Supported by APA to RJH, NHMRC program grant 400121 to SP, and NMHRC fellowship 1005050 to SP. CAR acknowledges the support of the ARC (FT0990628) and the DOWD fellowship scheme. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds.

All experiments on mice were approved by the Florey Institute animal ethics committee.
1. Setup for Cutting Brain Slices
<ol>
	<li>Prepare a cutting solution comprised of (mM) 125 Choline-Cl, 2.5 KCl, 0.4 CaCl2, 6 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 D-glucose saturated with carbogen gas (95% O2&#8211;5% CO2) and an artificial cerebrospinal fluid (aCSF) recording solution comprised of (mM) 125 NaCl, 2.5 KCl, 2&#160;CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose, saturated with carbogen. Place the cutting solution on ice to keep it cold.</li>
	<li>Freeze approximately 400 ml of the cutting solution and blend together with 100 ml of unfrozen cutting solution to create an ice slurry. Bubble with carbogen (95 % O2&#8211;5 % CO2) at a flow of approximately 0.5 L/min through small caliber tubing or sintered glass to produce a steady but gentle flow of bubbles.</li>
	<li>Prepare a 250 ml beaker with a raised nylon mesh insert on which the brain slices will be placed. Fill with aCSF to cover the mesh by approximately 2 cm and bubble with carbogen, ensuring that the bubbles do not directly disrupt the slice holding area. It is also important that there are no air bubbles in the nylon mesh, so if any are present remove them. This will be the holding chamber and is kept at RT (20 - 25 &#176;C).</li>
	<li>Layout the dissecting instruments including a large pair of scissors, a small pair of scissors, small and large micro spatulas, and large and small pairs of forceps and chill them on ice. Place the vibratome tissue-cutting block on ice on a square of aluminum foil. Obtain 2 pieces of 6 cm filter paper, a single edge razor blade, and a 25 ml beaker filled with the cutting solution slurry as well.</li>
	<li>Fill a second container with ice, and lay a piece of tissue paper on the ice and place a 12 cm culture dish on top. Fill the culture dish with cutting solution ice slurry and bubble with carbogen. This is the container in which the brain dissection with be performed.</li>
	<li>Prepare the vibratome. Remove a fresh double edged razor blade (use a fresh blade every time) from its wrapping and spray with 80% ethanol then deionized water. Cut a 3 ml plastic transfer pipette at the point where it starts to taper. This will be used to transfer brain slices. Bend a 27 G needle at the base by approximately 45&#176; and attach to a 1 ml syringe. This will be used to manipulate slices during cutting.</li>
</ol>
2. Cutting Brain Slices
<ol>
	<li>Anesthetize a mouse (P16 - P18) with 2% isoflurane or a locally approved method. Following induction, decapitate the animal with a large pair of scissors and drop the head into the 12 cm culture dish that contains bubbled cutting solution slurry. The slurry must totally immerse the head for rapid cooling.</li>
	<li>Hold the front of the head with one hand peeling the skin and connective tissue forward towards the nose. Using the small scissors cut the connective tissue to reveal the underlying skull. Then remove the muscles overlying dorsal aspect of the skull and neck.</li>
	<li>Remove the brain from the skull by first securing the front of the skull with the large forceps and then make two lateral cuts through the bone either side of the foramen magnum using the small scissors (Figure 1, cuts labeled A1 and A2).
	<ol>
		<li>Make another cut between the eyes (just anterior of the bregma) (Figure 1, cut labeled B) then carefully cut along the sagittal suture anteriorly and reflect the cut skull sections to reveal the brain (Figure 1, cut labeled C).</li>
		<li>Use the small spatula to scoop out the brain and place onto a culture dish. Be aware of the cranial nerves on the inferior aspect of the brain which will need to be severed, this can be done using the smaller sized micro spatula. Using the larger spatula transfer the brain to the 25 ml beaker filled with the cutting solution slurry.</li>
	</ol>
	</li>
	<li>Preparing the brain hemisphere for slicing.
	<ol>
		<li>Place one piece of the 6 cm filter paper in the bottom of a fresh culture dish then fill with fresh cutting solution slurry and bubble. Use the larger spatula to position the brain, ventral side down, onto the filter paper. Take a new and cleaned single edge razor blade and cut the brain to remove the cerebellum, then make a cut along the mid line to separate the brain into two hemispheres.</li>
	</ol>
	</li>
	<li>Preparing the vibratome tissue block.
	<ol>
		<li>Take the chilled vibratome tissue block, dry the surface, and place a drop of cyanoacrylate glue in the middle and spread evenly to the approximate size of the brain. Use the small spatula to manipulate one of the brain hemispheres onto the large micro spatula so the medial side of the brain is down.</li>
		<li>Touch the edge of the spatula at the interface of the brain on the second piece of filter paper to remove as much of the solution as possible. Slide the brain off the larger spatula using the smaller spatula to guide it onto the glue.</li>
		<li>Secure the cutting block into the vibratome chamber and fill the chamber with cutting solution slurry and bubble, ensuring the brain is completely immersed. Rotate the cutting block so the ventral side of the brain is facing the blade.</li>
	</ol>
	</li>
	<li>Slicing the brain. 
	Note: Each vibratome is unique so follow the manufacturer&#8217;s instructions.
	<ol>
		<li>Set the slice thickness to 450 &#181;m, and ensure the blade is vibrating as it moves through the brain at a speed of approximately 0.3 mm/s. Cut entirely through the brain from the ventral side to the cortical surface, this will produce whole brain sagittal slices that can be used for electrophysiological recordings. Slices will be produced laterally to medially and typically 3 - 4 slices can be cut from each hemisphere.</li>
		<li>If the base of the slice lifts use the bent 27 G needle to gentle press the slice back down. As each slice is cut, use the transfer pipette to move it onto the platform in the holding chamber where they are viable for up to 8 hrs.</li>
	</ol>
	</li>
</ol>
3. Extracellular Electrophysiology Recordings
<ol>
	<li>Mounting the slice in the recording chamber.
	<ol>
		<li>Using the transfer pipette, place a brain slice into a submerged recording chamber perfused with aCSF flowing at 1 - 2 ml/min and heated to 32&#160;&#176;C. The aCSF used for recordings differs from that in the holding chamber as the Mg2+ concentration is increased from 2 mM to 4 mM.</li>
		<li>Secure the slice with a &#8220;harp&#8221; (semi circular stainless steel with nylon strands stretched across at 2 - 3 mm spacing). Place the harp so that the strands run parallel to CA1. Increase the speed of the perfusion to 8 - 10 ml/min at 32 &#176;C.</li>
	</ol>
	</li>
	<li>Under a dissecting microscope place a stimulating electrode and a recording electrode (glass electrode filled with the aCSF recording solution) on the surface of the stratum radiatum of the CA1 (Figure 2A). Place the stimulating electrode first then place the recording electrode.</li>
	<li>Stimulate the Schaffer collaterals with a 120 - 150 &#181;A amplitude and 0.1 msec duration test pulse and observe the resulting field excitatory post synaptic potential (fEPSP) waveform to determine slice health (Figure 2B). The stimulating and recording electrodes may need to be moved into the slice approximately 50 - 100 &#181;m from the slice surface to get a fEPSP recording with a small fiber volley and large amplitude. The Schaffer collateral evoked fEPSPs are stereotypical and provide a good indicator of slice health. Healthy slices typically show a fiber volley to fEPSP amplitude ratio of less than 0.3.</li>
	<li>Repositioning the electrodes.
	<ol>
		<li>Using a dissecting microscope, move the stimulating electrode to the middle of the stratum oriens and move the recording electrode to the pyramidal cell layer as close to the recording electrode as possible as shown in Figure 3A. The stimulating and recording electrodes may need to be pushed into the slice approximately 50 - 100 &#181;m so that the fEPSP response amplitude to the 120 - 150 &#181;A test pulse is approximately 1 mV.</li>
	</ol>
	</li>
	<li>Generating &#947; oscillations.
	<ol>
		<li>To generate &#947; oscillations stimulate the tissue with a train of 20 x 0.1 msec pulses delivered at 200 Hz. This tetanic stimulus can yield reproducible responses when delivered every 5 min.</li>
	</ol>
	</li>
	<li>Use the following recording parameters.
	<ol>
		<li>Scale the gain of the output signal to match the input voltage range of the analog to digital converter. Make sure that maximum expected signal excursion uses a minimum of 30% of the input voltage range. Be careful when setting gains to high as signals might clip. The typical bandwidth needed for field recordings is 500 Hz with AC coupling at 0.1 Hz to remove baseline drift.</li>
		<li>Digitize at least 4 - 5 times faster than the corner frequency of the low pass filter to avoid signal aliasing.</li>
	</ol>
	</li>
	<li>Data analysis.
	<ol>
		<li>High pass filter data at 5 Hz to remove any baseline shifts. Identify spikes using a derivative threshold, with a minimum event separation of 10 msec, a typical event time to peak and regression interval of 7 ms, a threshold of ~100 mV/msec, and a separating valley of 100%. Calculate latency as the time taken for the first identified spike to occur after the end of the stimulation artifact. This analysis allows for characterization of the number of spikes, latency, inter-event interval, and duration calculated as follows:</li>
		<li>Calculate the number of spikes as the total number of spikes per oscillation for each sweep is determined.</li>
		<li>Calculate the oscillation latency. For each oscillation, subtract the time of the first identified spike from the end of the stimulation artifact.</li>
		<li>Calculate the average inter-event interval (ISI). Determine the time period between multiple detected spikes and average.</li>
		<li>Calculate the oscillation duration. Measure the time between the first and last detected spike within each oscillation.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

