Name: recording gamma band oscillations in pedunculopontine nucleus neurons
Uploaded: 2016-09-14T15:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

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 continuously stirring a volume of 500 ml, add 136.75 g of NaCl, 6.99 g of KCl, 2.89 g of MgSO4, and 2.83 g of NaH2PO4.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 117 mM NaCl, 4.69 mM KCl, 1.2 mM MgSO4, and 1.18 mM NaH2PO4.</li>
		<li>Keep refrigerated at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Preparation of Stock Solution B
	<ol>
		<li>Add 700 ml of distilled water to a clean 1L beaker before adding chemicals.</li>
		<li>While continuously stirring a volume of 500 ml, add 41.45 g of D-glucose and 41.84 g of NaHCO3.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 11.5 mM D-glucose and 24.9 mM NaHCO3.</li>
		<li>Keep refrigerated at 4 &#176;C for up to 2 weeks.</li>
		<li>Before each experiment mix 50 ml of stock A with 50 ml stock B and bring to 1 L with distilled water to obtain final concentration aCSF solution and leave at RT&#160;while continuously oxygenating it with carbogen (95% O2 - 5% CO2 mix) for at least 30 min. Prepare final concentration aCSF only at the beginning of each experiment and discard at the end of the day.</li>
	</ol>
	</li>
</ol>
2. Preparation of Sucrose-artificial Cerebrospinal Fluid (Sucrose-aCSF)
<ol>
	<li>Preparation of Stock Solution C
	<ol>
		<li>Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.</li>
		<li>While continuously stirring 500 ml of distilled water, add 240 g of sucrose, 6.55 g of NaHCO3, 0.671 g of KCl, 4.88 g of MgCl2, 0.22 g of CaCl2 and 0.21 g of ascorbic acid.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 701.1 mM sucrose, 78 mM NaHCO3, 9 mM KCl, 24 mM MgCl2, 1.5 mM CaCl2 and 1.2 mM ascorbic acid.</li>
		<li>Keep stock solution C at 4 &#176;C for up to one week.</li>
		<li>Before each experiment mix 300 ml of 50 ml of stock C with 600 ml distilled water to obtain the sucrose-aCSF solution at the final concentration and leave at RT while continuously oxygenating it with carbogen (95% O2 - 5% CO2 mix) for at least 30 min.</li>
	</ol>
	</li>
</ol>
3. Slice Preparation
<ol>
	<li>Place a clean beaker with 100 ml sucrose-aCSF solution in ice while oxygenating it with carbogen and fix the pH at 7.4 using a pH meter while adding few drops of 0.1 M NaOH solution (when pH &#60; 7.4) or 0.1 M HCl solution (when pH &#62; 7.4).</li>
	<li>Fill up a cutting chamber of a vibratome with sucrose-aCSF and oxygenate it. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 min to allow it to cool down to 0 - 4 &#176;C.</li>
	<li>Anesthetize rat pups (aged 9 to 12 from adult timed-pregnant Sprague-Dawley rats) with Ketamine (70 mg/kg, i.p.; using &#60; 50 &#181;l final volume). When pup is calm, double check that tail pinch reflex is absent.</li>
	<li>Decapitate pups.
	<ol>
		<li>Cut the head skin longitudinally from the front to back using a carbon steel scalpel blade, and pull the skin to the sides using forceps. Cut the bone covering the brain moving the scissors laterally and totally remove it to expose the brain.</li>
		<li>Then, rapidly remove the brain using a spatula (initially placed under the olfactory bulb in order to gently push out the brain from the most rostral towards the most caudal areas). Gently push the brain into ice-cooled oxygenated sucrose-artificial cerebrospinal fluid (sucrose-aCSF).</li>
	</ol>
	</li>
	<li>Make a parasagittal cut on the right hemisphere (removing approximately one third of the hemisphere), and glue the trimmed side of the brain onto a metallic disk that will be magnetically fixed to the cutting chamber of a vibroslicer to cut sagittal 400 &#181;m sections containing the pedunculopontine nucleus (PPN). Keep PPN slices at RT for 45 min prior to whole-cell patch-clamp recordings.</li>
</ol>
4. Recordings Gamma-band Oscillations in PPN Slices
<ol>
	<li>Preparation of Potassium-gluconate Intracellular Solution (High-K+ Solution)
	<ol>
		<li>Place in ice a clean beaker with 10 ml distilled water. While continuously stirring add: K+-gluconate, 90.68 mg phosphocreatine di Tris salt, 47.66 mg HEPES, 1.5 mg EGTA, 40.58 mg Mg2+-ATP, and 4 mg Na+2-GTP.</li>
		<li>Adjust pH to 7.3 with KOH (100 mM in distilled water). Add distilled water to reach a final volume of 20 mL. If needed, adjust osmolarity with sucrose (100 mM in distilled water) to be 280 - 290 mOsm. After dilution, the final concentration of each compound is 124 mM K+-gluconate, 10 mM phosphocreatine di Tris salt, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg2+-ATP, and 0.3 mM Na+2-GTP.</li>
		<li>Aliquot intracellular solutions in 1 ml tubes and freeze at -20 &#176;C. Use one aliquot per day and keep at 4 &#176;C during experiments.</li>
	</ol>
	</li>
	<li>Whole-cell Patch Clamp Recordings
	<ol>
		<li>Place slices in an immersion chamber and perfuse them (1.5 ml/min) with oxygenated (95% O2 -&#160; 5% CO2) aCSF containing the following receptor antagonists: selective NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 40 &#956;M), competitive AMPA/Kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10&#956;M), glycine receptor antagonist strychnine (STR, 10 &#956;M), the specific GABAA receptor antagonist gabazine (GBZ, 10 &#956;M), and sodium channel blocker tetrodotoxin (TTX, 3&#956;M) 
		NOTE: In the results and figures, these antagonists are collectively referred to as synaptic blockers or SBs.</li>
		<li>Fill the recording patch pipettes (Resistance 2-7 M&#937;; made from regular, commercially available thick wall borosilicate glass capillaries of 1.0 mm outer diameter and 0.6 mm inner diameter) with intracellular high-K+ solution using commercially available patch-pipette fillers with a solution filter. Insert the pipette in its amplifier&#39;s holder. Apply a small positive pressure using a 1 ml syringe connected to the pipette holder using a silicon tube connected to a three-way valve. Connect the back of the pipette holder to a patch clamp amplifier.</li>
		<li>Move the recording pipette using a mechanical micromanipulator near the PPN nucleus using a 4X objective combined to near-infrared differential interference contrast optics. 
		NOTE: PPN nucleus can be observed in slices dorsal to the superior cerebellar peduncle (SCP; observable as a thick bundle of axons). Recording pipettes were located in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle.</li>
		<li>Bring the recording pipette in contact with a PPN neuron while visualized using a 40X water immersion lens, and rapidly apply negative suction to form a seal with the cell.</li>
		<li>Use voltage-clamp seal software to monitor pipette resistance during negative suction using manufacturer&#39;s protocol.
		<ol>
			<li>When negative suction is slowly increased and resistance values reading by the patch-clamp monitor at the tip of the pipette reach 80 - 100 MOhm, rapidly change the holding potential to -50 mV and release the negative pressure. Start continuously applying negative suction until rupturing the neuron&#39;s membrane and electrical access is achieved in the whole-cell configuration.</li>
			<li>If access resistance values measured by the voltage-clamp seal software are 10 MOhm or higher, then continue applying negative suction in smaller amounts.</li>
		</ol>
		</li>
		<li>Compensate capacitance (i.e., slow and fast transients observed after rupturing the membrane of the cell) and series resistance in voltage-clamp mode. Switch recording mode to current clamp, and rapidly compensate bridge values (e.g., move amplifier knob or click on the automatic compensation menu using computer&#180;s mouse).
		<ol>
			<li>Continuously monitor resting membrane potential of PPN neuron being recorded using manufacturer&#39;s protocol. If resting membrane potential shift towards depolarizing or hyperpolarizing values, then use small amounts of direct current (up to 100 pA) to keep the optimum -50 mV final value. 
			NOTE: Keep only PPN neurons with a stable resting membrane potential (RMP) of -48 mV or more hyperpolarized (i.e., RMP &#60; -48 mV).</li>
		</ol>
		</li>
	</ol>
	</li>
</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.

