measurement of evoked potassium ion concentration dynamics in coronal hippocampal slices

Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices

To measure the potassium ion dynamics, gently place the brain slice in the bath using a Pasteur pipette and gently hold it in place with a platinum harp with nylon strings. Ensure the tips of the bipolar stimulating electrode are approximately parallel to one another, and are level with the plane of the slice. Over the course of 5 to 7 seconds, slowly insert the electrodes into CA3 stratum radiatum approximately 40 to 50 micrometers deep to stimulate Schaffer collaterals.
Then, carefully insert the potassium ion-selective electrode into CA1 stratum radiatum to approximately 50 micrometers deep by slowly lowering the electrode over approximately 3 to 4 seconds. Allow the potential to stabilize across the electrode before applying stimulations to the slice, which usually takes 5 to 10 minutes. If the slice exhibits excessive spontaneous changes in extracellular potassium ion concentrations, discard and repeat the process with a new slice.
To measure evoked potassium ion release, apply trains of electrical stimulation via the stimulus isolator while digitally recording responses. Apply stimulation at 10 Hertz and 1 millisecond per pulse starting at 10 microamps stimulus amplitude. Apply increasing stimulation amplitudes by a factor of 2 until a maximum potassium response amplitude is detected. If no response is observed, move the position of the potassium ion-selective electrode closer to the stimulation site in 100-micrometer increments.
To confirm that potassium ion concentration changes are mediated by action potential firing of the electrically activated Schaffer collaterals bath, apply 0.5 micromolar TTX in aCSF for 10 minutes and repeat the stimulation protocol. No evoked responses should be observed.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Hippocampal Brain Slices
<ol>
	<li>Preparation of slice solutions
	<ol>
		<li>Prepare 500 mL sucrose cutting solution composed of: 194 mM sucrose, 30 mM NaCl, 4.5 mM KCl, 10 mM D-glucose, 1 mM MgCl2, 1.2 mM NaH2PO4, and 26 mM NaHCO3, 290-300 mOsm, saturated with 95% O2&#160;and 5% CO2.</li>
		<li>Prepare 1-2 liters of recording solution (artificial cerebrospinal fluid, ACSF) composed of: 124 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 10 mM D-glucose, 2 mM CaCl2, 1.2 mM NaH2PO4, and 26 mM NaHCO3; pH 7.3 - 7.4 (after bubbling), 290 - 300 mOsm, saturated with 95% O2&#160;and 5% CO2. Fill a beaker containing a brain slice holder chamber with recording solution and keep it at 32-34 &#176;C. Fill the vibratome chamber with ice-water slush.</li>
	</ol>
	</li>
	<li>Acute slice preparation
	<ol>
		<li>Deeply anesthetize a mouse by placing it in a bell jar pre-charged with 2-3 mL isoflurane. Check for toe pinch reflex, and if non-responsive, rapidly decapitate it using a pair of sharp shears or guillotine as your animal protocol requires.</li>
		<li>Make a 2-3 cm incision using shears from the caudal portion of the skull to cut the scalp along the midline. While manually retracting the scalp, make two 1 cm horizontal incisions from the foramen magnum along the sides of the skull. Then, using fine shears, cut an incision the length of the skull, along the midline from the back of the skull to the nose.</li>
		<li>Using fine forceps inserted near the midline, retract the incised skull in two portions. Extract the mouse brain from the skull and use a blade to remove the cerebellum and olfactory bulbs, which are respectively located at the caudal and rostral portions of the brain. These can be identified by the large fissures, which separate them from the cortex.</li>
		<li>Mount the brain block onto the vibratome tray using super glue. Fill the vibratome tray with ice cold cutting solution.</li>
		<li>Cut tissue sections on the coronal plane at 300 &#181;m thickness. Usually, 6 coronal hippocampal slices can be collected.</li>
		<li>After each section is cut, immediately transfer the slices to the slice holding beaker warmed to 32-34&#176;C. Keep the sections at this temperature for 20 min before removing the beaker containing the sections and place this at room temperature for at least 20-30 minutes prior to recording.</li>
	</ol>
	</li>
</ol>
2. Measurement of Electrically Evoked K+&#160;Dynamics
<ol>
	<li>Setting up the slice preparation
	<ol>
		<li>Gently place the brain slice in the bath using a Pasteur pipette and gently hold it in place with a platinum harp with nylon strings.</li>
		<li>Ensure the tips of the bipolar stimulating electrode are parallel to one another and are level with the plane of the slice. Slowly, over the course of 6-7 seconds, insert the electrodes into CA3 stratum radiatum approximately 40-50 &#181;m deep to stimulate Schaffer collaterals. In coronal sections, CA3 can be approximately identified as the portion of the hippocampus proper lateral to the granule cell layer at the hippocampal genu, with the stratum radiatum falling medial and ventral to the pyramidal cell layer.</li>
		<li>Carefully insert the K+-selective electrode into CA1 stratum radiatum approximately 50 &#181;m deep, by slowly lowering the electrode over approximately 3-4 seconds. Allow the potential to stabilize across the electrode before applying stimulations to the slice: this usually takes 5 to 10 minutes. If the slice exhibits spontaneous changes in extracellular K+&#160;then discard and repeat this process with a new slice.</li>
	</ol>
	</li>
	<li>Measure evoked K+&#160;release
	<ol>
		<li>Apply trains of electrical stimulation (8 pulses) by manually depressing the trigger on the stimulator while digitally recording responses. Apply stimulation at 10 Hz and 1 ms pulse width, starting at 10 &#181;A stimulus amplitude.</li>
		<li>Apply increasing stimulation amplitudes by a factor of 2 until a maximum K+&#160;response amplitude is detected. If you do not see any response, move the position of the K+&#160;electrode closer to the stimulation site in 100 &#181;m in increments</li>
		<li>Determine the stimulus amplitude that produces the half maximal response. In our experience, this is between 40-160 &#181;A, depending upon the preparation quality, age of the animal, and the distance between stimulation electrodes and K+&#160;selective electrode.</li>
		<li>In the same slice, using a stimulus amplitude at one step lower than the amplitude that produces the half maximal response (e.g. if 80 &#181;A produces a half-maximal response, use 40 &#181;A) apply stimulus trains of increasing number of pulses. Initially, we have used trains of 1, 2, 4, 8, 16, 32, 64 and 128&#160;pulses.</li>
		<li>To confirm that K+&#160;signals are mediated by action potential firing of the electrically stimulated Schaffer collaterals bath, apply 0.5 &#181;M tetrodotoxin (TTX) in ACSF for 10 minutes and repeat the stimulation protocol. No evoked responses should be observed.</li>
		<li>After finishing the slice experiment, confirm the electrode has maintained its responsivity by re-calibrating the electrode in the calibration solutions and ensuring the response has not deviated by more than 10% from the initial calibration</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vibratome</td>
			<td>DSK</td>
			<td>Microslicer Zero 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6NTac inbred mice</td>
			<td>Taconic</td>
			<td>Stock#B6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>pCLAMP10.3</td>
			<td>Molecular Devices</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom microfil 28G tip</td>
			<td>World precision instruments</td>
			<td>CMF28G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten Rod</td>
			<td>A-M Systems</td>
			<td>716000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar stimulating electrodes</td>
			<td>FHC</td>
			<td>MX21XEW(T01)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus isolator</td>
			<td>World precision instruments</td>
			<td>A365</td>
			<td></td>
		</tr>
		<tr>
			<td>Grass S88 Stimulator</td>
			<td>Grass Instruments Company</td>
			<td>S88</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass pipettes</td>
			<td>World precision instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr>
			<td>A to D board</td>
			<td>Digidata 1322A</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Amplifier</td>
			<td>Multiclamp 700A or 700B</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>CV-7B Cat 1</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch computer</td>
			<td>Dell</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>21108</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>valinomycin</td>
			<td>Sigma</td>
			<td>V0627-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimethyl-3-nitrobenzene</td>
			<td>Sigma</td>
			<td>40870-25ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium tetrakis (4-chlorophenyl)borate</td>
			<td>Sigma</td>
			<td>60591-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>5% dimethyldichlorosilane in heptane</td>
			<td>Sigma</td>
			<td>85126-5ml</td>
			<td></td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Cayman Chemical Company</td>
			<td>14964</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H1758-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378-5kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Micromanipulator</td>
			<td>Sutter</td>
			<td>MP-285 / ROE-200 / MPC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>PlanAPO 10xW</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Octeau, J. C. et al. <a href="https://app.jove.com/v/57511">Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain.</a> J. Vis. Exp. (2018).
The video demonstrates evoked potassium ion concentration dynamics on electrical stimulation of mouse coronal hippocampal brain slices. A bipolar stimulating electrode is inserted into the stratum radiatum of the CA3 region in the hippocampus, and a potassium-ion selective electrode is placed in the CA1 stratum radiatum. The electrical stimulation causes an action potential, resulting in potassium ion release. Finally, the lack of evoked potassium ion response in the presence of an inhibitory molecule confirms that potassium ion concentration dynamics are due to action potential generation in the Schaffer collaterals.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Hippocampal ...

Secure a mouse coronal brain slice in a recording chamber filled with aCSF.
Insert a bipolar stimulating electrode into the stratum radiatum of the CA3 region in the hippocampus. The CA3 neurons project their axons, termed Schaffer collaterals, to CA1.
Position a potassium ion-selective electrode in the CA1 stratum radiatum.
Electrically stimulate CA3 neurons to open voltage-gated sodium channels. The sodium ion influx turns the membrane potential positive, termed depolarization, and generates an action potential.
After the peak of depolarization, sodium channels deactivate, and voltage-gated potassium channels allow potassium ion outflow, repolarizing the membrane. Subsequently, the membrane potential is restored, and the action potential propagates to CA1.
The potassium ion-selective electrode measures the outflow, shown by a response curve.
Add a blocker to inhibit voltage-gated sodium channel activity. Upon electrical stimulation, a lack of potassium ion response confirms that its dynamics are due to action potentials in the Schaffer collaterals.

18 조회수. Coronal Hippocampal Slices에서 유발된 칼륨 이온 농도 역학 측정

비디오: Coronal Hippocampal Slices에서 유발된 칼륨 이온 농도 역학 측정 - 실험

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.

두뇌 조각에서 신경 모 수석에서 2 광자 칼슘 이미지

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

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Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

생물학적 조직의 자극을 위한 전기 및 자기장 장치

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices

내레이션 대본

Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices