Financiamiento proporcionado por el Instituto Nacional sobre el Abuso de Alcohol y Alcoholismo (NIAAA, AA-016967 y AA016967 01S1; TAM), Wayne State University en marcha los fondos, y Wayne State University Programa de Becas de Investigación. El contenido es responsabilidad exclusiva de sus autores y no representan la opinión oficial del NIAAA y los Institutos Nacionales de Salud.

<ol>
	<li>Pike, C. M., Grabner, C. P., Harkins, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Amperometric+Electrodes.">Fabrication of Amperometric Electrodes.</a> J. Vis. Exp. (27), e1040-e1040 (2009).</li><li>Lack, A. K., Diaz, M. R., Chappell, A., DuBois, D. W., McCool, B. 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+ethanol+and+withdrawal+differentially+modulate+pre-+and+postsynaptic+function+at+glutamatergic+synapses+in+rat+basolateral+amygdala.">Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala.</a> J. Neuropyhysiol. 98, 3185-3196 (2007).</li><li>John, C. E., Jones, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+characterization+of+the+effect+of+monoamine+uptake+inhibitors+and+release+on+dopamine+and+serotonin+uptake+in+mouse+caudate-putamen+and+substantia+nigra+slices.">Voltammetric characterization of the effect of monoamine uptake inhibitors and release on dopamine and serotonin uptake in mouse caudate-putamen and substantia nigra slices.</a> Neuropharmacology. 52, 1596-1605 (2007).</li><li>Wightman, R. M., Amatore, C., Engstrom, R. C., Hale, P. D., Kistensen, E. W., Kuhr, W. G., May, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+characterization+of+dopamine+overflow+and+uptake+in+the+rat+striatum.">Real-time characterization of dopamine overflow and uptake in the rat striatum.</a> Neuroscience. 25, 513-523 (1988).</li><li>Wightman, R. M., Zimmerman, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+dopamine+extracellular+concentration+in+rat+striatum+by+impulse+flow+and+uptake.">Control of dopamine extracellular concentration in rat striatum by impulse flow and uptake.</a> Brain. Res. Brain. Res. Rev. 15, 135-144 (1990).</li><li>Jones, S. R., Garris, P. A., Kilts, C. D., Wightman, 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=Comparison+of+dopamine+uptake+in+the+basolateral+amygdaloid+nucleus,+caudate-putamen,+and+nucleus+accumbens+of+the+rat.">Comparison of dopamine uptake in the basolateral amygdaloid nucleus, caudate-putamen, and nucleus accumbens of the rat.</a> J. Neurochem. 64, 2581-2589 (1995).</li><li>Michael, D. J., Wightman, 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=Electrochemical+monitoring+of+biogenic+amine+neurotransmission+in+real+time.">Electrochemical monitoring of biogenic amine neurotransmission in real time.</a> J. Pharm. Biomed. Anal. 19, 33-46 (1999).</li><li>Pajski, M. L., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+release+evoked+by+short+electrical+stimulations+in+striatal+brain+slices+is+primarily+activity+dependent.">Adenosine release evoked by short electrical stimulations in striatal brain slices is primarily activity dependent.</a> A.C.S. Chem. Neurosci. 1, 775-787 (2010).</li><li>Sanford, A. L., Morton, S. W., Whitehouse, K. L., Oara, H. M., Lugo-Morales, L. Z., Roberts, J. G., Sombers, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+detection+of+hydrogen+peroxide+at+carbon+fiber+microelectrodes.">Voltammetric detection of hydrogen peroxide at carbon fiber microelectrodes.</a> Anal. Chem. 82, 5205-5210 (2010).</li><li>Park, J., Kile, B. M., Wightman, 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=In+vivo+voltammetric+monitoring+of+norepinephrine+release+in+the+rat+ventral+bed+nucleus+of+the+stria+terminalis+and+anteroventral+thalamic+nucleus.">In vivo voltammetric monitoring of norepinephrine release in the rat ventral bed nucleus of the stria terminalis and anteroventral thalamic nucleus.</a> Eur. J. Neurosci. 30, 2121-2133 (2009).</li><li>Hashemi, P., Dankoski, E. C., Petrovic, J., Keithley, R. B., Wightman, 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=Voltammetric+detection+of+5-hydroxytryptamine+release+in+the+rat+brain.">Voltammetric detection of 5-hydroxytryptamine release in the rat brain.</a> Anal. Chem. 81, 9462-9471 (2009).