Nous tenons à remercier les Drs. Jun Zhu Ping et Susumu Koyama pour leur aide lors de la configuration initiale de la technique, et le Dr Veronica Alvarez de l&#39;aide pour le formatage du manuscrit. Cette étude a été financée par la Division de la recherche clinique et biomédicale intra-muros des NIAAA.

<ol>
	<li>Zhu, P. J., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+potentiates+gabaergic+synaptic+transmission+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+basolateral+amygdala.">Ethanol potentiates gabaergic synaptic transmission in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.</a> J Neurophysiol. 96, 433-441 (2006).</li><li>Zhu, P. J., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+endocannabinoid+signaling+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+basolateral+amygdala.">Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.</a> J Neurosci. 25, 6199-6207 (2005).</li><li>Akaike, N., Moorhouse, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques:+Applications+of+the+nerve-bouton+preparation+in+neuropharmacology.">Techniques: Applications of the nerve-bouton preparation in neuropharmacology.</a> Trends Pharmacol Sci. 24, 44-47 (2003).</li><li>Akaike, N., Murakami, N., Katsurabayashi, S., Jin, Y. H., Imazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+stimulation+of+single+gabaergic+presynaptic+boutons+on+the+rat+hippocampal+neuron.">Focal stimulation of single gabaergic presynaptic boutons on the rat hippocampal neuron.</a> Neurosci Res. 42, 187-195 (2002).</li><li>Ye, J. H., Wang, F., Krnjevic, K., Wang, W., Xiong, Z. G., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+glycine+receptors+on+gabaergic+terminals+facilitate+discharge+of+dopaminergic+neurons+in+ventral+tegmental+area.">Presynaptic glycine receptors on gabaergic terminals facilitate discharge of dopaminergic neurons in ventral tegmental area.</a> J Neurosci. 24, 8961-8974 (2004).</li><li>Koyama, S., Matsumoto, N., Kubo, C., Akaike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+5-ht3+receptor-mediated+modulation+of+synaptic+gaba+release+in+the+mechanically+dissociated+rat+amygdala+neurons.">Presynaptic 5-ht3 receptor-mediated modulation of synaptic gaba release in the mechanically dissociated rat amygdala neurons.</a> J Physiol. 529, 373-383 (2000).</li><li>Deng, C., Li, K. Y., Zhou, C., Ye, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+enhances+glutamate+transmission+by+retrograde+dopamine+signaling+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+the+ventral+tegmental+area.">Ethanol enhances glutamate transmission by retrograde dopamine signaling in a postsynaptic neuron/synaptic bouton preparation from the ventral tegmental area.</a> Neuropsychopharmacology. 34, 1233-1244 (2009).</li><li>Murakami, N., Ishibashi, H., Katsurabayashi, S., Akaike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+subtypes+on+single+gabaergic+presynaptic+terminal+projecting+to+rat+hippocampal+neurons.">Calcium channel subtypes on single gabaergic presynaptic terminal projecting to rat hippocampal neurons.</a> Brain Res. 951, 121-129 (2002).</li><li>Zhu, P. J., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+synaptic+activity+produces+long-lasting+enhancement+of+endocannabinoid+modulation+and+alters+long-term+synaptic+plasticity.">Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity.</a> J Neurophysiol. 97, 4386-4389 (2007).</li><li>Inada, H., Maejima, T., Nakahata, Y., Yamaguchi, J., Nabekura, J., Ishibashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocannabinoids+contribute+to+metabotropic+glutamate+receptor-mediated+inhibition+of+gaba+release+onto+hippocampal+ca3+pyramidal+neurons+in+an+isolated+neuron/bouton+preparation.">Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of gaba release onto hippocampal ca3 pyramidal neurons in an isolated neuron/bouton preparation.</a> Neuroscience. 165, 1377-1389 (2010).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, 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=Visualizing+secretion+and+synaptic+transmission+with+ph-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with ph-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Li, Z., Burrone, J., Tyler, W. J., Hartman, K. N., Albeanu, D. F., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+recycling+studied+in+transgenic+mice+expressing+synaptophluorin.">Synaptic vesicle recycling studied in transgenic mice expressing synaptophluorin.</a> Proc Natl Acad Sci U S A. 102, 6131-6136 (2005).</li></ol>

