Name: assessment of long-term depression induction in adult cerebellar slices
Uploaded: 2019-10-16T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Long-term Depression Induction in Adult Cerebellar Slices at JoVE.com

We thank A. Oba for her technical assistance. This research was partially supported by Grant-in-Aid for Scientific Research (C) 17K01982 to K.Y.

All experimental procedures were approved by the RIKEN committee on the care and use of animals in experiments. Mice were kept in the animal facility of the RIKEN Center for Brain Science under well-controlled temperature (23&#8211;25 &#176;C) and humidity (45%&#8211;65%) conditions. Both male and female WT mice (C57BL/6, 3&#8211;6 months) were used.
1. Preparation of Solutions Used in the Experiments
NOTE: All solutions should be made in ultrapure wa...

<ol>
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Differences among the four protocols
In LTD-inducing protocols 1 and 2, Cjs 300 times at 1 Hz is sufficient to induce cerebellar LTD. Stimulation frequency of the CF seemed to be in a physiological range, because the complex spike firing rate in alert adult mice (P60) was reported to be 1.25 Hz36. However, the CF stimulation alone did not cause long-term plasticity in the PF-CF synapse, as used in protocols 1 and 2 (Figure 4, <strong class="x...

The synaptic organization of the elaborated neuronal networks of the cerebellar cortex, composed of PCs, molecular layer interneurons (basket and stellate cells), Golgi cells, PFs from granule cells, mossy fibers and climbing fibers (CFs), have been elucidated in terms of excitation/inhibition and divergence/convergence, and the well-organized circuitry diagram has suggested that the cerebellum is a &#8220;neuronal machine&#8221;1, though there was previously no idea about purpose of this &#8220;machine&#8221;. Later Marr proposed that the PFs input to PCs constitute a triple layer associative learning network2. He also suggested that each CF conveys a cerebral instruction for elemental movement2. He assumed that simultaneous activation of PFs and CF would enhance PF-PC synapse activity, and cause long-term potentiation (LTP) of the PF-PC synapse. On the other hand, Albus assumed that synchronous activation of PFs and CF resulted in LTD at the PF-PC synapses3. Both the above studies interpret the cerebellum as a unique memory device, the incorporation of which into the cerebellar cortical network leads to the formation of the Marr&#8211;Albus model learning machine model.
Following these theoretical predictions, two lines of evidence suggest the presence of synaptic plasticity in the cerebellum. The first line of evidence was suggested by the anatomical organization of the flocculus; here MF pathways of vestibular organ origin and CF pathways of retinal origin converge on the PCs4. This unique convergence pattern suggests that a synaptic plasticity occurring in the flocculus causes the remarkable adaptability of the vestibulo-ocular reflex. Second, the recording of the PCs response in the flocculus and the lesioning of the flocculus also supported the above hypothesis5,6,7. Furthermore, the PC discharge pattern during adaptation of a monkey&#8217;s hand movement8 supported the synaptic plasticity hypothesis, especially Albus&#8217;s LTD-hypothesis3.
To determine the nature of the synaptic plasticity directly, repeated conjunctive stimulation (Cjs) of a bundle of PFs and the CF that specifically innervates the PC in vivo was shown to induce LTD for the transmission efficacy of the PF&#8211;PC synapses9,10,11. In the subsequent in vitro explorations using a cerebellar slice12 and cultured PCs, conjunction of co-cultured granule cell stimulation and olive cell stimulation13 or conjunction of iontophoretically applied glutamate and somatic depolarization14,15 caused LTD. The signal transduction mechanism underlying the LTD-induction was also intensively investigated using in vitro preparations16,17.
Adaptations of the VOR and the OKR were often used for quantitative evaluation of gene-manipulation effects on cerebellar motor learning, because the vestibule-cerebellar cortex was proven to be the essential origin in the adaptive learning of the VOR18,19,20 and the OKR19,21 The correlation between failure of LTD-induction and impairment of behavioral motor learning has been taken as evidence that LTD plays an essential role in motor learning mechanisms22. These views are collectively referred to as the LTD hypothesis of motor learning, or Marr-Albus-Ito hypothesis23,24,25,26.
Adaptive learning of eye movement was measured using similar protocols, while various experimental conditions were used to induce LTD in slice preparation27,28,29,30,31. Recently, Schonewille et al.26 reported that some gene-manipulated mice demonstrated normal motor learning, but the cerebellar slices did not show LTD, and thereby concluded that LTD was not essential for motor learning. However, the induction of LTD was only attempted using one type of protocol at room temperature. Hence, we used several types of LTD-inducing protocols under recording conditions at around 30 &#176;C, and we confirmed that the LTD was reliably induced in the gene-manipulated mice by using these protocols at near physiological temperatures32.
However, there remain some questions regarding the basic properties of conjunctive stimulation. The first is the relationship between the complex spike&#8217;s shape and the amplitude of LTD. Second, in conjunction with PF-stimulation and somatic depolarization, whether the number of stimuli used were necessary or not was elusive. In the present study, these questions were investigated using wild type (WT) mice.

<table>
	<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>

assessment of long-term depression induction in adult cerebellar slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

Four protocols were used in this study to induce cerebellar LTD. In the first two protocols (protocol 1 and 2), the conjunction of the PF-stimulation and the CF-stimulation was applied under current-clamp conditions. In the other two protocols (protocol 3 and 4), somatic depolarization was substituted for the CF-stimulation under voltage-clamp conditions. Voltage-traces or current-traces during conjunctive stimulation were compared (Figure 2).
Conjunction of 1 PF-...

