Diese Arbeit wurde von MH084494 (CJL) und MH079276 und NS060658 (GAP) unterstützt.

1. Slice-Vorbereitung <ol><li> Cut Hirnschnitten mit Hilfe eines vibrierenden Mikrotom. Wir bereiteten 240 um horizontale Mittelhirn Scheiben aus einer Isofluran-anethetized Sprague-Dawley Ratten (Charles River Laboratories) mit einem vibrierenden Mikrotom (Microm HM 650V) im Einklang mit der University of Texas at San Antonio Institutional Animal Care und Verwenden Committee. </li><li> Halten Sie die Scheiben in eine Inkubationskammer, bis Sie zur Aufnahme bereit sind. Wir verwenden eine Inkubationszeit Behälter erwärmt auf 32 ° C und mit künstlicher Liquor (aCSF; in mM): 126 NaCl, 2,5 KCl, 1,25 NaH 2 PO 4, 4 MgCl 2, 2 CaCl 2, 10 Dextrose, 25 NaHCO 3, 1,3 Ascorbinsäure, 2,4 Natriumpyruvat und 0,05 Glutathion. </li></ol> 2. Elektrophysiologische Recording <ol><li> Übertragen Sie die Scheibe, um die intrazelluläre Aufnahme rig, in denen eine künstliche Liquor (aCSF) bei 35 ° C wird durchblutet ist. Wir benutzen die gleichen aCSF wie in 1.2, außer dass 2 mM MgCl 2 verwendet wurde und Glutathion wurde verzichtet. Für horizontal vorbereitet Scheiben, wir in der Regel halbieren die Scheibe entlang der Mittellinie. </li><li> Visualisieren Sie die Zielneuron. Wir visualisieren einzelnen Substantia nigra dopaminergen Neuronen mit einem Gradienten Gegensatz Imaging-System. </li><li> Ziehen Sie eine Elektrode mit einem elektronischen Elektrode Abzieher. Wir ziehen Elektroden mit einer Spitze Widerstand von 4-10 MOhm mit einer P97 Micropipette Puller (Sutter Instrument Company). </li><li> Füllen Sie eine Elektrode mit der gewünschten internen Lösung. Wir verwenden eine Lösung, die (in mM): 138 K-Gluconat, 10 HEPES, 2 MgCl 2, 0,2 EGTA, 0,0001 CaCl 2, 4 Na-ATP, 0,4 Na-GTP. Die interne Lösung wurde auf einen pH von 7,3 mit 1 M KOH und einer Osmolarität von 270-275 mOsms angepasst. </li><li> Machen Sie eine Gigaohm Dichtung auf die gewünschte Neuron. Rupture die Dichtung mit Absaugung. Dies stellt eine ganze Zelle Aufnahme. Ein Multiclamp 700B Verstärker wurde in unserer Konfiguration verwendet. Der Verstärker sollte dann in Stromzange die &quot;I = 0&quot;-Modus versetzt werden. </li></ol> 3. Leitwert Anwendung mit Dynamic Clamp <ol><li> RTXI (www.rtxi.org) wurde auf dem dynamischen Clamp-Computer ausgeführt. Eine benutzerdefinierte geschrieben Modell mit einem NMDA-Rezeptor wurde in den Speicher geladen. Der Strom in der Zelle in Echtzeit injiziert wird durch die folgende Gleichung berechnet: Ich NMDA =-g NMDA * [1 / (1 ​​+ ([Mg] / 3,57) * e (-V m * 0,062))] * (V m - E NMDA), wo g NMDA ist die gewünschte Leitfähigkeit (in nS; standardmäßig auf 0 gesetzt nS), [Mg] ist die Magnesium-Konzentration (auf 1,5 mm in unserem Beispiel unten), ist E NMDA die Umkehrung Potenzial für den NMDA-Rezeptor (auf 0 mV) und V m wird das Membranpotential der Zelle aus dem Verstärker gemessen (in Millivolt). </li><li> Der Ausgang aus der dynamischen Clamp-Computer wurde die Command-Eingang des Verstärkers über einen Analog-Digital-Wandler verbunden. </li><li> Der Verstärker wurde in Stromzange der &quot;IC&quot;-Modus versetzt. </li><li> Geben Sie die gewünschte NMDA-Rezeptor-Leitwert in RTXI (zB 40ns). Sie sollten eine phasische Burst von Aktionspotentialen. Alternativ kann eine Leitfähigkeit bis RTXI über einen analogen Ausgang (Abbildung 1A, &#39;g (t)&#39;) angegeben werden. Eine entsprechende Skalierungsfaktor muss innerhalb RTXI verwendet werden, um das Signal von Volt an Siemens zu konvertieren. </li></ol> 4. Repräsentative Ergebnisse Ein erfolgreiches Setup für die Anwendung einer Leitfähigkeit unter Verwendung der dynamischen Klammer ist in Abbildung 1A dargestellt. Mit diesem Setup haben wir eine ganze Zelle somatischen Aufnahme von einem dopaminergen Neuronen in der Substantia nigra pars compacta. Dopaminergen Zellen in der Regel spontan Feuer zu günstigen Preisen mit einem Herzschrittmacher-ähnliches Muster. Ein Burst von Aktionspotentialen kann durch phasische Anwendung eines NMDA-Rezeptor-Leitwert mit dynamischen Klemme (Abbildung 1B) hervorgerufen werden. <img src="/files/ftp_upload/2275/2275fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Anwendung eines NMDA-Rezeptor-Leitwert mit dem dynamischen Clamp-Technik. A. Hardware-Setup, welches die Verbindungen zwischen den intrazellulären Aufnahme rig und dynamische Clamp-Computer. B. Ein Burst von Aktionspotentialen wird durch Anwendung eines 40ns NMDA-Rezeptor-Leitwert in einer ganzen Zelle Aufnahme von einem Substantia nigra pars compacta dopaminergen Neuronen hervorgerufen werden.

