><li>Hefendehl, J. 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=Mapping+synaptic+glutamate+transporter+dysfunction+in+vivo+to+regions+surrounding+Abeta+plaques+by+iGluSnFR+two-photon+imaging.">Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Abeta plaques by iGluSnFR two-photon imaging.</a> Nat Commun. 7, 13441 (2016).</li><li>Hinzman, J. M., Thomas, T. C., Quintero, J. E., Gerhardt, G. A., Lifshitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruptions+in+the+regulation+of+extracellular+glutamate+by+neurons+and+glia+in+the+rat+striatum+two+days+after+diffuse+brain+injury.">Disruptions in the regulation of extracellular glutamate by neurons and glia in the rat striatum two days after diffuse brain injury.</a> J Neurotrauma. 29 (6), 1197-1208 (2012).</li><li>Thomas, T. C., Hinzman, J. M., Gerhardt, G. A., Lifshitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypersensitive+glutamate+signaling+correlates+with+the+development+of+late-onset+behavioral+morbidity+in+diffuse+brain-injured+circuitry.">Hypersensitive glutamate signaling correlates with the development of late-onset behavioral morbidity in diffuse brain-injured circuitry.</a> J Neurotrauma. 29 (2), 187-200 (2011).</li><li>Hinzman, 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=Diffuse+brain+injury+elevates+tonic+glutamate+levels+and+potassium-evoked+glutamate+release+in+discrete+brain+regions+at+two+days+post-injury:+an+enzyme-based+microelectrode+array+study.">Diffuse brain injury elevates tonic glutamate levels and potassium-evoked glutamate release in discrete brain regions at two days post-injury: an enzyme-based microelectrode array study.</a> J Neurotrauma. 27 (5), 889-899 (2010).</li><li>Stephens, M. L., Quintero, J. E., Pomerleau, F., Huettl, P., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+glutamate+release+in+the+CA3+and+dentate+gyrus+of+the+rat+hippocampus.">Age-related changes in glutamate release in the CA3 and dentate gyrus of the rat hippocampus.</a> Neurobiol Aging. 32 (5), 811-820 (2009).</li><li>Nickell, J., Salvatore, M. F., Pomerleau, F., Apparsundaram, S., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+plasma+membrane+surface+expression+of+GLAST+mediates+decreased+glutamate+regulation+in+the+aged+striatum.">Reduced plasma membrane surface expression of GLAST mediates decreased glutamate regulation in the aged striatum.</a> Neurobiol Aging. 28 (11), 1737-1748 (2006).</li><li>Hascup, E. 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=An+allosteric+modulator+of+metabotropic+glutamate+receptors+(mGluR(2)+)+(+)-TFMPIP,+inhibits+restraint+stress-induced+phasic+glutamate+release+in+rat+prefrontal+cortex.">An allosteric modulator of metabotropic glutamate receptors (mGluR(2) ) (+)-TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex.</a> J Neurochem. 122 (2), 619-627 (2012).</li><li>Rutherford, E. C., Pomerleau, F., Huettl, P., Stromberg, I., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+second-by-second+measures+of+L-glutamate+in+the+central+nervous+system+of+freely+moving+rats.">Chronic second-by-second measures of L-glutamate in the central nervous system of freely moving rats.</a> J Neurochem. 102 (3), 712-722 (2007).</li><li>Matveeva, E. A., 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=Reduction+of+vesicle-associated+membrane+protein+2+expression+leads+to+a+kindling-resistant+phenotype+in+a+murine+model+of+epilepsy.">Reduction of vesicle-associated membrane protein 2 expression leads to a kindling-resistant phenotype in a murine model of epilepsy.</a> Neuroscience. 202, 77-86 (2011).</li><li>Matveeva, E. A., 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=Kindling-induced+asymmetric+accumulation+of+hippocampal+7S+SNARE+complexes+correlates+with+enhanced+glutamate+release.">Kindling-induced asymmetric accumulation of hippocampal 7S SNARE complexes correlates with enhanced glutamate release.</a> Epilepsia. 53 (1), 157-167 (2012).</li><li>Hunsberger, H. C., Rudy, C. C., Batten, S. R., Gerhardt, G. A., Reed, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P301L+tau+expression+affects+glutamate+release+and+clearance+in+the+hippocampal+trisynaptic+pathway.">P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway.</a> J Neurochem. 132 (2), 169-182 (2015).</li><li>Hunsberger, H. 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=Riluzole+rescues+glutamate+alterations,+cognitive+deficits,+and+tau+pathology+associated+with+P301L+tau+expression.">Riluzole rescues glutamate alterations, cognitive deficits, and tau pathology associated with P301L tau expression.</a> J Neurochem. 135 (2), 381-394 (2015).</li><li>Hunsberger, H. 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=Peripherally+restricted+viral+challenge+elevates+extracellular+glutamate+and+enhances+synaptic+transmission+in+the+hippocampus.">Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.