Watch this Scientific Journal Video about Assessment of Long-term Depression Induction in Adult Cerebellar Slices at JoVE.com

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

When a particular protein function is modified by gene manipulation, coordination of irritative failure and motor running impairment is considered a necessary condition for causal relationship between LTD and motor learning. Here we demonstrate the use of multiple protocols to assess LTD which can be induced via compensatory mechanisms in gene-manipulated animals. Before harvesting the brain, chill and oxygenate two 50 milliliter beakers of ACSF on ice.When the solution temperature reaches below four degrees celsius, add 50 microliters of one millimolar tetrodotoxin to one of the ice cold beakers. To harvest the brain, hold the head and use ophthalmological scissors to cut the superficial skin along the midline. Manually retract the skin to widely expose the skull surface and use the scissors to cut along the skull horizontally from the major spinocerebellar hole to just above the eyes and ears before cutting the skull along a line above both eyes.Use a scalpel to cut the brain at the middle of the cerebrum and isolate the caudal part of the brain, including the cerebellum, from the skull. Immerse the sample in the ice cold beaker of ACSF and adjust the bubble tubing so that it does not stir the brain block in the beaker. After at least seven minutes, use a spatula to pick up the brain block and use a piece of filter paper to absorb any excess ACSF.Mount the tissue ventral side down onto a two by two centimeter piece of agar with an appropriate medical adhesive. Use a blade to cut out the right hemisphere of the brain tissue as parallel to the dendritic plane of the Purkinje cells as possible. Cut and remove the other side of the hemisphere and cut the brain between the superior and inferior colliculi.Cut off the spinal cord and glue the right side of the trimmed cerebellum with the agar block onto the pre-chilled specimen tray. Then, tilting the specimen tray, pour ACSF onto the sample to fix the tissue and to wash away any excess glue. To slice the brain sample, orient the specimen such that the dorsal side of the cerebellum is at the front and pour enough of the ice cold cutting ACSF supplemented with tetrodotoxin to completely immerse the cerebellum.Place a gas tube into the cutting solution and initiate bubbling with an oxygen-carbon dioxide gas mixture. Using fine tweezers and magnifiers, remove the arachnoid mater and excise the cerebellar peduncle. After removing the brainstem and agar block, rotate the tray 180 degrees so that the dorsal surface of the cerebellum faces the razor blade of the vibratome and adjust the first cutting location.Set the vibratome amplitude to 5.5 and frequency to 85 Hertz, the speed to three to four, and the slice thickness to 300 micrometers. As the cerebellar slices are obtained, use a nylon net to transfer the sections to an acrylic incubator in a 26 degree celsius water bath and completely immerse the samples in fresh oxygenated ACSF for at least one hour. For whole cell patch clamp reporting, dissolve one micromolar picrotoxin in ACSF by ultasonication for three minutes before perfusing a 30 degree celsius recording chamber with the toxin solution at a two milliliters per minute flow rate.After a few minutes, transfer a cerebellar slice to the recording chamber and fix the tissue with a platinum weight and nylon threads. Then fill a stimulating electrode with fresh ACSF. To stimulate the parallel fibers, place the stimulating electrode on the surface of the molecular layer about 50 micrometers from the Purkinje cell layer.For stimulation of the climbing fibers, place the stimulating electrode at the bottom of the Purkinje cell layer and use a microloader to fill a recording electrode with eight microliters of 0.45 micrometer filtered potassium or cesium based internal solution. Apply a weak positive pressure to the recording electrode before immersing the electrode into the ACSF. The electrode resistance should be two to four megaohms and the liquid junctional potential should be corrected.Approach the healthy bright cell body of a Purkinje cell with the recording electrode and push the surface of the Purkinje cell slightly. Then stop applying the positive pressure and apply negative pressure until a gigaohm seal is formed. Then use negative pressure to establish a whole cell configuration, maintaining the membrane potential at minus 70 millivolts and applying a minus two millivolt 100 millisecond pulses at 0.1 hertz, continuous monitoring of the input resistance, series resistance, and input capacitance.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 post-synaptic 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 post-synaptic 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. In this representative experiment, a conjunction of one parallel fiber stimulation and one climbing fiber stimulation under current clamp conditions were used for the slice preparation.The shape of the complex spike elicited by the conjunctive stimulation was similar to that elicited by the climbing fiber stimulation alone with the first steep spikelet followed by two to three spikelets. A similarly shaped complex spike was observed when one parallel fiber stimulation was followed 50 milliseconds later by a conjunctive second parallel and climbing fiber stimulation. In this test under voltage clamp conditions using a cesium-based internal solution, a parallel fiber stimulation was followed 50 milliseconds later by a concomitant application of a second parallel fiber stimulation and somatic depolarization.An inward current was elicited upon somatic depolarization from minus 70 to zero millivolts and the tail current was also evoked after repolarization. Finally, five parallel fiber stimuli at 100 hertz were given simultaneously with the somatic depolarization under voltage clamp conditions. Again, a repetitive generation of inward currents was elicited during depolarization and the tail current was induced after the repolarization.To assess the relationship between cerebellar LTD and motor learning in gene-manipulated animals, multiple protocols should be used to induce LTD under cozy physiological conditions. If cerebellar LTD is visualized in gene-manipulated animals after motor learning, the causal relationship between them can be more directly examined.

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