</li><li>Borue, X., Cooper, S., Hirsh, J., Condron, B., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+serotonin+release+and+clearnece+in+drosophila.">Quantitative evaluation of serotonin release and clearnece in drosophila.</a> J. Neuroscience. Methods. 179, 300-308 (2009).</li><li>Vickrey, T. L., Condron, B., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+endogenous+dopamine+changes+in+drosophila+melanogaster+using+fast-scan+cyclic+voltammetry.">Detection of endogenous dopamine changes in drosophila melanogaster using fast-scan cyclic voltammetry.</a> Anal. Chem. 81, 9306-9313 (2009).</li><li>Makos, M. A., Han, K. A., Heien, M. L., Ewing, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+in+vivo+electrochemistry+to+study+the+physiological+effects+of+cocaine+and+other+stimulants+on+the+drosophila+melanogaster+dopamine+transporter.">Using in vivo electrochemistry to study the physiological effects of cocaine and other stimulants on the drosophila melanogaster dopamine transporter.</a> A.C.S. Chem Neurosci. 1, 74-83 (2009).</li><li>Budygin, E. A., John, C. E., Mateo, Y., Daunais, J. B., Friedman, D. P., Grant, K. A., Jones, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ethanol+exposure+alters+presynaptic+dopamine+function+in+striatum+of+monkeys:+a+preliminary+study.">Chronic ethanol exposure alters presynaptic dopamine function in striatum of monkeys: a preliminary study.</a> Synapse. 50, 266-268 (2003).</li><li>Jones, S. R., Gainetdinov, R. R., Jaber, M., Giros, B., Wightman, R. M., Caron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+neuronal+plasticity+in+response+to+inactivation+of+the+dopamine+transporter.">Profound neuronal plasticity in response to inactivation of the dopamine transporter.</a> PNAS. 95, 4029-4034 (1998).</li><li>Kennedy, R. T., Jones, S. R., Wightman, 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=Dynamic+observation+of+dopamine+autoreceptor+effects+in+rat+striatal+slices.">Dynamic observation of dopamine autoreceptor effects in rat striatal slices.</a> J. Neurochem. 59, 449-445 (1992).</li><li>Rice, M. E., Cragg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+amplifies+reward-related+dopamine+signals+in+striatum.">Nicotine amplifies reward-related dopamine signals in striatum.</a> Nat. Neurosci. 7, 583-584 (2004).</li><li>Zhang, L., Doyon, W. M., Clark, J. J., Phillips, P. E., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+tonic+and+phasic+dopamine+transmission+in+the+dorsal+and+ventral.">Controls of tonic and phasic dopamine transmission in the dorsal and ventral.</a> 76, 396-404 (2009).</li><li>Paredes, D., Grnaholm, A. C., Bickford, P. C. <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+NGF+and+BDNF+on+baseline+glutamate+and+dopamine+release+in+the+hippocampal+formation+of+the+adult+rat.">Effects of NGF and BDNF on baseline glutamate and dopamine release in the hippocampal formation of the adult rat.</a> Brain. Res. 11141, 56-64 (2007).</li><li>Goggi, J., Puller, I. A., Carney, S. L., Bradford, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+the+short-term+potentiation+of+dopamine+release+by+BDNF.">Signalling pathways involved in the short-term potentiation of dopamine release by BDNF.</a> Brain. Res. 968, 156-161 (2003).</li><li>Cheer, J. F., Wassum, K. M., Heien, M. L., Phillips, P. E., Wightman, 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=Cannabinoids+enhance+subsecond+doapmine+relese+in+the+nucleus+accumbens+of+awake+rats.">Cannabinoids enhance subsecond doapmine relese in the nucleus accumbens of awake rats.</a> J. Neurosci. 24, 4393-4400 (2004).</li><li>Phillips, P. E., Stuber, G. D., Heien, M. L., Wightman, R. M., Carelli, 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=Subsecond+dopamine+release+promotes+cocaine+seeking.">Subsecond dopamine release promotes cocaine seeking.</a> Nature. 422, 614-618 (2003).</li><li>Robinson, D. L., Heien, M. L., Wightman, 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=Frequency+of+dopamine+concentration+transients+increases+in+dorsal+and+ventral+striatum+of+male+rats+duing+introduction+of+conspecifics.">Frequency of dopamine concentration transients increases in dorsal and ventral striatum of male rats duing introduction of conspecifics.</a> J. Neurosci. 22, 10477-10486 (2002).</li></ol>

No hay conflictos de interés declarado.

