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 water free of metals (resistivity &#62; 18.2 M&#937;) and other impurities (total organic carbon (TOC) &#60; 5.0 ppb). Working artificial cerebrospinal fluid (ACSF) for slice-cutting and recording are made freshly on the day of experiment from a 10 times (x10) stock of ACSF. Bubble the solutions with 5% CO2/ 95% O2 gas mixture before use. The pH of ACSF is adjusted to 7.4 &#177; 0.1, and osmolarity is adjusted 315 &#177; 5 mOsm/kg by adding ultrapure water.
<ol>
	<li>Prepare 10x stock of ACSF containing 1250 mM NaCl, 30 mM KCl, 12.5 mM NaH2PO4, and 260 mM NaHCO3. This solution can be stored at 4 &#176;C.</li>
	<li>Prepare working ACSF containing 125 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM Mg2SO4, 1.25 mM NaH2PO4, 26 mM NaHCO3 and 20 mM glucose.
	<ol>
		<li>First, add 1 mL of 2 M CaCl2 solution followed by 1 mL of 1 M Mg2SO4 solution into around 800 mL of ultrapure water, to avoid precipitation. Then add 100 mL of 10x ACSF and glucose. Finally, make up to a total volume of 1,000 mL by adding ultrapure water.</li>
	</ol>
	</li>
	<li>Prepare 3.3% agar for brain handling. Dissolve 1 g of agar in 30 mL of 0.9% NaCl solution, and heat in a microwave until just boiling. Stir to mix, then pour it into a sterile 4 cm x 10 cm plastic box and allow to solidify. Store the agar plate (~8 mm thickness) in a refrigerator.</li>
	<li>Prepare the internal solution.
	<ol>
		<li>Prepare the K+-based internal solution containing 60 mM KCl, 60 mM K-gluconate, 0.3 mM EGTA, 4 mM MgCl2, 4 mM ATP, 0.4 mM GTP and 30 mM HEPES (pH 7.2). 
		NOTE: Low concentration (0.3 mM) of EGTA, a slow Ca2+-chelator, is added to chelate possibly contaminated Ca2+ in pure water, but this low concentration of EGTA in the internal solution never blocks the induction of LTD (Figure 3, Figure 4, Figure 5) during whole-cell recording. Measured osmotic pressure is 285 mOsm/kg.</li>
		<li>Prepare the Cs+-based internal solution containing 60 mM CsCl, 46 mM D-gluconate, 27 mM tetraethylammonium chloride (TEA-Cl), 0.3 mM EGTA, 4 mM MgCl2, 4 mM ATP, 0.4 mM GTP and 30 mM HEPES (pH 7.2, adjusted using CsOH). 
		NOTE: Cs+ blocks voltage-dependent K-channels, and improves space-clamp conditions at remote dendrites by increasing the length-constant. Measured osmotic pressure is 285 mOsm/kg.</li>
		<li>Prepare 200 &#181;L aliquots of the solutions and store at -30 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Brain Dissection and Trimming
<ol>
	<li>Chill and oxygenate two 50 mL beakers of ACSF on ice until the temperature is lower than 4 &#176;C. Add 50 &#181;L of tetrodotoxin (TTX, 1 mM) into one of the ice-cold beakers of ACSF and reserve it for slices cutting. To obtain mouse cerebellar slice reserving LTD-inducing ability, the addition of TTX to the normal ACSF is necessary.</li>
	<li>Cool down the metal specimen tray by filling an ice-bath area of the slicing chamber with ice.</li>
	<li>Pour 1 mL of isoflurane into an anesthetizing jar (~1000 mL) then place a mouse in it for 30&#8211;45 s. Ensure that the mouse is deeply anesthetized by confirming its inability to respond to mechanical stimulation.</li>
	<li>Decapitate the mouse using surgical scissors. Hold the head and cut the superficial skin along the midline using an ophthalmological scissor. Pull the skin by holding with fingers to widely expose the skull&#8217;s surface.</li>
	<li>Cut the skull horizontally along a line from the major spinocerebellar hole just above the ear and eye using an ophthalmological scissor. Cut the skull along a line above both eyes and remove to isolate the skull.</li>
	<li>Cut the brain at the middle of cerebrum by using a scalpel, then isolate the caudal part of the brain including the cerebellum from the skull. Immerse it into an ice-cold beaker of ACSF. Usually, the total time from decapitation to immersion of the brain block into the pre-chilled beaker of ACSF should be less than 60 s.</li>
	<li>Position of bubbling tubing should be adjusted so as not to stir the brain block in the beaker. Mechanical damage might cause swelling of the slice during recording. Leave it for at least 7 min and allow the brain to cool down.</li>
	<li>To trim the brain block, cut a rectangular agar piece (2 cm x 2 cm) from a large agar plate (4 cm x 10 cm, stored at 4 &#176;C) and put it on a filter paper to absorb the excess liquid.</li>
	<li>Turn the agar piece upside-down on the filter paper, then place the agar piece on a filter paper on a pre-chilled metal specimen tray (16 cm x 20 cm). Pick up the brain block using a spatula and absorb excessive liquid around it with a piece of filter paper.</li>
	<li>Mount the brain block onto the agar block using glue (medical cyanoacrylate instant adhesive). Make sure to attach the bottom (ventral side) of the brain block to the agar.</li>
	<li>Cut out the right hemisphere with a blade. Be sure that the side of the cutting plane is as parallel as possible to the dendritic plane of the PC because this side is attached to the surface of the specimen tray. Cut and remove the other side of the hemisphere. Then, cut the brain between the superior and inferior colliculi, and cut off the spinal cord.</li>
	<li>Glue the right side of trimmed cerebellum with the agar block onto the pre-chilled specimen tray. Spread excess glue around the cerebellum with the flat part of a spatula, to prevent excess glue from attaching to the cerebellar surface. Tilt the metal tray and pour ACSF in order to fix the glue and wash away the excess glue.</li>
</ol>
3. 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, containing 1 &#181;M TTX, sufficient to immerse the cerebellum completely. Place a gas tube into the ACSF and start bubbling with 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 into the oxygenated ACSF. The incubator should be placed in a water-bath that maintains a temperature at 26 &#176;C.</li>
	<li>Store the slices for at least 1 h to allow recovery from the damage during slicing.</li>
</ol>
4. Whole-cell Patch-clamp Recording
NOTE: A patch-clamp recording requires 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 5.3, 5.4).</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>
5. Induction of LTD
<ol>
	<li>Stimulate the molecular layer with a pulse (duration, 0.1 ms). Identify the PF-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
	<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
	<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>

