This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences (Protocol number #3593) and were in agreement with the National Institutes of Health guidelines for the care and use of laboratory animals.
1. Preparation of Standard-artificial Cerebrospinal Fluid (aCSF)
<ol>
	<li>Preparation of Stock Solution A
	<ol>
		<li>Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.</li>
		<li>While cont...

<ol>
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The authors declare that they have no competing financial interests.

PPN neurons have intrinsic properties that allow them to fire action potentials at beta/gamma band frequencies during in vivo recordings from animals that are awake or during REM sleep, but not during slow wave sleep2,3,5,13-17. Other authors have showed that brainstem transections at more anterior levels than PPN reduced gamma frequencies during EEG recordings. However, when brainstem lesions posterior to where this nucleus is located, the direct stimulation of PPN allowed the manifestation of cortic...

PPN nucleus is anatomically included in the caudal mesencephalic tegmentum. The PPN is a key component of RAS1. The PPN participates in the maintenance of behavioral activated states (i.e., waking, REM sleep)2. Electrical stimulation of the PPN in vivo induced fast oscillation (20 - 40 Hz) in the cortical EEG3, while bilateral PPN lesions in the rat reduced or eliminated REM sleep4. While a majority of PPN neurons fire action potentials at beta/gamma-band frequency (20 - 80 Hz), some neurons presented low rates of spontaneous firing (&#60; 10 Hz) 5. Furthermore, the PPN seems to be involved in other aspects of behavior such as motivation and attention6. Direct high frequency (40 - 60 Hz)7 electrical stimulation of PPN nucleus in decerebrate animals can promote locomotion. In recent years, deep brain stimulation (DBS) of PPN has been used to treat patients suffering from disorders involving gait deficits such as Parkinson&#39;s disease (PD)8.
Previous reports demonstrated that almost all PPN neurons can fire action potentials at gamma band frequency when depolarized using square current pulses9. Because of the drastic activation of voltage-gated potassium channels during square pulses depolarizations up to or under -25 mV. As a consequence, no robust gamma oscillations were observed after blocking action potentials generation using tetrodotoxin10. In an effort to bypass such a problem, 1 - 2 sec long depolarizing current ramps were used. Ramps gradually depolarized the membrane potential from resting values up to 0 mV, while partially inactivating voltage-gated potassium channels. Clear gamma band membrane oscillations were evident within the voltage dependence window of high threshold calcium channels (i.e., between -25 mV and -0 mV) 10. In conclusion, gamma band activity was observed in PPN neurons9, and both P/Q- and N-type voltage-gated calcium channels need to be activated in order to generate gamma band oscillations in the PPN10.
A series of studies determined the location of high threshold calcium channels in PPN neurons. Injecting the combination of dyes, ratiometric fluorescence imaging showed calcium transients through voltage-gated calcium channels that are activated in different dendrites when depolarized using current ramps11.
Intrinsic properties of PPN neurons have been suggested to allow simultaneous activation of these cells during waking and REM sleep, thus inducing high-frequency oscillatory neuronal activity between the RAS and thalamocortical loops. Such long-reaching interaction is considered to support a brain state capable of reliably assessing the world around us on a continuous basis12. Here, we describe the experimental conditions necessary to generate and maintain gamma band oscillation in PPN cells in vitro. This protocol has not been described previously, and would help a number of groups to study intrinsic membrane properties mediating gamma band activity at other brain areas. Moreover, current steps might lead to the erroneous conclusion that gamma band activity cannot be generated in these cells.

