Name: assessment of long-term depression induction in adult cerebellar slices
Uploaded: 2019-10-16T13:00:00.000+00:00
Duration: 9 min 30 s
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 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-...

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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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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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

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.

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

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.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Long-Term Engraftment of h-SC-NES Cells in Spinal Cord Organotypic Cultures

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

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Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury