induction of long-term depression in cerebellar purkinje neurons in a mouse brain slice

Induction of Long-Term Depression in Cerebellar Purkinje Neurons in a Mouse Brain Slice

For long term depression induction, stimulate the molecular layer with a 0.1 millisecond pulse, and apply a double pulse stimulus to identify the parallel fiber excitatory postsynaptic currents. A paired pulse facilitation and gradual increase in amplitude relative to the increase in stimulation intensity should be observed. To record the test response, apply a single 0.1 hertz pulse, and adjust the intensity of the stimulus so that the evoked amplitude is around 200 picoamps.
Stimulate the climbing fibers at the bottom of the Purkinje cell layer, and to identify the parallel fiber excitatory postsynaptic currents elicited by the climbing fiber activation, apply a double pulse stimulus. A paired pulse depression should be observed in an all or none manner, in correlation with the increase in stimulation intensity.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Brain Slicing
<ol>
	<li>Orient the sample such that the dorsal side of the cerebellum is on the front side. Pour ice-cold cutting ACSF (Artificial cerebrospinal fluid), containing 1 &#181;M TTX(Tetrodotoxin), sufficient to immerse the cerebellum completely. Place a gas tube into the ACSF and start bubbling with the O2/CO2 gas mixture.
</li>
	<li>Remove the arachnoid mater using a fine tweezer under binoculars. Cut the cerebellar peduncle with a blade, and remove the brainstem and agar block. Rotate the tray 180&#176;, so that the dorsal surface of the cerebellum faces a razorblade.</li>
	<li>Set the blade, and adjust the first cutting location. Set the vibratome slicing parameters to the following: amplitude to 5.5, frequency to 85 Hz, speed to 3&#8211;4, and slice thickness to 300 &#181;m.</li>
	<li>Transfer the cerebellar slice on a nylon net into an acrylic incubator and immerse the slice completely in the oxygenated ACSF. The incubator should be placed in a water bath that maintains a temperature of 26 &#176;C.</li>
	<li>Store the slices for at least 1 h to allow recovery from the damage during slicing.</li>
</ol>
2. Whole-cell Patch-clamp Recording
NOTE: A patch-clamp recording requires the following equipment: an upright microscope with infrared differential interference contrast (IR-DIC) optics, a patch-clamp amplifier, data digitizer, digital stimulator, isolator, computer, software for data acquisition and analysis, motorized manipulator, microscope platform, vibration isolation table, Faraday cage, solution heating system, peristaltic pumps, and electrode puller.
<ol>
	<li>Add picrotoxin (0.1 mM) to ACSF and resolve it using ultra-sonication for 3 min.</li>
	<li>Perfuse a recording chamber with picrotoxin-containing, O2-CO2-saturated ACSF at rate of 2 mL/min. Maintain the temperature of the recording chamber at around 30 &#176;C.</li>
	<li>Make a recording electrode by pulling a borosilicate glass capillary with filament (outer diameter = 1.5 mm) using a puller with 4 steps. The tip-diameter should be around 1 &#181;m.</li>
	<li>Make a stimulating electrode by pulling the same capillary using the puller with 2 steps, then break to produce a fine tip by striking the tip against an iron block under a binocular microscope. The final diameter should be 3&#8211;5 &#181;m.</li>
	<li>Transfer the cerebellar slice to the recording chamber and fix it with a Pt-weight with nylon threads. Fill a stimulating electrode with ACSF.</li>
	<li>For stimulation of the PFs, place the stimulating electrode on the surface of the molecular layer, around 50 &#181;m away from the Purkinje cell layer.</li>
	<li>For stimulation of the CF, place the stimulating electrode at the bottom of the Purkinje cell layer (steps 3.3, 3.4).</li>
	<li>Filter K+-based or Cs+-based internal solution with a 0.45 &#181;m filter. Use a micro-loader to fill a recording electrode with 8 &#181;L of internal solution.</li>
	<li>Apply a weak positive pressure to the recording electrode before immersing it into the ACSF. Its resistance should be 2&#8211;4 M&#937; and the liquid junctional potential should be corrected.</li>
	<li>Approach the healthy, bright cell body of the PC with the recording electrode. Push the surface of Purkinje cell slightly, stop applying positive pressure, next apply negative pressure until forming a giga-ohm seal. Then establish the whole-cell configuration using negative pressure.</li>
	<li>Hold the membrane potential at -70 mV, and apply -2 mV pulse (duration, 100 ms) at 0.1 Hz to monitor input resistance, series resistance and input capacitance, continuously. Do not use the series resistance compensation. Discard data when the series resistance varies by more than 15%.</li>
</ol>
3. Induction of LTD (Long-term depression)
<ol>
	<li>Stimulate the molecular layer with a pulse (duration, 0.1 ms). Identify the PF(Parallel fibers)-excitatory postsynaptic currents (EPSC) by applying a double pulse stimulus (interspike interval (ISI) of 50 ms). The PF-EPSC should show paired-pulse facilitation and gradual increase in amplitude relative to increase in stimulation intensity.</li>
	<li>Record the test response of the PF-EPSC by applying a single pulse at 0.1 Hz. Adjust the intensity of the stimulus so that the evoked EPSC amplitude is around 200 pA. Avoid contamination of current through the voltage-dependent ionic channel.</li>
	<li>Stimulate the CF at the bottom of the Purkinje cell layer, and identify the EPSC elicited by the CF activation (by applying a double pulse stimulus). The CF-EPSC should show paired-pulse depression and an all-or-none manner according to the increase in stimulation intensity. For LTD induction, a single stimulus should be used.</li>
	<li>LTD-inducing protocol 1&#8203;
	<ol>
		<li>Using an electrode containing K+-based internal solution under current-clamp conditions, apply a single PF-stimulus and a single CF-stimulus simultaneously at 1 Hz for 5 min (300 pulses) (Figure 1A).</li>
	</ol>
	</li>
	<li>LTD-inducing protocol 2
	<ol>
		<li>Using an electrode-containing K+-based internal solution under current-clamp conditions, apply double PF-stimuli (ISI of 50 ms) and single CF-stimulus as the second PF-stimulus is coincident with CF-stimuli at 1 Hz for 5 min (Figure 1B).</li>
	</ol>
	</li>
	<li>LTD-inducing protocol 3
	<ol>
		<li>Using an electrode containing Cs+-based internal solution under voltage-clamp conditions, apply a double PF-stimulus (ISI of 50 ms) and a single depolarizing voltage-step (-70 to 0 mV, 50 ms) to the soma at 1 Hz for 3 min, so that the second PF-stimulus is equivalent to the beginning of the depolarizing voltage step (Figure 1C).</li>
	</ol>
	</li>
	<li>LTD-inducing protocol-4&#8203;
	<ol>
		<li>Using an electrode containing Cs+-based internal solution under voltage-clamp conditions, apply the PF-stimuli (5x at 100 Hz) and a single depolarizing voltage-step (-70 to 0 mV, 50 ms) to the soma at 0.5 Hz for 3 min, simultaneously (Figure 1D).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices-Axon</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Sutter</td>
			<td>BF150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular Devices-Axon</td>
			<td>Digidata1322A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Sutter</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolator</td>
			<td>A.M.P.I.</td>
			<td>ISOflex</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear slicer</td>
			<td>Dosaka EM</td>
			<td>PRO7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>MP1 Single Channel Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water maker</td>
			<td>Merck-Millipore</td>
			<td>MilliQ 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for experiment</td>
			<td>Molecular probe-Axon</td>
			<td>pClamp 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for statistics</td>
			<td>KyensLab</td>
			<td>KyPlot 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>WPI</td>
			<td>DS8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1078</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Yamaguchi, K., et al.&#160; <a href="https://app.jove.com/v/59859">Assessment of Long-term Depression Induction in Adult Cerebellar Slices.</a> J. Vis. Exp. (2019)
In this video, a mouse cerebellar brain slice was used to investigate synaptic interactions between parallel fibers, climbing fibers, and Purkinje cells, which are essential for motor control. Stimulation of climbing fibers induced long-term depression (LTD) in Purkinje cells, weakening synaptic strength and resulting in reduced response to parallel fiber stimulation.