El protocolo que aquí se presenta muestra cómo preparar y usar rodajas de cerebro de ratón coronal para experimentos FSCV. Aunque este método es específico para la obtención y medición de la dinámica de la dopamina, neurotransmisores tales como la adenosina, el peróxido de hidrógeno, norepinefrina y serotonina han sido controlados en vivo o in vitro con FSCV 3, 8 - 11. FSCV se puede utilizar para controlar algunos de estos neuroquímicos otras modificaciones simples de la forma de ...

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> CAS </td></tr><tr><td width="274"> Reactivo </td><td></td><td></td></tr><tr><td width="274"> Cloruro de Potasio </td><td> Pescador </td><td width="142" valign="top"> 7447407 </td></tr><tr><td width="274"> De cloruro de sodio </td><td> EMD Chemicals </td><td width="142" valign="top"> 7647145 </td></tr><tr><td width="274"> Cloruro de magnesio </td><td width="194" valign="top"> Pescador </td><td width="142" valign="top"> 7791186 </td></tr><tr><td width="274"> Cloruro de calcio </td><td width="194" valign="top"> Pescador </td><td width="142" valign="top"> 10035048 </td></tr><tr><td width="274"> De bicarbonato de sodio </td><td width="194" valign="top"> EMD Chemicals </td><td width="142" valign="top"> 144558 </td></tr><tr><td width="274"> Fosfato de sodio, dibásico </td><td width="194" valign="top"> EMD Químicas </td><td width="142" valign="top"> 7558794 </td></tr><tr><td width="274"> D-glucosa </td><td width="194" valign="top"> Pescador </td><td width="142" valign="top"> 50997 </td></tr><tr><td width="274"> Ácido ascórbico </td><td width="194" valign="top"> Pescador </td><td width="142" valign="top"> 50817 </td></tr><tr><td width="274"> Sacarosa </td><td width="194" valign="top"> Pescador </td><td> 57501 </td></tr><tr><td width="274">&nbsp;</td><td width="194" valign="top">&nbsp;</td><td>&nbsp;</td></tr><tr><td width="274"> Materiales </td><td width="194" valign="top"> Vendedor </td><td> Número de catálogo </td></tr><tr><td width="274"> De fibra de carbono </td><td> Goodfellow Oakdale, PA </td><td></td></tr><tr><td width="274"> Capilar de vidrio </td><td> AM Sistemas Carlsborg WA. </td><td> 602000 </td></tr><tr><td width="274"> Alambre de plata </td><td> A-M Sistemas Carlsborg WA. </td><td> 787000 </td></tr><tr><td width="274"> Tungsteno electrodos estimulantes </td><td> Uno de los plásticos, Roanoke, VA </td><td></td></tr><tr><td width="274"> De alambre de platino </td><td></td><td></td></tr><tr><td width="274"> Cable conductor </td><td> Squires Electronics, Cornelius, OR </td><td></td></tr><tr><td width="274"> Loctite 404 adhesivos instantáneos </td><td> Hankal Corp. Rocky Hill CT. </td><td></td></tr><tr><td width="274"> Hoja de afeitar </td><td> World Precision Instruments Inc. FL. </td><td></td></tr><tr><td width="274"> BD aguja espinal </td><td> BD Medical Systems, Franlin Lake, NJ </td><td> REF 405234 </td></tr><tr><td width="274"> Hoja de bisturí </td><td> Plumas para maquinillas de afeitar Co. LTD. Japón </td><td></td></tr><tr><td width="274"></td><td></td><td></td></tr><tr><td width="274"> Software </td><td width="194" valign = &quot;top&quot;&gt; Vendedor </td><td> Número de catálogo </td></tr><tr><td width="274"> TH software </td><td> ESA Inc., de Chelmsford, MA </td><td></td></tr><tr><td width="274"> Instrumento </td><td width="194" valign="top"> Vendedor </td><td> Número de catálogo </td></tr><tr><td width="274"> Cámara de grabación de la inmersión </td><td> Costumbre Científico, Denver, CO </td><td></td></tr><tr><td width="274"> Neorolog aislador de estímulo </td><td> Digitmeter, Hertfordshire, Inglaterra </td><td></td></tr><tr><td width="274"> Controlador automático de temperatura </td><td> Warner Instrument Corporation </td><td></td></tr><tr><td width="274"> Microscopio (SZX7) </td><td> Olimpo </td><td></td></tr><tr><td width="274"> Microscopio </td><td> Pescador </td><td></td></tr><tr><td width="274"> Vibratome 3000 seccionamiento del sistema </td><td> San LouEs decir, MO. </td><td></td></tr><tr><td width="274"> Perfusión de la bomba </td><td> Watson Marlow Limited, Falmouth Cornwall - Inglaterra </td><td> H110708 </td></tr><tr><td width="274"> Micropipeta extractor </td><td> Narishige, Tokio, Japón </td><td></td></tr><tr><td width="274"> ChemClamp potenciostato </td><td> Dagan de Sociedades Anónimas, Minneapolis, MN. </td><td></td></tr></tbody></table>