A robust method to generate CA1 &#947; oscillations in acute brain slices is described. The oscillations generated arise from a local circuit enabling a better opportunity for controlling and understanding the neurophysiological basis of network oscillations12. AMPA receptors, GABAA receptors, Ih and T-type Ca2+ channels are all required for &#947; oscillations in this model. While the local CA1 oscillations described here can be robustly generated this is dependent on ensuring that the brain slices are healthy. A critical step is rapid removal of the brain from the skull, taking care to not penetrate the brain during removal and then rapid immersion in ice cold solution. Ideally the time between decapitation and removal of the brain from the skull should be no more than 30 - 60 sec&#160;to preserve slice health21.
By using tetanic stimulation local CA1 &#947; oscillations can be generated in standard physiological recording solutions without reliance on pharmacological agents. This is advantageous for characterizing pathologies in disease models where addition of pharmacological agents may confound interpretation. For example, using this model local CA1 activity and sensitivity to anti-epileptic drugs was enhanced in a mouse model of human genetic epilepsy22. The use of this model to investigate network activity is not limited to epilepsy and may easily be applied to other diseases such as Alzheimer&#8217;s disease, autism and schizophrenia. While pharmacological agents are not required to generate the local CA1 oscillations sufficient tissue oxygenation is, due to the metabolic demand of the brain slice23. High perfusion rates improve oxygen and pH balance within the slice creating a more physiological environment for the brain slices24,25.
A further advantage of using tetanic stimulation to generate network oscillations is that an episodic experimental design can be used that enables more ready quantitation of network activity parameters, such as delay to onset, duration and spike numbers, in a repeatable manner. In contrast, chemically induced network activity is spontaneous13,15,16,26-28 and more difficult to quantify. Tetanic stimulation, however, can cause changes in NMDA receptor-dependent synaptic plasticity, which can cause changes in network activity over time. To control for this and enable stable oscillation generation over multiple tetanic stimulations plasticity changes can be limited by elevating Mg2+ levels. Although this protocol enhances reproducibility it is opaque to any influence of NMDA receptor function.
Interface recording chambers can improve slice health, yield better signal to noise ratios and more focal lower intensity stimulation with the added benefit of smaller stimulation artifacts. Perforated chamber recording methods would also improve slice health for long term experiments. Using micro electrode array recording chambers or patch clamp recording enables wide field and single neuron function to be measured during recordings to better investigate mechanisms underlying &#947; oscillations and their impact on broader network function. Incorporation of voltage and calcium sensors as well as optogenetic methods would introduce additional experimental flexibility in both recording and stimulating paradigms. Similar protocols developed for other brain regions that may be more relevant to the disease pathology under study. A final use for the protocol describe here may be for building and testing computation models of oscillations by combining field recordings with single neuron patch clamp and imaging studies.

Brain network oscillations occur within distinct frequency bands that correlate to behavioral states. In rodents, hippocampal &#952; oscillations (5 - 10 Hz) are observed during exploratory behaviors1,2, while &#947; oscillations (20 - 80 Hz) associate with various cognitive processes, including perception and attention3,4. Synchronous &#947; network activity is also implicated in the pathology of disorders such as epilepsy and schizophrenia5,6. For example, &#947; oscillations are thought to correspond to areas of cortical epileptic foci5,7,8 and could be used as markers of pharmacosensitivity or resistance, two important areas of investigation in epilepsy research9.
The hippocampal brain slice is a model that has been widely used to investigate network activity10-12. Various protocols have been developed to generate &#947; oscillations in brain slices that typically involve pharmacological modulation such as low Mg2+, 4-aminopyridine (4AP), bicuculline, and kainic acid12-17. Shortcomings of pharmacologically triggered oscillations are that they occur randomly after drug application and are not reliably generated or stable over time. Electrically triggered &#947; oscillations overcome many of these problems and also have the advantage of being temporally locked to the stimulating event allowing for episodic recording and analysis. Here a protocol is described for generating CA1 &#947; oscillations by delivering a tetanic stimulation to the stratum oriens in the hippocampal slice.

<table><tbody><tr><td>4-(N-Ethyl-N-phenylamino)-1,2- dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288)</td><td>Sigma-Aldrich</td><td>Z3777</td><td></td></tr><tr><td>Biuculline</td><td>Sigma-Aldrich</td><td>14340</td><td></td></tr><tr><td>6-cyano-7-nitroquinoxa- line-2,3-dione (CNQX)</td><td>Sigma-Aldrich</td><td>C127</td><td></td></tr><tr><td>Nickel</td><td>Sigma-Aldrich</td><td>266965</td><td></td></tr><tr><td>Carbamazepine</td><td>Sigma-Aldrich</td><td>C4024</td><td></td></tr><tr><td>(2R)-amino-5-phosphonopentano-ate (APV)</td><td>Tocris Bioscience</td><td>0105</td><td></td></tr><tr><td>Retigabine</td><td>ChemPacific</td><td>150812-12-7</td><td></td></tr><tr><td>Choline-Cl</td><td>Sigma-Aldrich</td><td>C1879-5KG</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638-250G</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6297-250G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653-5KG</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>223506-500G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670-500G</td><td></td></tr><tr><td>Electrode glass</td><td>Harvard Apparatus&amp;nbsp;</td><td>GC150F-10</td><td></td></tr><tr><td>Concentric bipolar stimulating metal electrode&amp;nbsp;</td><td>FHC</td><td>CBBPF75</td><td></td></tr><tr><td>Digital Isolator</td><td>Getting Instruments</td><td>Model BJN8-9V1&amp;nbsp;</td><td></td></tr><tr><td>Model 1800 amplifier</td><td>A-M systems</td><td>Model 1800 amplifier</td><td></td></tr><tr><td>Digitizer</td><td>National Intruments</td><td>NI USB-6211</td><td></td></tr><tr><td>Vibrotome</td><td>Leica</td><td>VT1200s</td><td></td></tr></tbody></table>

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.

Tetanic stimulation of the stratum oriens generated robust and reproducible &#947; oscillations (35.4 &#177; 2.2&#160;Hz), see Figure 3B. To demonstrate that the oscillations were generated within the local CA1 network the inputs from CA3 were severed by cutting the slice in the CA2 region using a bent 32&#160;G needle. The oscillation properties in the cut slices did not differ from the uncut slices (p = 0.85; cut slices 6.16 &#177; 1.1 spikes, n = 6; uncut slices 5.89 &#177; 0.8 spikes, n = 6), indicating that the oscillations are generated locally.
An important advantage of this method is the stability of recordings. When the tetanus was delivered at 5-min&#160;intervals the oscillations were stable for at least 30 min&#160;for any given slice (p = 0.26 for number of spikes at 15 min&#160;vs. 30 min). There is variability between slices in the total number of spikes within an oscillation and other parameters. For drug studies paired experiments can be done so that differences in baseline properties are minor factors. For intra-slice comparisons variability can be a concern and can be overcome by sufficiently powering comparisons.
Next, the ion channels and receptors that are required for the generation of these tetanically stimulated CA1 &#947; oscillations were investigated. Pharmacological blockade of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors with 20 &#181;M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Figure 4A, B), GABAA receptors with 20 &#181;M bicuculline (Figure 4C), Ih current with 20 &#181;M 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288) (Figure 4D), and T-type Ca2+ channels with 100 &#181;M Ni2+ (Figure 4E) were each independently able to reduce the number of spikes generated (p &#60; 0.05; CNQX n = 6; bicuculline n = 7; ZD7288 n = 6; Ni2+ n = 6). Application of (2R)-amino-5-phosphonopentanoate (AP5, 20 &#181;M) did not affect the number of spikes (p = 0.22; n = 5; AP5, 5.9 &#177; 3.1 spikes; control, 8.3 &#177; 2.2 spikes), suggesting that NMDA receptors are not involved in &#947; oscillation generation in this preparation.
This preparation is ideal for determining the network scale action of drugs. Retigabine is a clinically used anti-epileptic medication that opens potassium channels and reduces membrane excitability18-20. Bath application of retigabine produced a dose-dependent reduction in the number of spikes in and duration of the oscillation (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/52877/52877fig1.jpg" /> 
Figure 1.&#160;Schematic of the skull and overlying skin showing location of cuts to reveal the brain. A1, A2, B and C mark the locations of cuts that need to be made to open up the skull for the removal of the brain. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52877/52877fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52877/52877fig2.jpg" /> 
Figure 2.&#160;Placement of the stimulating and recording electrodes in the CA1 Schaffer collaterals to test slice health. (A) Shows placement of the stimulating electrode (Stim) and the recording electrode (Rec). (B) A representative example of a fEPSP evoked by a 120 &#181;A stimulation (fiber volley marked by *). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52877/52877fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52877/52877fig3.jpg" /> 
Figure 3.&#160;Configuration of recording and stimulation electrodes used to evoke oscillation. (A) Location of the stimulating electrode (Stim) in the stratum oriens and recording electrode (Rec) in the stratum pyramidale of the CA1. (B) A representative example of the &#947; oscillations induced by tetanic stimulation (artifact marked by &#8220;Stim&#8221;). Representative trace showing quantifiable outputs. ISI inter-spike interval. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52877/52877fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52877/52877fig4.jpg" /> 
Figure 4.&#160;Pharmacological characterization of tetanically generated &#947; oscillations. (A) Representative trace demonstrating the effect of CNQX. Graphs demonstrating the effect of (B) CNQX (20 &#181;M; n = 6), (C) bicuculline (20 &#181;M; n = 7), (D) ZD7288 (20 &#181;M; n = 6) and (E) Ni2+ (100 &#181;M; n = 6) on the number of spikes. Data are presented as paired comparisons. *p &#60; 0.05, **p &#60; 0.01 and, ***p &#60; 0.001. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52877/52877fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52877/52877fig5.jpg" /> 
Figure 5.&#160;Retigabine effects on network properties. Representative examples showing the reduced network activity of induced oscillations (A) in control conditions and (B) and (C) with retigabine. Summary of the effects shown in (D) spike count, (E) oscillation duration, (F) latency to oscillation onset, and (G) inter-spike interval (ISI). Data are presented as paired comparisons. *p &#60; 0.05. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52877/52877fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Local CA1 ÃŽÂ³ Oscillations by Tetanic Stimulation at JoVE.com

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

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 ...

generation-of-local-ca1-oscillations-by-tetanic-stimulation

Nanyang Technological University

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Neuroscience

9.1K Views.  University of Melbourne. 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 γ oscillations.

Video: Generation of Local CA1 γ Oscillations by Tetanic Stimulation

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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.

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 ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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 ...

Inducing Long-Term Plasticity of Intrinsic Neuronal Excitability in Neurons of the Dorsal Lateral Geniculate Nucleus

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for visual information traveling from the retina to cortical areas, but recent findings suggest largely underestimated functional plasticity of dLGN principal cells. However, the cellular mechanisms supporting these changes have not been fully explored. Here, we report a protocol to induce long-term potentiation of intrinsic neuronal excitability (LTP-IE) in dorsal dLGN relay cells from acute brain slices of young rats. Intrinsic plasticity is generally induced in parallel with synaptic plasticity. However, in dLGN neurons, LTP-IE is reliably induced by spiking activity at a frequency of 40 Hz for 10 min. LTP-IE in dLGN relay neurons is long-lasting as it can be followed up to 40 min after the induction protocol. In conclusion, the results of this study provide the first evidence for the induction of intrinsic plasticity in dLGN relay cells, thus further pointing to the role of thalamic neurons in activity-dependent visual plasticity.

Here, we describe a protocol for inducing long-term plasticity of neuronal intrinsic excitability in relay neurons from the dorsal lateral geniculate nucleus maintained in ex vivo brain slices.

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for visual information traveling from the retina to cortical ...

Generation of Spontaneous and On-Demand Ictal Events in Mouse Brain Cortical Slices

Source: Chang, M. et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57952">Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue.</a> J. Vis. Exp. (2019)
This video demonstrates the induction of ictal events in coronal cortical slices from a transgenic mouse expressing light-activated cation channels in cortical excitatory pyramidal neurons. The slice is placed in a recording chamber, and a recording electrode is inserted into the superficial cortical layer. Upon perfusion with a seizure-inducing drug that blocks potassium channels, the neurons become hyperexcitable and generate spontaneous ictal events. To generate on-demand ictal events, a brief light pulse is used to activate the cation channels in pyramidal neurons, triggering the release of excitatory neurotransmitters, which induce the postsynaptic neurons to generate ictal events for a short duration.

Source: Chang, M. et al. Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue. J. Vis. Exp. (2019)
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JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice

Source: Hatch, R. J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52877">Generation of Local Ca1 &#947; Oscillations by Tetanic Stimulation.</a> J. Vis. Exp. (2015).
This video demonstrates the procedure of inducing local CA1 gamma oscillations in a CA1 hippocampal mouse brain slice using tetanic simulations. This method is important to understand the neuronal network activity.

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

Neurophysiology

Stimulation and Modulation Techniques

Optogenetic Control of Hippocampal Network Dynamics

Source: Manseau, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017)
This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

	Optogenetic control in the ...

Generation of Local CA1 γ Oscillations by Tetanic Stimulation

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Chapters in this video

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

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

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

Inducing Long-Term Plasticity of Intrinsic Neuronal Excitability in Neurons of the Dorsal Lateral Geniculate Nucleus

Generation of Spontaneous and On-Demand Ictal Events in Mouse Brain Cortical Slices

Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice

Optogenetic Control of Hippocampal Network Dynamics