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M., Bean, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+mechanisms+of+burst+firing+in+dissociated+Purkinje+neurons.">Ionic mechanisms of burst firing in dissociated Purkinje neurons.</a> Journal of Neuroscience. 23 (29), 9650-9663 (2003).</li><li>Fukuda, J., Kameyama, M., Yamaguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakdown+of+cytoskeletal+filaments+selectively+reduces+Na+and+Ca+spikes+in+cultured+mammal+neurones.">Breakdown of cytoskeletal filaments selectively reduces Na and Ca spikes in cultured mammal neurones.</a> Nature. 294 (5836), 82-85 (1981).</li><li>Arancillo, M., White, J. J., Lin, T., Stay, T. L., Silltoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+Purkinje+cell+firing+properties+during+postnatal+mouse+development.">In vivo analysis of Purkinje cell firing properties during postnatal mouse development.</a> Journal of Neurophysiology. 113, 578-591 (2015).</li><li>Ishikawa, T., Shimuta, M., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+sensory+integration+in+single+cerebellar+granule+cell+in+vivo.">Multimodal sensory integration in single cerebellar granule cell in vivo.</a> eLife. 4, e12916 (2015).</li><li>Tempia, F., Minlaci, M. C., Anchisi, D., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+current+mediated+by+metabotropic+glutamate+receptors+in+cerebellar+Purkinje+cells.">Postsynaptic current mediated by metabotropic glutamate receptors in cerebellar Purkinje cells.</a> Journal of Neurophysiology. 80, 520-528 (1998).</li><li>Wang, S. S., Denk, W., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coincidence+detection+in+single+dendritic+spines+mediated+by+calcium+release.">Coincidence detection in single dendritic spines mediated by calcium release.</a> Nature Neuroscience. 3, 1266-1273 (2000).</li><li>Kuroda, S., Schweighofer, N., Kawato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+signal+transduction+pathways+in+cerebellar+long-term+depression+by+kinetic+simulation.">Exploration of signal transduction pathways in cerebellar long-term depression by kinetic simulation.</a> Journal of Neuroscience. 21 (15), 5693-5702 (2001).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+cerebellar+engrams+in+short-term+and+long-term+motor+learning.">Distinct cerebellar engrams in short-term and long-term motor learning.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (1), E188-E193 (2014).</li><li>Inoshita, T., Hirano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+long-term+depression+in+the+cerebellar+flocculus+during+adaptation+of+optokinetic+response.">Occurrence of long-term depression in the cerebellar flocculus during adaptation of optokinetic response.</a> eLife. 27, 36209 (2018).</li><li>Belmeguenai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+plasticity+complements+long-term+potentiation+in+parallel+fiber+input+gain+control+in+cerebellar+Purkinje+cells.">Intrinsic plasticity complements long-term potentiation in parallel fiber input gain control in cerebellar Purkinje cells.</a> Journal of Neuroscience. 30 (41), 13630-13643 (2010).</li><li>Ohtsuki, G., Piochon, C., Adelman, J. P., Hansel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SK2+channel+modulation+contributes+to+compartment+specific+dendritic+plasticity+in+cerebellar+Purkinje+cells.">SK2 channel modulation contributes to compartment specific dendritic plasticity in cerebellar Purkinje cells.</a> Neuron. 75, 108-120 (2012).</li></ol>

The authors have nothing to disclose.

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 ...

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Neuroscience

6.9K Views.  RIKEN. 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.

Video: Assessment of Long-term Depression Induction in Adult Cerebellar Slices

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.