<ol>
	<li>Robinson, H. P., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> J Neurosci Methods. 49, 157-165 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp:+artificial+conductances+in+biological+neurons.">The dynamic clamp: artificial conductances in biological neurons.</a> Trends Neurosci. 16, 389-394 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> J Neurophysiol. 69, 992-995 (1993).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends Neurosci. 27, 218-224 (2004).</li><li>Deister, C. A., Teagarden, M. A., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intrinsic+neuronal+oscillator+underlies+dopaminergic+neuron+bursting.">An intrinsic neuronal oscillator underlies dopaminergic neuron bursting.</a> J Neurosci. 29, 15888-15897 (2009).</li><li>Lobb, C. J., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+role+for+GABA+receptors+on+the+firing+pattern+of+midbrain+dopaminergic+neurons.">A dynamic role for GABA receptors on the firing pattern of midbrain dopaminergic neurons.</a> J Neurophysiol. 104, 403-413 (2010).</li></ol>

Die dynamische Clamp-Technik demonstriert hier eine Verbesserung gegenüber der traditionellen Technik der direkten Stromeinspeisung, indem der Experimentator die elektrischen Effekte der Aktivierung eines Rezeptors zu imitieren. In diesem Video haben wir gezeigt, dass man die Effekte hinzufügen Aktivierung von NMDA-Rezeptor, die spontane Aktivität der dopaminergen Neuronen, dh ein Platzen der Aktionspotentiale hervorgerufen werden. Durch die Flexibilität der Hardware / Software-Implementierung kann eine Vielzahl von Erweiterungen verwendet werden. Das Zeichen des injizierten Strom von negativ auf positiv geschaltet werden, was einem Szenario, in dem die Auswirkungen der aktivierten Rezeptor von einem Neuron entfernt wird. Modell Neuronen, in Form einer Reihe von Differentialgleichungen dargestellt werden, können auch numerisch gelöst werden und erlauben es dem Forscher, kleine Netzwerke zu untersuchen.

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application of a nmda receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique

Neurowissenschaftler untersuchen die Funktion des Gehirns durch die Untersuchung, wie Nervenzellen im Gehirn kommunizieren. Viele Forscher betrachten Veränderungen in der elektrischen Aktivität eines oder mehrerer Neuronen in Reaktion auf eine experimentell-Eingang auf. Die elektrische Aktivität der Neuronen kann in isolierten Hirnschnitten mittels Patch-Clamp-Techniken mit Glasmikropipetten aufgezeichnet werden. Traditionell können Experimentatoren neuronalen Input durch direkte Injektion der aktuellen imitieren durch die Pipette, die elektrische Stimulation des anderen Zellen oder verbleibende axonalen Verbindungen in der Scheibe, oder pharmakologische Manipulation durch Rezeptoren auf der neuronalen Membran der aufgenommenen Zelle. Gleichstrom Injektion hat den Vorteil, daß man eine vorgegebene Stromform mit hoher zeitlicher Präzision auf dem Gelände der Aufnahme (in der Regel die Soma). Allerdings ist es nicht ändern, der Widerstand der neuronalen Membran, da keine Ionenkanäle physisch geöffnet werden. Aktuelle Injektion in der Regel beschäftigt Rechteckimpulse und damit nicht-Modell die Kinetik der Ionenkanäle. Schließlich können Stromeinspeisung nicht imitieren die chemischen Veränderungen in der Zelle, die mit der Öffnung von Ionenkanälen auftritt. Rezeptoren können physikalisch durch elektrische oder pharmakologische Stimulation aktiviert werden. Der Versuchsleiter hat eine gute zeitliche Präzision der Rezeptoraktivierung mit elektrischer Stimulation der Scheibe. Allerdings gibt es nur begrenzte räumliche Präzision der Rezeptor-Aktivierung und die genaue Natur dessen, was nach Stimulation aktiviert ist unbekannt. Das letztere Problem kann teilweise durch spezifische pharmakologische Wirkstoffe gelindert werden. Leider ist der zeitliche Verlauf der Aktivierung von pharmakologischen Wirkstoffen in der Regel langsam und die räumliche Genauigkeit der Eingänge auf die aufgenommenen Zelle ist unbekannt. Die dynamische Clamp-Technik ermöglicht ein Experimentator auf die aktuelle Veränderung gingen direkt in die Zelle basierend auf Echtzeit-Feedback der Membran der Zelle (Robinson und Kawai 1993, Sharp et al, 1993a, b;. Für eine Übersicht siehe Prinz et al. 2004). Dies ermöglicht ein Experimentator auf der elektrischen Veränderungen, die auf dem Gelände der Aufnahme treten als Reaktion auf die Aktivierung des Rezeptors zu imitieren. Real-time Veränderungen in der angewandten Strom werden durch eine mathematische Gleichung in Hardware implementiert bestimmt. Wir haben vor kurzem die dynamische Clamp-Technik verwendet, um die Erzeugung von Bursts von Aktionspotentialen durch phasische Aktivierung von NMDA-Rezeptoren in dopaminergen Neuronen der Substantia nigra pars compacta (Deister et al, 2009.; Lobb et al, 2010.) Zu untersuchen. In diesem Video zeigen wir die Verfahren benötigt, um eine NMDA-Rezeptor-Leitwert in einer dopaminergen Neuronen gelten.

Watch this Scientific Journal Video about Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique at JoVE.com

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

In diesem Video zeigen wir, wie man einen Leitwert in einem dopaminergen Neuronen in der ganzen Zelle Konfiguration in Hirnschnitten der Ratte festgestellt werden. Diese Technik nennt man das dynamische Klemme.

Die Anwendung eines NMDA-Rezeptor-Leitwert in Rat Mittelhirn dopaminergen Neuronen Mit der Dynamic-Clamp-Technik

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

application-nmda-receptor-conductance-rat-midbrain-dopaminergic

Research

JoVE Journal

Neuroscience

12.0K Aufrufe.  University of Texas San Antonio - UTSA. In diesem Video zeigen wir, wie man einen Leitwert in einem dopaminergen Neuronen in der ganzen Zelle Konfiguration in Hirnschnitten der Ratte festgestellt werden. Diese Technik nennt man das dynamische Klemme.

Serie: Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Forschung

Neurowissenschaften

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorant-induzierten Reaktionen aus olfaktorischen Rezeptor-Neuronen mit der Saugpipette Technik aufgenommen

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Elektroporation in Mittelhirn-Küken für Studien über Genfunktion in dopaminergen Neuronen Entwicklung

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementieren Dynamische Clamp mit Synaptic und künstliche Leitfähigkeiten in Maus retinalen Ganglienzellen

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Primärkultur von Maus dopaminergen Neuronen

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Umwelt Modulationen der Anzahl der Mittelhirn Dopamin-Neuronen in erwachsenen Mäusen

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

Zuverlässige Identifikation von Wohn dopaminerge Neurone in Mittelhirns Kulturen unter Verwendung von RNA-Sequenzierung und TH-Promotor getriebene eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

Die gleichzeitige Auswertung der zerebralen Hämodynamik und Lichtstreuungseigenschaften der<em&gt; In Vivo</em&gt; Rattenhirn Mit Multispektral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

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.

Anwendung von automatisierten Bild-geführten Patch-Clamp für die Studie von Neuronen in Gehirn-Scheiben

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 ...