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	<tbody>
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			<td height="40" style="height:40px;width:619px;">Sucrose</td>
			<td style="width:335px;">Sigma-Aldrich</td>
			<td style="width:307px;">S8501</td>
			<td style="width:405px;">C12H22O11, molecular weight = 342.30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td>NaHCO3, molecular weight = 84.01</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td>KCl, molecular weight = 74.55</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnesium Chloride Hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>MgCl2 &#183; 6H2O, molecular weight =&#160; 203.30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>CaCl2 &#183; 2H2O, molecular weight =147.02</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>C6H12O6, molecular weight = 180.16</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td>C6H8O6, molecular weight =176.12</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride</td>
			<td>Acros Organics</td>
			<td>327300025</td>
			<td>NaCl, molecular weight =&#160; 58.44</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G4500</td>
			<td>C6H11KO7, molecular weight =&#160; 234.25</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Phosphocreatine di(tris) salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1937</td>
			<td>C4H10N3O5P &#183; 2C4H11NO3, molecular weight =&#160; 453.38</td>
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		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>C8H18N2O4S, molecular weight = 238.30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E0396</td>
			<td>[-CH2OCH2CH2N(CH2CO2H)2]2, molecular weight = 380.40</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Adenosine 5&#39;-triphosphate magnesium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>&#160;C10H16N5O13P3 &#183; xMg2+, molecular weight = 507.18</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Guanosine 5&#39;-triphosphate sodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td>C10H16N5O14P3 &#183; xNa+, molecular weight = 523.18</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Tetrodotoxin citrate</td>
			<td>Alomone Labs</td>
			<td>T-550</td>
			<td>C11H17N3O8, molecular weight = 319.27</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;DL-2-Amino-5-Phosphonovaleric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5282</td>
			<td>&#160;C5H12NO5P, molecular weight = 197.13</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CNQX disodium salt hydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>C9H2N4Na2O4 &#183; xH2O, molecular weight = 276.12</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Strychnine</td>
			<td>Sigma-Aldrich</td>
			<td>S0532</td>
			<td>C21H22N2O2, molecular weight = 334.41</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mecamylamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>M9020</td>
			<td>&#160;C11H21N &#183; HCl, molecular weight = 203.75</td>
		</tr>
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			<td height="40" style="height:40px;">Gabazine (SR-95531)</td>
			<td>Sigma-Aldrich</td>
			<td>S106</td>
			<td>C15H18BrN3O3, molecular weight = 368.23</td>
		</tr>
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			<td height="40" style="height:40px;">Ketamine hydrochloride</td>
			<td>Mylan</td>
			<td>67457-001-00</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Nikon</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>ROE-200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heater</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pump cartridge</td>
			<td>Cole-Parmer</td>
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		<tr height="33">
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		<tr height="33">
			<td height="33" style="height:33px;">Refrigeration system</td>
			<td>Vibratome Instruments</td>
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			<td>Sutter Instruments</td>
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		<tr height="23">
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			<td>Sutter Instruments</td>
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			<td>Molecular Devices</td>
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			<td>Molecular Devices</td>
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		<tr height="23">
			<td height="23" style="height:23px;">heater</td>
			<td>Warner Instruments</td>
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		<tr height="23">
			<td height="23" style="height:23px;">pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
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			<td height="23" style="height:23px;">pump cartridge</td>
			<td>Cole-Parmer</td>
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recording gamma band oscillations in pedunculopontine nucleus neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

Initially, gamma oscillations were evoked using square current pulses. Current clamp recording of PPN neurons in the presence of synaptic blockers and TTX was continuously monitored to assure that resting membrane potential was kept stable at ~-50 mV (Figure 1A). Two second long square current pulses were injected intracellularly by the patch clamp amplifier through the recording pipette, increasing their amplitude from 200 pA to 600 pA (Figure 1A). Membr...

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Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

