This work was supported by MH084494 (CJL), and MH079276 and NS060658 (CAP).

1. Slice Preparation
<ol>
	<li>Cut brain slices using a vibrating microtome. We prepared 240 &mu;m horizontal midbrain slices from an isoflurane-anethetized Sprague-Dawley rat (Charles River Laboratories) using a vibrating microtome (Microm HM 650V) in accordance with the University of Texas at San Antonio Institutional Animal Care and Use Committee.</li>
	<li>Keep the slices in an incubation chamber until you are ready to record. We use an incubation container heated to 32&deg;C and filled with artificial cerebrospinal fluid (aCSF; in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 MgCl2, 2 CaCl2, 10 dextrose, 25 NaHCO3, 1.3 ascorbic acid, 2.4 sodium pyruvate and 0.05 glutathione.</li>
</ol>
2. Electrophysiological Recording
<ol>
	<li>Transfer the slice to the intracellular recording rig in which an artificial cerebrospinal fluid (aCSF) at 35&deg;C is being perfused. We use the same aCSF as in 1.2 except that 2mM MgCl2 was used and glutathione was omitted. For horizontally prepared slices, we typically bisect the slice along the midline.</li>
	<li>Visualize the target neuron. We visualized individual substantia nigra dopaminergic neurons with a gradient contrast imaging system.</li>
	<li>Pull an electrode using an electronic electrode puller. We pull electrodes with a tip resistance of 4-10 M&Omega; using a P97 Micropipette puller (Sutter Instrument Company).</li>
	<li>Fill an electrode with the desired internal solution. We use a solution containing (in mM): 138 K-gluconate, 10 HEPES, 2 MgCl2, 0.2 EGTA, 0.0001 CaCl2, 4 Na-ATP, 0.4 Na-GTP. The internal solution was adjusted to a pH of 7.3 using 1M KOH and an osmolarity of 270-275 mOsms.</li>
	<li>Make a gigaohm seal onto the desired neuron. Rupture the seal with suction. This constitutes a whole cell recording. A Multiclamp 700B amplifier was used in our configuration. The amplifier should then be placed in the 'I=0' current clamp mode.</li>
</ol>
3. Conductance Application with Dynamic Clamp
<ol>
	<li>RTXI (www.rtxi.org) was executed on the dynamic clamp computer. A custom written model containing an NMDA receptor was loaded into memory. The current to be injected into the cell in real-time is calculated by the following equation: 
INMDA = -gNMDA * [1/(1+([Mg]/3.57) * e(-Vm*0.062))] * (Vm - ENMDA)
where gNMDA is the desired conductance (in nS; by default set to 0 nS), [Mg] is the magnesium concentration (set to 1.5 mM in our example below), ENMDA is the reversal potential for the NMDA receptor (set to 0 mV), and Vm is the membrane potential of the cell measured from the amplifier (in millivolts).</li>
	<li>The output from the dynamic clamp computer was connected to the Command input of the amplifier via an analog-to-digital converter.</li>
	<li>The amplifier was placed in the 'IC' current clamp mode.</li>
	<li>Enter your desired NMDA receptor conductance into RTXI (e.g. 40nS). You should see a phasic burst of action potentials. Alternatively, a conductance can be given to RTXI via an analog output (Figure 1A, 'g(t)'). An appropriate scaling factor must be used within RTXI to convert the signal from volts to Siemens.</li>
</ol>
4. Representative Results
A successful setup for application of a conductance using the dynamic clamp is shown in Figure 1A. Using this setup, we made a whole cell somatic recording from a dopaminergic neuron in the substantia nigra pars compacta. Dopaminergic cells typically fire spontaneously at low rates with a pacemaker-like pattern. A burst of action potentials can be evoked by phasic application of a NMDA receptor conductance with dynamic clamp (Figure 1B).
<img src="/files/ftp_upload/2275/2275fig1.jpg" alt="Figure 1" /> Figure 1: Application of a NMDA receptor conductance using the dynamic clamp technique. A. Hardware setup illustrating the connections between the intracellular recording rig and dynamic clamp computer. B. A burst of action potentials is evoked by application of a 40nS NMDA receptor conductance in a whole cell recording from a substantia nigra pars compacta dopaminergic neuron.

<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>

No conflicts of interest declared.

The dynamic clamp technique demonstrated here improves upon the traditional technique of direct current injection by allowing the experimenter to mimic the electrical effects of the activation of a receptor. In this video, we have shown that one can add the effects of activation of an NMDA receptor to the spontaneous activity of the dopaminergic neuron, i.e. a burst of action potentials are evoked.
Due to the flexibility of the hardware/software implementation, a variety of extensions can be used. The sign of the injected current can be switched from negative to positive, representing a scenario where the effects of activated receptor is removed from a neuron. Model neurons, represented in the form a series of differential equations, can also be numerically solved and allow the experimenter to investigate small networks.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>K-gluconate anhydrous</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 X 2H2O</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis(B-aminoethyl ether)-N,N,N&#x2019;,N&#x2019;-tetraacetic acid</td>
<td>Reagent</td>
<td>EGTA; Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgATP</td>
<td>Reagent</td>
<td>MP Biomedicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaGTP</td>
<td>Reagent</td>
<td>MP Biomedicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaH2PO4, Anhydrous</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CholCl</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ascorbic Acid</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX51WI Microscope (with 40x objective)</td>
<td>Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2 A/D converters</td>
<td>Equipment</td>
<td>e.g. Heka Instruments Inc. ITC-18, National Instruments BNC-2090A</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Multiclamp 700B with CV-7B headstage</td>
<td>Equipment</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>Equipment</td>
<td>Sutter Instrument Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microfil syringe needles</td>
<td>Equipment</td>
<td>World Precision Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>Equipment</td>
<td>Siskiyou, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monitor</td>
<td>Equipment</td>
<td>Triview</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

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 this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

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 vues.  University of Texas San Antonio - UTSA. Dans cette vidéo, nous démontrons comment appliquer une conductance dans un neurone dopaminergique enregistrés dans la configuration cellule entière dans les tranches de cerveau de rat. Cette technique est appelée le collier dynamique.

Vidéo: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ...