Nous remercions le Dr Chirwa Sanika à l&#39;examen critique de ce manuscrit. Ce travail a été soutenu par les Instituts nationaux de la santé (DA026947, DA021471 et NS071122).

1. Matériel et fournitures <ol><li> Monter une cage de Faraday sur le dessus de la table anti-vibration (TMI) pour diminuer le bruit de fond. </li><li> La pince simultanée correctif d&#39;enregistrement ampérométrie système nécessite un microscope inversé avec d&#39;excellentes optiques DIC et une lentille longue distance de travail. Branchez le microscope phare à une batterie de voiture. Cette source de lumière continue pour le système va encore réduire le bruit électrique. </li><li> Micromanipulateurs hydrauliques (Siskiyou) diminution supplémentaire du bruit. Dans notre configuration, nous utilisons un manipulateur droitier pour l&#39;enregistrement de cellules entières, et le gaucher pour ampérométrie. </li></ol> 2. Préparer électrodes pour l&#39;enregistrement <ol><li> Tirez électrodes de patch en utilisant les pipettes de quartz sur un extracteur P-2000 (Sutter). Notre traction dure environ 5 secondes, avec deux cycles thermiques. Cette fois, la chaleur a provoqué une résistance constante (3-4 MQ) dans nos pipettes de patch ensemble de cellules. </li><li> Remplir l&#39;électrode avec l&#39;solution de la pipette contenant 2 mM de dopamine et le monter sur le manipulateur droit. Envelopper le tube contenant la solution contenant DA pipette avec une feuille d&#39;aluminium. Garder sur la glace. La dopamine est oxydable. Garder la solution sur de la glace, à l&#39;abri de la lumière diminue le taux d&#39;oxydation de la dopamine. </li><li> Retirez délicatement un ProCFE (Dagan) électrode basse en fibre de carbone ampérométrique de bruit à partir de la boîte de remisage, remplir avec du mercure, montage sur l&#39;adaptateur ampérométrique (comme le montre la figure 1), puis monter sur le maniupulator droite. Protéger la pointe de la fibre de carbone contre les dommages en maintenant l&#39;extrémité de la fibre de carbone. Vérifiez l&#39;électrode avec un microscope de laboratoire pour garantir la pointe est propre et intacte. </li><li> Examiner l&#39;intégrité de l&#39;électrode ampérométrique en mettant l&#39;électrode dans une boîte de Petri en verre contenant une solution de fond externe. Enregistrer un courant de référence en l&#39;absence de la dopamine. Ajouter 10 ul d&#39;une solution 1 mM DA au plat. Une nouvelle électrode ampérométrique bonnecâblés d&#39;une augmentation du courant d&#39;oxydation. Répétez cette étape au début et à la fin de chaque expérience afin de s&#39;assurer de l&#39;électrode ampérométrique fonctionne correctement. </li></ol> 3. Préparer la culture primaire de neurones à dopamine des neurones ou des cellules modifiées génétiquement pour exprimer transporteur de la dopamine dans les plats de Pétri en verre de fond <ol><li> Lavez délicatement les cellules appelées neurones dopaminergiques trois fois avec une solution externe au chaud. </li><li> Monter le fond en verre de Pétri sur la platine du microscope. </li></ol> 4. Visualisez cellulaire et exécuter l&#39;expérience <ol><li> Trouver le bon point focal de visualiser clairement les cellules. Exercer une pression positive sur l&#39;électrode de patch. Ensuite, amener doucement vers le bas les deux électrodes dans la solution, et à proximité de la cellule. </li><li> Positionner l&#39;électrode ampérométrique à côté de la cellule (à gauche), et l&#39;électrode de patch sur la droite. </li><li> Réaliser un joint d&#39;étanchéité gigaohm sur la cellule de l&#39;électrode de patch. La rupture du joint avec aspirationatteindre configuration cellule entière. </li><li> Permettre 5-8 min pour la dialyse de la solution interne contenant la dopamine dans la cellule. </li><li> Utiliser désiré échelon de tension ou d&#39;un protocole de rampe. Acquérir simultanément les données provenant à la fois de la pipette de patch et l&#39;électrode ampérométrique pour mesurer les courants de cellules entières et inverser le transport de la dopamine par le transporteur de la dopamine tandis que le potentiel de la membrane est contrôlée par l&#39;intermédiaire de la pipette de patch. </li></ol>