presynaptic dopamine dynamics in striatal brain slices with fast-scan cyclic voltammetry

Una amplia investigación se ha centrado en el neurotransmisor dopamina debido a su importancia en el mecanismo de acción de las drogas de abuso (cocaína y anfetaminas, por ejemplo), el papel que desempeña en las enfermedades psiquiátricas (esquizofrenia, por ejemplo, y déficit de atención e hiperactividad), y su participación en degenerativos trastornos como el Parkinson y la enfermedad de Huntington. En condiciones fisiológicas normales, la dopamina se sabe que regulan la actividad locomotora, la cognición, el aprendizaje, afecta emocional y la secreción de hormonas neuroendocrinas. Una de las mayores densidades de las neuronas de dopamina en el estriado es, que se pueden dividir en dos regiones distintas neuroanatómicas conocida como el núcleo accumbens y el núcleo caudado putamen. El objetivo es ilustrar un protocolo general para la voltametría cíclica corte rápido escaneo (FSCV) en el cuerpo estriado del ratón. FSCV es una técnica bien definida electroquímico proporciona la oportunidad de medir la liberación de dopamina y la captación en tiempo real en discrete regiones del cerebro. Microelectrodos de fibra de carbono (diámetro de aproximadamente 7 micras) se utilizan en FSCV para detectar la oxidación de la dopamina. La ventaja de la utilización de análisis para detectar FSCV dopamina es su mejor resolución temporal de 100 milisegundos y una resolución espacial de menos de diez micras, proporciona información complementaria a la microdiálisis in vivo.

Watch this Scientific Journal Video about Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry at JoVE.com

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

El uso rápido de exploración voltametría cíclica para medir la dinámica evocada eléctricamente presináptica de dopamina estriatal en rodajas de cerebro.

Dinámica presináptica de dopamina estriatal en rodajas de cerebro de voltametría cíclica rápida de exploración

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

presynaptic-dopamine-dynamics-striatal-brain-slices-with-fast-scan

Research

JoVE Journal

Neuroscience

21.7K vistas.  Wayne State University. El uso rápido de exploración voltametría cíclica para medir la dinámica evocada eléctricamente presináptica de dopamina estriatal en rodajas de cerebro.

Video: Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de las neuronas del cerebro de roedores Rodajas para el Estudio de la transmisión sináptica y la imagen terminales presinápticos

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Investigación

Neurociencias

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

Tensión de polarización, voltametría cíclica, y Espectroscopia de Impedancia Eléctrica para las interfaces neuronales

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pareadas Grabaciones de células enteras en organotípicos de hipocampo rebanadas

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Las grabaciones de células enteras Patch-clamp en rodajas de cerebro

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

Modelado de datos de voltametría cíclica de exploración rápida de datos de neurotransmisión de dopamina estimulada eléctricamente usando QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

En Vivo Una sola molécula de seguimiento en la Terminal presináptica del nervio Motor del Drosophila

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirugía estereotáctica para manipulación genética en las células estriatales de cerebro de ratón Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...