Vibrodissociation réussie exige que les tranches contiennent neurones sains et que les espaces interstitiels sont suffisamment souples pour permettre aux neurones pour sortir de la tranche sans dégâts toxiques. Ainsi, la technique fonctionne de manière optimale au début âges postnataux (P1-21) lors des tranches saines peuvent être faites avec moins de matières gliales / interstiatial que se trouve dans des tranches de cerveau adulte. Dans notre expérience, cependant, l&#39;optimisation de la préparation pour la tranche de la survie neuronale dans la tranche elle-même peut être contre-productif pour la technique vibrodissociation. Alors que nous avons l&#39;habitude de préparer des tranches pour l&#39;enregistrement in situ en utilisant froide aCSF modifié dans lequel le saccharose a été remplacé par une grande partie de la matrice extracellulaire de Na + et Ca 2 +, les tranches préparées pour vibrodissociation peut être préparé (coupure par exemple) dans notre aCSF enregistrement normal. Lors de la préparation des tranches pour l&#39;enregistrement à partir de neurones individuels dans les tranches elles-mêmes, nous aussi placer les tranches de aCSF normale à 35 ° C, juste après la coupe et les laisser pendant 30-60 min à cette température avant de les retourner à la température ambiante. Toutefois, les tranches sont préparées pour vibrodissociation déplacé immédiatement à la température ambiante, juste après le tranchage. Ces procédures fournissent un rendement plus élevé de neurones sains après vibrodissociation, peut-être à cause du manque de tissu interstitiel qui permet cabinet neurones pour être plus facilement détaché de son support de la tranche de lui-même. Nous n&#39;avons pas examiné de manière exhaustive les conditions de préparation de tranches, comme nous avons l&#39;habitude d&#39;obtenir neurones suffisant pour notre enregistrement et des expériences d&#39;imagerie sur un jour donné. Il est possible que des modifications supplémentaires, comme des changements dans l&#39;épaisseur de coupe ou de procédures preincubaton, pourraient être apportées pour accroître le rendement des neurones sains. Très doux traitements protéase ont été essayées dans un effort pour augmenter le rendement des cellules et maîtriser la technique de travailler dans des animaux plus âgés. Toutefois, invariablement, même le traitement de la protéase douce semble pas perturber le fonctionnement des synapses. Ainsi, alors que la combinaison de la protéase et la dissociation mécanique peut encore être trouvée pour le travail qu&#39;il n&#39;a pas fait ses preuves dans les neurones du cerveau antérieur à ce jour. Il convient également de noter le rendement relativement faible des neurones sains après vibrodissociation signifie que la technique est principalement utile pour étudier les sous-types neuronaux abondante, principalement les neurones de projection. La disponibilité des souris exprimant la GFP dans lequel de petites sous-populations neuronales peuvent être facilement identifiés augmente la chance d&#39;étudier ces neurones plus rares. Cependant, à moins de rendement neuronale peut être sensiblement améliorée accumulation de données sur ces neurones est susceptible d&#39;être relativement lente. Plusieurs étapes se sont avérées cruciales pour le chargement réussi de neurones avec du calcium-sensing colorants. L&#39;exposition aux PM-estérifiés teinture est réalisée à 37 ° C, et nous n&#39;avons pas réussi à essayer de colorant de charge à basse température. La concentration des colorants devraient être optimisées considérant à la fois le temps de chargement et de la température car il semble que plus la concentration de colorant de chargement diminue la concentration de calcium dans les boutons présynaptiques. Nous avons observé que des concentrations plus élevées de chargement avec une diminution de la fréquence et l&#39;amplitude de sIPSCs. Lors du chargement de calcium-sensing colorants dans les terminaux présynaptiques des neurones vibrodissociated, il faut prendre soin, car la capacité tampon de l&#39;boutons petits présynaptique peut être différente de celle dans le soma et la taille des boutons ne sont pas compatibles. Neuron contenant les plats sont lavées avec tamponné HEPES solution externe suivants colorant de chargement, et le temps de récupération après le lavage est essentiel. En outre, pour faire une bonne étanchéité des cellules entières d&#39;enregistrement, les cellules doivent être soigneusement lavés à se débarrasser de non internalisés molécules de colorant de la membrane cellulaire extérieure. En plus d&#39;utiliser des neurones vibrodissociated d&#39;imagerie cellulaire électrophysiologie et en direct, des techniques immunocytochimiques peuvent également être appliquées à ces cellules 11,12. Les cellules peuvent être facilement fixés et colorés avec une variété d&#39;anticorps, et autres marqueurs cellulaires. L&#39;utilisation de ces cellules permet une préparation propre pour la visualisation de l&#39;expression des protéines dans les terminaisons présynaptiques GABAergique. En utilisant des souris qui expriment des protéines fluorescentes sous le contrôle de promoteurs spécifiques aux neurones GABAergiques permet à l&#39;enquêteur d&#39;identifier individuellement terminaisons synaptiques dans la préparation de la vibrodissociated (figure 1). Marqueurs fluorescents imagerie de ce type peut permettre des mesures morphologiques dans les terminaux utilisant la microscopie à base de laser et de nouvelles techniques comme la STED (Microscopie épuisement stimulation d&#39;émission). Visualisation avec des colorants que le rapport de vélo vésicules synaptiques est également possible avec cette préparation. Akaike et ses collègues ont qualifié terminaux GABAergiques avec le colorant styryle, FM1-43 pour visualiser les terminaux dans les cellules vivantes 4. Il devrait également être possible d&#39;effectuer une seule cellule de la transcriptase inverse PCR pour l&#39;ARN le profil d&#39;expression dans vibrodissociatedneurones. Cette technique est couramment appliquée à enzymatiquement dissocié les neurones et les neurones en culture cellulaire.

<table border="1"><tbody><tr><td> Item </td><td> Société </td><td> Catalogue # </td><td> Commentaires </td></tr><tr><td> Vibrant Slicer tissus </td><td> Leica Microsystems Inc </td><td> VT1200S </td><td></td></tr><tr><td> Boîte de culture cellulaire (35 mm) </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> Plats à fond de verre </td><td> Willco-vaisselle </td><td> GWSB-5040-3522 Ou GWSB </td><td> 0.16 à 0.19 mm d&#39;épaisseur de verre pour l&#39;imagerie </td></tr><tr><td> Manipulateur piézoélectriques </td><td> Exfo-Burleigh </td><td> LSS-3000 </td><td> Pourrait aussi utiliser le relais, etc </td></tr><tr><td> SD9 Place Pulse stimulateur </td><td> Technologies Herbe </td><td> SD9K </td><td> Pour le déclenchement manipulateur piézoélectriques </td></tr><tr><td> Dissection Stéréoscope </td><td> Wild Heerbrugg </td><td> TYP 374 590 </td><td> Pourrait aussi utiliser n&#39;importe quel stéréoscope </td></tr><tr><td> Flaming / Brown micropipette extracteur </td><td> Instrument Sutter </td><td> P-97 </td><td></td></tr><tr><td> Paroi mince capillaires en verre </td><td> Instruments de précision du monde </td><td> TW-150F-4 </td><td> Pour le verre scellées à la flamme micropipette et patch micropipette </td></tr><tr><td> Six systèmes de perfusion Canal Vanne de régulation </td><td> Warner Instruments </td><td> VC-6 ou VC-6M </td><td></td></tr><tr><td> Perfusion rapide Étape </td><td> Warner Instruments </td><td> SF-77B </td><td></td></tr><tr><td> Microscope inversé avec 20-63x objectifs </td><td> Nikon </td><td> TS200 Diaphot </td><td></td></tr><tr><td> Caméra EMCCD </td><td> ANDOR technologie </td><td> ixon EM + UA-888 </td><td></td></tr><tr><td> Excite Source de lumière à éclairage par fluorescence </td><td> EXFO Solutions Photonics Inc </td><td> X-Cite 120PC </td><td></td></tr><tr><td> Fluo-4, AM indicateur de calcium </td><td> Molecular Probes </td><td> F14201 </td><td></td></tr></tbody></table>

vibrodissociation of neurons from rodent brain slices to study synaptic transmission and image presynaptic terminals

Dissociation mécanique des neurones du système nerveux central a l&#39;avantage de boutons présynaptiques restent attachés à la neurone isolé de l&#39;intérêt. Cela permet à l&#39;examen de la transmission synaptique dans des conditions où les environnements extracellulaires et intracellulaires postsynaptique peut être bien contrôlé. Une technique basée sur les vibrations sans l&#39;utilisation de protéases, connu sous le nom vibrodissociation, est la technique la plus populaire pour l&#39;isolation mécanique. D&#39;une micropipette, avec la pointe polie au feu à la forme d&#39;une petite balle, est placé dans une tranche de cerveau fait à partir d&#39;un P1-P21 rongeurs. La micropipette est vibré parallèle à la surface tranche et abaissé grâce à l&#39;épaisseur de coupe résultant de la libération de neurones isolés. Les neurones isolés sont prêts pour des études au sein de quelques minutes de vibrodissociation. Cette technique a des avantages sur l&#39;utilisation de cultures primaires de neurones, des tranches de cerveau et les neurones isolés enzymatiquement, notamment: la production rapide de viabilité, les neurones relativement matures appropriés pour des études électrophysiologiques et d&#39;imagerie, le contrôle supérieur de l&#39;environnement extracellulaire libre de l&#39;influence des cellules voisines; adéquation pour le bien-contrôlé expériences pharmacologiques en utilisant l&#39;application rapide du médicament et de surfusion total de cellules, et l&#39;amélioration espace-clamp dans la cellule entière par rapport à des enregistrements de neurones dans des préparations ou des tranches de culture cellulaire. Cette préparation peut être utilisée pour examiner synaptique physiologie, la pharmacologie, la modulation et la plasticité. Imagerie en temps réel de ces deux éléments pré-et post-synaptiques dans les cellules vivantes et boutons est également possible en utilisant les neurones vibrodissociated. Caractérisation des constituants moléculaires des éléments pré-et post-synaptiques peuvent également être obtenus avec des approches immunologiques et fondés sur l&#39;imagerie.

Watch this Scientific Journal Video about Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals at JoVE.com

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

Ce rapport démontre une technique d&#39;isolation mécanique de neurones individuels viables conservant attachés boutons présynaptiques. Vibrodissociated neurones ont l&#39;avantage d&#39;une production rapide, un excellent contrôle pharmacologique et amélioré l&#39;espace-pince sans influence des cellules voisines. Cette méthode peut être utilisée pour l&#39;imagerie des éléments synaptiques et patch-clamp.

Vibrodissociation des neurones à partir de tranches de cerveau de rongeurs pour étudier la transmission synaptique et de l&#39;image terminaisons présynaptiques

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

vibrodissociation-neurons-from-rodent-brain-slices-to-study-synaptic

Research

JoVE Journal

Neuroscience

15.4K vues.  National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism. Ce rapport démontre une technique d'isolation mécanique de neurones individuels viables conservant attachés boutons présynaptiques. Vibrodissociated neurones ont l'avantage d'une production rapide, un excellent contrôle pharmacologique et amélioré l'espace-pince sans influence des cellules voisines. Cette méthode peut être utilisée pour l'imagerie des éléments synaptiques et patch-clamp.

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Préparation des oligomères β-amyloïde 1-42 Et induction de la plasticité synaptique Dépréciation sur les coupes d&#39;hippocampe

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Recherche

Neurosciences

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

Étiquetage DiOLISTIC des neurones à partir de rongeurs et non-humains tranches de cerveau des primates

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Enregistrement colorant sensible à la tension d&#39;axones, des dendrites et des épines dendritiques des neurones individuels dans des tranches de cerveau

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Aiguë de dissociation de la lamproie réticulo axones pour permettre l&#39;enregistrement de la sortie de visage membrane des terminaux présynaptiques fonctionnels individuels

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolement, Culture et maintenance à long terme des mésencéphalique primaire dopaminergiques neurones à partir de cerveaux embryonnaires de rongeurs

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analyse Synaptic Modulation de<em&gt; Drosophila melanogaster</em&gt; Photorécepteurs après exposition à la lumière prolongée

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Application de la pincée de patch automatisée guidée par image pour l&#39;étude des neurones dans les tranches de cerveau

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Évaluation de la densité de la Synapse dans des tranches de cerveau de rongeurs hippocampe

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Alliant Optogenetics artificiel microARN pour caractériser les effets du gène Knockdown fonction présynaptique dans les Circuits de neurones intacts

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...