<img alt="Figure 1" src="/files/ftp_upload/23202/23202_Figure1.jpg" /> 
Figure 1: Schematic illustration of protocols to induce LTD in PF-PC synapse. (A) Protocol 1 Cj. 1 PF and 1 CF stimuli are applied simultaneously 300 times at 1 Hz (5 min) under current-clamp conditions. Electrode for whole-cell recording contains K+-based internal solution. (B) Protocol 2 Cj. 2 PF and 1 CF stimuli are applied simultaneously 300 times ...

Source: Yamaguchi, K., et al.&#160; Assessment of Long-term Depression Induction in Adult Cerebellar Slices. J. Vis. Exp. (2019)
In this video, a mouse ...

Begin with a mouse cerebellar brain slice containing parallel fibers, climbing fibers and Purkinje cells, essential for motor control.
The slice is submerged in an artificial cerebrospinal fluid, with a recording electrode attached to a Purkinje cell and stimulating electrodes near the parallel and climbing fibers.
Apply electrical stimuli to the parallel fibers, prompting the release of glutamate, an excitatory neurotransmitter near the Purkinje cell.
The neurotransmitters bind to the glutamate receptors, leading to ion influx that activates the Purkinje cells and generates an electrical signal.
Adjust the stimulus intensity for an optimum response from the Purkinje cells.
Next, deliver repetitive stimulations to the climbing and parallel fibers.
The climbing fiber stimuli activate voltage-gated calcium channels on the Purkinje cell, causing a significant calcium influx.
This triggers the internalization of glutamate receptors, reducing the cell&#39;s sensitivity to glutamate.
This induces long-term depression, characterized by a diminished response to parallel fiber stimulation.

12 ビュー. Induction of Long-Term Depression in Cerebellar Purkinje Neurons in a Mouse Brain Slice

ビデオ: Induction of Long-Term Depression in Cerebellar Purkinje Neurons in a Mouse Brain Slice - 実験

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

電気生理学的フィールド記録によるインターラメラ海馬CA1における長期シナプス可塑性の調査

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

JoVE Journal

神経科学

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

オルガ鼻記小脳スライス培養におけるプルキンジェ細胞生存

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

シナプス可塑性を用いた樹状脊椎の3Dモデリング

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...