</a> J Neurochem. , (2016).</li><li>Obrenovitch, T. P., Urenjak, J., Zilkha, E., Jay, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+in+neurological+disorders--the+glutamate+paradox.">Excitotoxicity in neurological disorders--the glutamate paradox.</a> Int J Dev Neurosci. 18 (2-3), 281-287 (2000).</li><li>Hillered, L., Vespa, P. M., Hovda, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+neurochemical+research+in+acute+human+brain+injury:+the+current+status+and+potential+future+for+cerebral+microdialysis.">Translational neurochemical research in acute human brain injury: the current status and potential future for cerebral microdialysis.</a> J Neurotrauma. 22 (1), 3-41 (2005).</li><li>Burmeister, J. J., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-referencing+ceramic-based+multisite+microelectrodes+for+the+detection+and+elimination+of+interferences+from+the+measurement+of+L-glutamate+and+other+analytes.">Self-referencing ceramic-based multisite microelectrodes for the detection and elimination of interferences from the measurement of L-glutamate and other analytes.</a> Anal Chem. 73 (5), 1037-1042 (2001).</li><li>Burmeister, 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=Improved+ceramic-based+multisite+microelectrode+for+rapid+measurements+of+L-glutamate+in+the+CNS.">Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS.</a> J Neurosci Methods. 119 (2), 163-171 (2002).</li><li>Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes:+transmitter+uptake+gets+faster+during+development.">Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.</a> J Neurosci. 25 (11), 2906-2916 (2005).</li><li>Greene, J. G., Borges, K., Dingledine, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+transcriptional+neuroanatomy+of+the+rat+hippocampus:+evidence+for+wide-ranging,+pathway-specific+heterogeneity+among+three+principal+cell+layers.">Quantitative transcriptional neuroanatomy of the rat hippocampus: evidence for wide-ranging, pathway-specific heterogeneity among three principal cell layers.</a> Hippocampus. 19 (3), 253-264 (2009).</li><li>Borland, L. M., Shi, G., Yang, H., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+study+of+extracellular+dopamine+near+microdialysis+probes+acutely+implanted+in+the+striatum+of+the+anesthetized+rat.">Voltammetric study of extracellular dopamine near microdialysis probes acutely implanted in the striatum of the anesthetized rat.</a> J Neurosci Methods. 146 (2), 149-158 (2005).</li><li>Jaquins-Gerstl, A., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+brain+penetration+injury+associated+with+microdialysis+and+voltammetry.">Comparison of the brain penetration injury associated with microdialysis and voltammetry.</a> J Neurosci Methods. 183 (2), 127-135 (2009).</li><li>Azbill, R. D., Mu, X., Springer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+increases+high-affinity+glutamate+uptake+in+rat+spinal+cord+synaptosomes.">Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes.</a> Brain Res. 871 (2), 175-180 (2000).</li><li>Gourley, S. L., Espitia, J. W., Sanacora, G., Taylor, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antidepressant-like+properties+of+oral+riluzole+and+utility+of+incentive+disengagement+models+of+depression+in+mice.">Antidepressant-like properties of oral riluzole and utility of incentive disengagement models of depression in mice.</a> Psychopharmacology (Berl). 219 (3), 805-814 (2011).</li><li>Frizzo, M. E., Dall'Onder, L. P., Dalcin, K. B., Souza, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+enhances+glutamate+uptake+in+rat+astrocyte+cultures).">Riluzole enhances glutamate uptake in rat astrocyte cultures).</a> Cell Mol Neurobiol. 24 (1), 123-128 (2004).</li><li>Fumagalli, E., Funicello, M., Rauen, T., Gobbi, M., Mennini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+enhances+the+activity+of+glutamate+transporters+GLAST,+GLT1+and+EAAC1.">Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1.</a> Eur J Pharmacol. 578 (2-3), 171-176 (2008).</li><li>Day, B. K., Pomerleau, F., Burmeister, J. J., Huettl, P., Gerhardt, G. 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The MEA technique allows for measurement of fast kinetics of neurotransmitter release and uptake in vitro and in vivo. Hence, the technology produces a wide variety of data output including tonic neurotransmitter levels, evoked neurotransmitter release, and neurotransmitter clearance. However, because use of MEAs is a relatively complex procedure, there are numerous factors that may need to be optimized for successful use. For example, during calibration, one may note that there are no signal waveforms ...

Measuring neurotransmitter alterations in the brain is an essential tool for neuroscientists studying diseases of the central nervous system (CNS) that are often characterized by neurotransmitter dysregulation. Though microdialysis in combination with high pressure liquid chromatography (HPLC/EC) has been the most widely used method to measure changes in extracellular neurotransmitter levels1,2,3,4, the spatial and temporal resolution of microdialysis probes may not be ideal for neurotransmitters, such as glutamate, that are tightly regulated in the extracellular space5,6. Because of the recent advances in genetics and imaging, there are additional methods that can be used to map glutamate in vivo. Using genetically encoded glutamate fluorescent reporters (iGluSnFR) and two-photon imaging, researchers are able to visualize glutamate release by neurons and astrocytes both in vitro and in vivo7,8,9. Notably, this allows for recording from a larger field of view and does not disrupt the intrinsic connections of the brain. While these new optical techniques allow for visualization of glutamate kinetics and measurement of sensory evoked responses and neuronal activity, they lack the ability to quantify the amount of glutamate in the extracellular space in discrete brain regions.
An alternative method is the enzyme-linked microelectrode array (MEA) that can selectively measure extracellular neurotransmitter levels, such as glutamate, through the use of a self-referenced recording scheme. The MEA technique has been used to study alterations in extracellular glutamate following traumatic brain injury10,11,12, aging13,14, stress15,16, epilepsy17,18, Alzheimer&#39;s disease19,20, and injection of a viral mimic21 and represents an improvement over the spatial and temporal limitations inherent in microdialysis. Whereas microdialysis restricts the ability to measure near the synapse22,23, MEAs have a high spatial resolution that allows for selective measures of extracellular glutamate spillover near synapses24,25. Second, the low temporal resolution of microdialysis (1 - 20 min) limits the ability to investigate the fast dynamics of glutamate release and clearance occurring in the millisecond to second range26. Because differences in the release or clearance of glutamate may not be evident in measures of tonic, resting glutamate levels, it may be essential that glutamate release and clearance be directly measured. MEAs allow for such measures due to their high temporal resolution (2 Hz) and low limits of detection (&#60; 1 &#181;M). Third, MEAs allow for examination of subregional variations in neurotransmitters within a particular brain region, such as the rat or mouse hippocampus. For example, using MEAs we can separately target the dentate gyrus (DG), cornu ammonis 3 (CA3) and cornu ammonis 1 (CA1) of the hippocampus, which are connected via a trisynaptic circuit27, to examine subregional differences in extracellular glutamate. Because of the size of microdialysis probes (1 - 4 mm length) and the damage caused by implantation28,29, subregional differences are difficult to address. Furthermore, the optical systems only allow stimulation through external stimuli, such as a whisker stimulation or light flicker, which does not permit subregional stimulation7. A final benefit of MEAs over other methods is the ability to study these subregions in vivo without disrupting their extrinsic and intrinsic connections.
Here, we describe how a recording system (e.g., FAST16mkIII) in combination with MEAs, consisting of a ceramic-based multisite microelectrode, can be differentially coated on the recording sites to allow for interfering agents to be detected and removed from the analyte signal. We also demonstrate these arrays can be used for amperometry-based studies of in vivo glutamate regulation within the DG, CA3, and CA1 hippocampal subregions of anesthetized rTg(TauP301L)4510 mice, a commonly used mouse model of Alzheimer&#39;s disease. In addition, we provide confirmation of the sensitivity of the MEA system to the fast dynamics of glutamate release and clearance by treating the mice with riluzole, a drug shown in vitro to decrease glutamate release and increase glutamate uptake30,31,32,33, and demonstrating these respective changes in vivo in the TauP301L mouse model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FAST-16mkIII-8 channel</td>
			<td>Quanteon</td>
			<td>16mkIII</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode arrays</td>
			<td>CenMet</td>
			<td>W4 or 8-TRK</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A-3059</td>
			<td>10 g (expires after 1 month)</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G-6257</td>
			<td>100 mL (expires after 6 months)</td>
		</tr>
		<tr>
			<td>Glutamate Oxidase</td>
			<td>US Biological or Sigma Aldrich</td>
			<td>G4001-01 or 100646</td>
			<td>50 UI (expires after 6 months)</td>
		</tr>
		<tr>
			<td>Hamilton Syringes</td>
			<td>Hamilton</td>
			<td>#701</td>
			<td>2 syringes</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>UN1230</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>m-Phenylenediamine dihydrochloride (mPD)</td>
			<td>ACROS Organics</td>
			<td>1330560250</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Reference Electrodes (RE-5B)</td>
			<td>BAS</td>
			<td>MF-2079</td>
			<td>3 electrodes</td>
		</tr>
		<tr>
			<td>Magnetic stir plate</td>
			<td>Cole-parmer</td>
			<td>EW-04804-01</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G-1626</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>TCI</td>
			<td>50-81-7</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>62-31-7</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>VWR</td>
			<td>UN2920</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Postassium chloride</td>
			<td>VWR</td>
			<td>7447-40-7</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>7647-40-7</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>MP</td>
			<td>153502</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>1310732</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Glass pressure ejection pipettes</td>
			<td>CenMet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sticky wax</td>
			<td>Kerrlab</td>
			<td>625</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Microsyringe</td>
			<td>World Precision Instruments</td>
			<td>MF28G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Modeling clay</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Parker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>AM systems</td>
			<td>782000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>BDH</td>
			<td>7647010</td>
			<td>2.5 L</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>AM Systems</td>
			<td>778000</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder gun</td>
			<td>Lowes or Home Depot</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>SomnoSuite</td>
			<td>Kent Scientific</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Stereotaxic device</td>
			<td>Stoelting</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Digital Lab Standard</td>
			<td>Stoelting</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Meiji EMZ microscope</td>
			<td>Meiji</td>
			<td>EMZ-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>Micro</td>
			<td></td>
		</tr>
		<tr>
			<td>Metricide</td>
			<td>Metrex</td>
			<td>102800</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Surgery scissors</td>
			<td>VWR</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile cotton swabs</td>
			<td>Puritan</td>
			<td>25806</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Puralube Vet Ointment</td>
			<td></td>
			<td>Obtain from the vet</td>
		</tr>
		<tr>
			<td>Iodine swabs</td>
			<td>VWR</td>
			<td>S48050</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Local drug store</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile surgery drape</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Teknova</td>
			<td>S5815</td>
			<td>Can make own soltuion using filters</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (3%)</td>
			<td>Local drug store</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
	</tbody>
</table>

using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

While this technology can be used to measure alterations in glutamatergic signaling in many types of animal models, such as traumatic brain injury, aging, stress, and epilepsy, here we demonstrate how the MEA technology can be used to examine glutamatergic alterations in transgenic mouse model of human tauopathy19,20. The rTg(TauP301L)4510 mouse expresses the P301L mutation in tau associated with frontotemporal dementia and parkin...

Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

The overall goal of this procedure is to measure tonic and phasic extracellular glutamate changes in vivo using enzyme linked MicroElectrode Arrays. This method can help answer key questions in the neuroscience field, such as whether extracellular neurotransmitter levels and the fast dynamics of neurotransmitter release and clearance are altered in disease states. The main advantage of this technique is that the MicroElectrode Arrays can measure specific neurotransmitter changes within discrete brain regions, making it a spatially and temporally precise approach while being minimally invasive.Demonstrating the procedure will be Holly Hunsberger and Ryan Heslin from my laboratory. To begin this procedure, coat a pair of recording sites on an MEA with the desired enzyme and a different pair of recording sites with the inactive protein matrix. Then, draw up the glutamate oxidase or protein matrix using a 10 microliter Hamilton syringe.Gently press the plunger to dispense a small bead of solution at the syringe tip. To target the MEA recording sites, under a dissecting microscope, apply the solution to the appropriate recording sites by briefly contacting the recording sites with the solution droplet, which represents one layer. Set a timer for one minute between coating applications to the recording sites.Now, place the reference electrode through the opening in the plastic arm. Lower the reference electrode into a beaker containing PBS and ensure that it does not contact the bottom of the beaker. Next, connect the reference electrode to the head stage and connect the MEA to the mPD plated to the head stage.Lower the MEA into the beaker of solution such that only the MEA tip is submerged in solution. Do not submerge the MEA tip in liquid beyond the black bubble. Doing so may damage the MEA.Then, select the electroplating icon on the desktop. In the electroplating tool menu, verify the correct settings are entered. Afterward, select exit software to finish electroplating.Rinse the mPD plated electrode tips with DI water and store for 24 hours before calibrating. In this procedure, turn on a heating pad at 37 degrees Celsius and connect the head stage to the recording control system. Then, insert the MEA tip into 40 milliliters of stirred 0.5 molar PBS.Next, connect the reference electrode to the head stage. Flick the reference electrode tip to ensure there are no air bubbles. After that, open the recording program, make sure the settings are correct, choose the MEA number, and click Calibration, and press start.Allow five to 10 minutes for equilibration and begin calibration once the baseline has stabilized. Next, select Baseline and add 500 microliters of ascorbic acid. Then, select Interferent once the current has reached a new steady plateau.If the MEA is properly electroplated, no change should be observed. Next, add 40 microliters of 20 millimolar L-glutamate. Once the current has reached a new steady plateau, mark first addition as analyte.Repeat three times for a total of three glutamate additions. Then, add 40 microliters of dopamine. And select Test Substance.Subsequently, add 40 microliters of peroxide. Select Test Substance and click the stop button once the calibration is finished. In this step, place the micropipette centrally among all four platinum recording sites and mount it 50 to 100 micrometers above the MEA using sticky wax and modeling clay.Using a DC adapter, clamp the red positive wire to the prepared silver wire on the gold pin side, and clamp the black negative wire to the platinum bath cathode. Next, plug in the DC adapter and look for the correct plating process. Bubbles should appear at the bath electrode, and the reference electrode should turn a silver gray color.In this procedure, place the animal in the stereotaxic device and use the ear bars to stabilize its head. Make sure the animal is secured and the head does not move. Then, apply eye ointment using a sterile cotton applicator.After that, shave the head with a trimmer. And remove the fur near the ears using small surgical scissors. Subsequently, apply iodine and then alcohol to the scalp three times alternatively.Following this, make an incision in the middle of the scalp and spread the skin. Soak up any blood with a sterile cotton tip and apply hydrogen peroxide to facilitate the appearance of bregma and lambda. Make sure the head is properly positioned by measuring the dorsal-ventral and medial-lateral of bregma and lambda.Set the coordinates at bregma to zero and mark the target coordinates. Next, drill around the mark. Afterward, attach the MEA to the head stage.Back fill the pipette with the first desired solution. Be sure to leave a gap in the pipette without solution so that the solution being expelled can be examined. Then, attach the tubing to the glass micropipette.Find bregma with the MEA attached and set the coordinates to zero. Move the MEA to the desired coordinates and slowly lower the MEA until the tip touches the brain. Next, zero the dorsal-ventral coordinate and then slowly lower the MEA into the brain.Place the reference electrode in a remote location from the MEA such that it's still in liquid contact with the brain to complete the circuit. On the recording program, click on the desired calibrated electrode and then click perform experiment to start recording. After obtaining a stable baseline, set the pressure ejector to 0.6 seconds and 0.5 psi and press the red button on the pressure ejector to eject.Record the time and pressure as well as volume ticks moved on the injection sheet and make any notes if necessary. After that, remove the MEA with the micropipette attached, rinse it with DI water, and soak it in PBS overnight until all the blood is gone. In the CA3 and CA1 regions of the hippocampus, tonic glutamate levels were significantly increased in the vehicle treated TauP301L mice, an effect attenuated by riluzole treatment.Here, the baseline matched representative recordings of potassium chloride evoked glutamate release in the CA3 showed riluzole treatment attenuated the significant increase in the amplitude of glutamate release observed in Vehicle-TauP301L mice. Local application of potassium chloride produced a robust increase in extracellular glutamate that rapidly returned to tonic levels. The significantly increased potassium chloride evoked glutamate release in the DG, CA3, and CA1 observed in the Vehicle treated TauP301L mice after local application of potassium chloride was attenuated with riluzole treatment.This cresyl violet stained section of hippocampus confirms the location of MEA tracks in CA3 and CA1. While attempting this procedure, it's important to remember the troubleshooting steps listed in the manuscript. For instance, to reduce noisy signals, remember to ground the system and follow the steps in a timely manner.

using-enzyme-based-biosensors-to-measure-tonic-phasic-glutamate

Research

JoVE Journal

Neuroscience

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

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 ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...