The overall goal of this demonstration is to describe a method for detecting high threshold and subthreshold oscillations in pedunculopontine neurons. This method can help answer questions about, for example, how cells maintain their firing frequency. How do populations maintain their firing frequency?We know that circuits cannot maintain gamma band or 40 to 60 per second oscillations. Therefore, you require intrinsic membrane properties to maintain that kind of firing frequency. Demonstrating the procedure will be Susan Mahaffey and Dr.Stasia D'Onofrio.To prepare standard aCSF, first prepare stock solutions A and B and keep them refrigerated at four degrees Celsius for up to two weeks. Before each experiment, mix 50 milliliters of stock A with 50 milliliters of stock B.Then bring the mixture to one liter with distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. Transfer to a beaker for stirring and oxygenate.After bubbling, add the calcium chloride. Next, prepare sucrose aCSF by preparing stock solution C.Before each experiment, mix 300 milliliters of stock C with 600 milliliters of distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. After that, transfer 100 milliliters of sucrose aCSF solution to a clean beaker, place it on ice, and keep it oxygenated with carbogen.Now, fill up a vibratome cutting chamber with sucrose aCSF and keep it oxygenated. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 minutes to allow it to cool down to about four degrees Celsius. Next, remove the pup's brain by placing a spatula under the olfactory bulb and gently pushing it out from the most rostral area towards the most caudal area.Subsequently, place the brain into the ice-cold oxygenated sucrose aCSF. Make a parasagittal cut on the right hemisphere to remove approximately 1/3 of the hemisphere. Then, glue the trimmed side of the brain onto the metal disk.Subsequently, fix the metal disk to the vibratome cutting chamber and slice 400 micron sagittal sections containing the PPN. Keep the PPN slices at room temperature for 45 minutes prior to whole-cell patch clamp recording. In this procedure, transfer a slice to the submersion chamber, place a screen on top of it to hold the slice down, and perfuse it at 1.5 milliliters per minute with oxygenated aCSF containing the selected receptor antagonist.In order to isolate intrinsic membrane properties, what you need to do is block the fast synaptic transmission around the cell and also block the ability to generate action potentials, so you have to use synaptic blockers and tetrodotoxin, or TTX, in order to block action potentials. That way, the cell that you are patching is the one that generates those properties, and no synaptic input is responsible for those, and that's what we're studying here, intrinsic membrane property. After that, fill the recording patch pipette with intracellular high potassium solution.Insert the pipette in the headstage holder. Next, apply a small positive pressure using a one milliliter syringe connected to the pipette holder. Using a 4x objective together with the near infrared differential interference contrast optics, locate the PPN nucleus, which is dorsal to the superior cerebellar peduncle.Slowly lower the recording pipette to the PPN nucleus using a mechanical micromanipulater. Then position the recording pipette in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle. Using a 40x water immersion lens, bring the recording pipette in contact with the PPN neuron, and rapidly apply negative suction to form a seal with the cell.Use voltage clamp seal software to monitor the pipette resistance during negative suction. Then slowly increase the negative pressure. When the resistance value of the pipette reaches 80 to 100 megaohms, rapidly change the holding potential to 50 millivolts and release the negative pressure.Apply negative pressure continuously until the cell membrane is ruptured and electrical access is achieved in the wholesale configuration. Before suction, the resistance is low, in this case, 2.5 megaohms. As suction is applied, step amplitude decreases towards holding.When the seal is formed, note the resistance increases to 167 megaohms. With additional suction, a gigaseal is formed. Resistance briefly increases to 1.2 gigaohms before acquiring wholesale access.Continuously monitor the resting membrane potential of the PPN neuron being recording. If the resting membrane potential shifts towards depolarizing or hyperpolarizing values, apply a small amount of direct current to keep the optimal potential at 50 millivolts. The reason why we use current ramps is so that we can slowly depolarize the membrane without activating potassium channels that keep you from depolarizing the membrane sufficiently high to reach the threshold of these high-threshold calcium channels that we're studying.Shown here are the representative membrane potentials responding to the depolarizing two-second-long square steps of increasing current injected intracellularly through the recording pipette in the presence of synaptic blockers and TTX. This figure shows a power spectrum of the membrane oscillations induced by the indicated current steps in three recordings. The frequencies induced were all low in power and low in frequency.This figure shows the current ramps of 200, 400, and 700 picoamps and the membrane responses. Note that the membrane is sufficiently depolarized to reach 30 millivolts, at which high-threshold calcium channels begin to open, as in the blue recording, and the 10 millivolt level at which their oscillation amplitude is maximal, as in the red recording. This graph shows a power spectrum of the membrane oscillations induced by the current ramps in the corresponding three recordings.The frequencies induced by the 400 picoamp ramp were in the beta range, and by the 700 picoamp ramp were in the gamma range. Once mastered, this technique can be done in about an hour or less. When you're doing your recording, it's really important to make sure that you have a tight seal and that you have capacity compensation and series resistance well-monitored to make sure that there are no major changes during the recording procedure.Once you've isolated these oscillations, what you can do is test to determine if they're really mediated by N-type or P/Q-type calcium channels. You can use conotoxin to block N-type channel oscillations and agatoxin to block P/Q-type channel oscillations. As we developed this technique, it became much easier for you to demonstrate that indeed, high threshold calcium channels were dependent for intrinsic membrane oscillations, and it became a breakthrough in the field of sleep research to determine that the reticular activating system could actually generate gamma band oscillations by itself.

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Research

JoVE Journal

Neuroscience

8.5K Views.  University of Buenos Aires. The overall goal of this demonstration is to describe a method for detecting high threshold and subthreshold oscillations in pedunculopontine neurons. This method can help answer questions about, for example, how cells maintain their firing frequency. How do populations maintain their firing frequency?We know that circuits cannot maintain gamma band or 40 to 60 per second oscillations. Therefore, you require intrinsic membrane properties to maintain that kind of firing frequency. Demon...