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, 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=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, 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=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, 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=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. 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=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Simultanée voltage-clamp et ampérométrie présente les avantages suivants. Tous les types de cellules sont accessibles et peuvent être utilisés pour l&#39;enregistrement. L&#39;identification des cellules ou neurones où les enregistrements sont effectués est simple et direct. En particulier, si la cellule est marquée par fluorescence par addition d&#39;un marqueur fluorescent pour la protéine d&#39;intérêt à l&#39;expérimentateur peut facilement sélectionner la cellule cible ou des neurones. La configurati...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Équipement </td></tr><tr><td> Anti-vibration table w / cage de Faraday </td><td> Technique Manufacturing Corporation </td><td> 63-500 série </td><td> nous utilisons le modèle 63-543 </td></tr><tr><td> Microscope inversé Nikon TE-2000 </td><td> Nikon </td><td> interrompu </td><td> maintenant Eclipse Ti </td></tr><tr><td> Deux amplificateurs à faible bruit Axopatch 200B </td><td> Molecular Devices </td><td></td><td> 800-635-5577 </td></tr><tr><td> 1-203 CV headstage BU </td><td> Molecular Devices </td><td></td><td> 800-635-5578 </td></tr><tr><td> HL-1-U porte-pipette </td><td> Molecular Devices </td><td></td><td> 800-635-5579 </td></tr><tr><td> Digidata 1440A convertisseur A / N </td><td> Molecular Devices </td><td></td><td> 800-635-5580 </td></tr><tr><td> Deux manipulateurs Siskyou, gaucheet droitier </td><td> Siskiyou </td><td> MX6600R MX6600L </td><td> 877-313-6418 </td></tr><tr><td> Laser pipette extracteur </td><td> Sutter Instruments </td><td> P-2000 </td><td> 888-883-0128 </td></tr><tr><td> Faible bruit de fibres de carbone électrode ampérométrique </td><td> ProCFE </td><td></td><td> <a href="http://www.dagan.com" target="_blank">www.dagan.com</a> </td></tr><tr><td> Faible bruit de quartz pipette </td><td> Sutter Instruments </td><td> QF100-70-7.5 </td><td> 888-883-0128 </td></tr><tr><td> Batterie de voiture de 12 volts </td><td></td><td></td><td> largement disponibles </td></tr><tr><td> Chargeur de batterie de voiture </td><td></td><td></td><td> largement disponibles </td></tr><tr><td></td><td></td><td></td><td> Réactif </td></tr><tr><td> Chlorure de sodium (NaCl) </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td><td></td></tr><tr><td> Dextrose </td><td> Sigma </td><td> G7528 </td><td></td></tr><tr><td> Le sulfate de magnésium (MgSO 4) </td><td> Sigma </td><td> M2643 </td><td></td></tr><tr><td> Phosphate de potassium monobasique (KH 2 PO 4) </td><td> Sigma </td><td> P5655 </td><td></td></tr><tr><td> Le chlorure de potassium (KCl) </td><td> Sigma </td><td> P9333 </td><td></td></tr><tr><td> Dihydrate de chlorure de calcium (CaCl 2 • 2H 2 0) </td><td> Sigma </td><td> 223506 </td><td></td></tr><tr><td> Hexahydrate de chlorure de magnésium (MgCl 2 • 6H 2 0) </td><td> Sigma </td><td> M2670 </td><td></td></tr><tr><td> EGTA </td><td> Sigma </td><td> E0396 </td><td></td></tr></tbody></table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

Après sa libération dans la fente synaptique, la dopamine exerce ses propriétés biologiques par l&#39;intermédiaire de ses objectifs pré-et post-synaptique 1. Le signal de la dopamine se termine par la diffusion de 2-3, enzymes extracellulaires 4 et transporteurs membranaires 5. Le transporteur de la dopamine, située dans la fente péri-synaptique des neurones dopaminergiques efface les amines libérées par un flux entrant de dopamine (absorption). Le transporteur de la dopamine peut aussi fonctionner dans le sens inverse pour libérer des amines de l&#39;intérieur vers l&#39;extérieur dans un processus appelé transport vers l&#39;extérieur ou l&#39;efflux de dopamine 5. . Plus de 20 ans, Sulzer et al ont rapporté le transporteur de la dopamine peut fonctionner en deux modes d&#39;activité: avant (absorption) et inverse (efflux) 5. Le neurotransmetteur libéré par efflux par le transporteur peut transporter une grande quantité de dopamine dans l&#39;espace extracellulaire, et il a été montré à jouer un rôle majeur dans la régulation extracellulaire de dopamine homeostasis 6. Nous décrivons ici comment patch-clamp et l&#39;enregistrement simultanés ampérométrie peut être utilisé pour mesurer la dopamine libérée par l&#39;intermédiaire du mécanisme d&#39;efflux avec une résolution temporelle millisecondes lorsque le potentiel de la membrane est contrôlée. Pour cela, l&#39;ensemble de la cellule courante et à l&#39;oxydation (ampérométrique) signaux sont mesurés simultanément en utilisant un amplificateur Axopatch 200B (Molecular Devices, avec un ensemble de Bessel passe-bas du filtre à 1.000 Hz pour l&#39;ensemble des cellules d&#39;enregistrement en cours). Pour l&#39;enregistrement d&#39;une électrode ampérométrique fibre de carbone est reliée à un second amplificateur (200B Axopatch) et est placé à côté de la membrane plasmique et maintenu à +700 mV. Les cellules entières et à l&#39;oxydation (ampérométrique) courants peuvent être enregistrées et la relation courant-tension peut être généré à l&#39;aide d&#39;un protocole échelon de tension. Contrairement à l&#39;étalonnage d&#39;habitude ampérométrique, qui exige la conversion de la concentration, le courant est directement signalé sans tenir compte de la 7 fois le volume efficace. Ainsi, les données qui en résultentreprésentent une limite inférieure à efflux de dopamine parce que certains émetteur est perdu à la solution en vrac.

Patch-clamp combinée avec ampérométrie peut mesurer la tension en fonction de DAT DA induite par efflux. Figure 2A montre une configuration représentative expérimentale et l&#39;enregistrement des DAT DA induite par efflux lorsque le milieu intracellulaire et le potentiel de membrane sont serrées par une pipette de patch de cellule entière. En utilisant cette technique, les cellules exprimant les protéines YFP-DAT sont serrées en tension avec une pipette de patch pendant toute cellule d&#39;une...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

La technique de mesure ampérométrique la libération de dopamine à partir d&#39;une seule cellule en détectant le courant oxydatif produit par oxydation spontanée dopamine. Pince simultanée de la tension et de la méthodologie ampérométrie révéler la relation mécanique entre l&#39;ensemble «activité» du transporteur de la dopamine et le rôle régulateur de l&#39;activité sur le transport inverse de la dopamine.

Mesure Single Cell de la libération de dopamine avec Voltage-clamp simultanée et Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

single-cell-measurement-dopamine-release-with-simultaneous-voltage

Research

JoVE Journal

Neuroscience

13.7K vues.  University of Florida. La technique de mesure ampérométrique la libération de dopamine à partir d'une seule cellule en détectant le courant oxydatif produit par oxydation spontanée dopamine. Pince simultanée de la tension et de la méthodologie ampérométrie révéler la relation mécanique entre l'ensemble «activité» du transporteur de la dopamine et le rôle régulateur de l'activité sur le transport inverse de la dopamine.

Vidéo: Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

Cartographie fonctionnelle avec MEG et EEG simultanée

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Recherche

Neurosciences

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

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 amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Dynamique présynaptique de la dopamine dans le striatum avec des tranches de cerveau de voltampérométrie cyclique à balayage rapide

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 ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Une seule cellule de profilage de développer et d&#39;âge mûr neurones de la rétine

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

D&#39;enregistrement optique de l&#39;activité neuronale supraliminaire avec une résolution à cellule unique et un seul pic

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Électroencéphalographie simultanée, en temps réel Mesure de la concentration de lactate et manipulation optogenetic de l&#39;activité neuronale dans le cortex cérébral des rongeurs

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La stimulation cérébrale profonde avec simultanée IRMf chez les rongeurs

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Préparation de neurones co-cultures avec l&#39;unité portable de précision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

Détection simultanée de c-Fos Activation de Sites de récompense dopaminergique mésolimbique et mésocortical Après Naive sucre et de graisse Ingestion chez le rat

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Méthode électrophysiologique pour les enregistrements de la pince à tension intégrale à partir de<em&gt; Drosophila</em&gt; Photorécepteurs

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus Préparation pour l’examen de la Transmission neuromusculaire mammifères dans des Conditions de tension de serrage

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...