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;, molecular weight = 342.30</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 84.01</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td>KCl, molecular weight = 74.55</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =&amp;nbsp; 203.30</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =147.02</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight = 180.16</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight =176.12</td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td>NaCl, molecular weight =&amp;nbsp; 58.44</td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;KO&lt;sub&gt;7&lt;/sub&gt;, molecular weight =&amp;nbsp; 234.25</td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td>C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;P &amp;middot; 2C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, molecular weight =&amp;nbsp; 453.38</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S, molecular weight = 238.30</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>[-CH&lt;sub&gt;2&lt;/sub&gt;OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;N(CH&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H)&lt;sub&gt;2&lt;/sub&gt;]&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 380.40</td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td>&amp;nbsp;C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;13&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xMg2+, molecular weight = 507.18</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;14&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xNa+, molecular weight = 523.18</td></tr><tr><td>Tetrodotoxin citrate</td><td>Alomone Labs</td><td>T-550</td><td>C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;, molecular weight = 319.27</td></tr><tr><td>&amp;nbsp;DL-2-Amino-5-Phosphonovaleric Acid</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P, molecular weight = 197.13</td></tr><tr><td>CNQX disodium salt hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C239</td><td>C&lt;sub&gt;9&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt; &amp;middot; xH2O, molecular weight = 276.12</td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>C&lt;sub&gt;21&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 334.41</td></tr><tr><td>Mecamylamine hydrochloride</td><td>Sigma-Aldrich</td><td>M9020</td><td>&amp;nbsp;C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;21&lt;/sub&gt;N &amp;middot; HCl, molecular weight = 203.75</td></tr><tr><td>Gabazine (SR-95531)</td><td>Sigma-Aldrich</td><td>S106</td><td>C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;BrN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 368.23</td></tr><tr><td>Ketamine hydrochloride</td><td>Mylan</td><td>67457-001-00</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>Pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>&amp;nbsp;Leica VT1200 S</td><td></td></tr><tr><td>Refrigeration system</td><td>Vibratome Instruments</td><td>900R</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr></tbody></table>

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 ...

recording-gamma-band-oscillations-in-pedunculopontine-nucleus-neurons

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Neuroscience

8.6K Views.  University of Buenos Aires. 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.

Video: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Biology

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Source: Urbano, F. J., et al. <a href="https://app.jove.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

Rejestrowanie oscylacji pasma gamma z neuronów jądra szypułkowego w wycinku mózgu

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Source: Haumesser, J. K., et al. <a href="https://app.jove.com/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

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Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

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