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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 “neuronal machine”1, though there was previously no idea about purpose of this “machine”. Later Marr proposed that the PFs input to PCs constitute a triple layer associative learning network2. He also ....

Protocol

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 (23–25 °C) and humidity (45%–65%) conditions. Both male and female WT mice (C57BL/6, 3–6 months) were used.

1. Preparation of Solutions Used in the Experiments

NOTE: All solutions should be made in ultrapure wa.......

Representative Results

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-.......

Discussion

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,

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
AmplifierMolecular Devices-AxonMulticlamp 700B
Borosilicate glass capillarySutterBF150-110-10
DigitizerMolecular Devices-AxonDigidata1322A
Electrode pullerSutterModel P-97
IsofluraneFUJIFILM Wako Pure Chemical26675-46-7
IsolatorA.M.P.I.ISOflex
Linear slicerDosaka EMPRO7N
MicroscopeNIKONEclipse E600FN
Peristaltic pumpGilsonMP1 Single Channel Pump
PicrotoxinSigma-AldrichP1675
Pure water makerMerck-MilliporeMilliQ 7000
Software for experimentMolecular probe-AxonpClamp 10
Software for statisticsKyensLabKyPlot 5.0
StimulatorWPIDS8000
Temperature controllerWarnerTC-324B
TetrodotoxinTocris1078

References

  1. Eccles, J. C., Ito, M., Szentagothai, J. . The Cerebellum as a Neuronal Machine. , (1967).
  2. Marr, D. A theory of cerebellar cortex. Journal of Physiology. 202 (2), 437-470 (1969).
  3. Albus, J. S.

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