Brain Structure and Function

, Volume 223, Issue 6, pp 2575–2587 | Cite as

Developmental abnormality contributes to cortex-dependent motor impairments and higher intracortical current requirement in the reeler homozygous mutants

  • Mariko Nishibe
  • Yu Katsuyama
  • Toshihide Yamashita
Original Article


The motor deficit of the reeler mutants has largely been considered cerebellum related, and the developmental consequences of the cortex on reeler motor behavior have not been examined. We herein showed that there is a behavioral consequence to reeler mutation in models examined at cortex-dependent bimanual tasks that require forepaw dexterity. Using intracortical microstimulation, we found the forelimb representation in the motor cortex was significantly reduced in the reeler. The reeler cortex required a significantly higher current to evoke skeletal muscle movements, suggesting the cortical trans-synaptic propagation is disrupted. When the higher current was applied, the reeler motor representation was found preserved. To elucidate the influence of cerebellum atrophy and ataxia on the obtained results, the behavioral and neurophysiological findings in reeler mice were reproduced using the Disabled-1 (Dab1) cKO mice, in which the Reelin-Dab1 signal deficiency is confined to the cerebral cortex. The Dab1 cKO mice were further assessed at the single-pellet reach and retrieval task, displaying a lower number of successfully retrieved pellets. It suggests the abnormality confined to the cortex still reduced the dexterous motor performance. Although possible muscular dysfunction was reported in REELIN-deficient humans, the function of the reeler forelimb muscle examined by electromyography, morphology of neuromuscular junction and the expression level of choline acetyltransferase were normal. Our results suggest that the mammalian laminar structure is necessary for the forepaw skill performance and for trans-synaptic efficacy in the cortical output.


Reeler Cortex Motor deficits Development Skilled reach 



This work was supported by the Japan Society for the Promotion of Science Kakenhi Grant-in Aid for Scientific Research B Grant number T1SK199660 to M.N., and a Grant-in-Aid for Scientific Research S from the Japan Society for the Promotion of Sciences 17H06178 to T.Y.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the Care and Use of Laboratory Animals of Osaka University Graduate School of Medicine.

Informed consent

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

429_2018_1647_MOESM1_ESM.tif (2.6 mb)
Online Source 1. Morphology of the cortex A. Coronally cut motor cortical sections (acquired from posterior 1.5mm to bregma), each of WT and reeler, were stained with Cresyl Violet. B. WT and reeler sections were stained with antibodies against Neurofilament (Alexa-568). C. Similarly, coronally cut motor cortical sections of Dab1 control and Dab1 cKO were stained with Cresyl Violet. D. Motor cortical sections of Dab1 control and Dab1 cKO were stained with antibodies against Neurofilament (Alexa-488). Note that the cortical layers are disrupted in reeler and Dab1 cKO. Scale bar= 200 µm (TIF 2664 KB)
429_2018_1647_MOESM2_ESM.tif (2.3 mb)
Online Source 2. Morphology of the cerebellum. Coronally cut cerebellum sections each (posterior ~5.8mm to bregma) of WT, reeler, Dab1 control and Dab1 cKO were stained with Cresyl Violet. While reeler mice show ectopic, no laminated structure (upper right), the cerebellum of Dab1 cKO mice (lower right) contains a typical cytoarchitecture of cerebellum layers indistinguishable from the layers observed in the control (lower left). Scale bar= 1 mm (TIF 2318 KB)
429_2018_1647_MOESM3_ESM.tif (2 mb)
Online Source 3 Ketamine volume. Ketamine/xylazine used volume during intracortical microstimulation mapping, per body weight, per operation hour (±SEM). The ketamine use was ensured consistent throughout the experiments for both groups. A. in reeler ICMS experiments (quantified from n=5) and B. Dab1 ICMS experiments (quantified from n=3). (TIF 2032 KB)
429_2018_1647_MOESM4_ESM.tif (3.7 mb)
Online Source 4 Neurophysiological results in Dab1 cKO A. Color-coded bilateral maps of movements evoked by ICMS at the current threshold of 90 µA in Dab1 control (Left), of 90 µA in Dab1 cKO (Middle), and of 300 µA in Dab1 cKO (Right). Each representative case is illustrated on a dorsolateral view of the brain. All forelimb maps were bordered either by face, whisker or trunk, tail, hindlimb movements, or non-responsive sites. Dots reflect the ICMS penetration sites, each 500 µm2 apart. The numbers in mm2 are the total forelimb area (±SEM, illustrated in blue, data from n=3). B. Movement representation areas of Dab1 cKO and control (±SEM, quantified from n=3). The forelimb motor representation area was significantly reduced in Dab1 cKO bilaterally (right cortical representation *p=0.002 and left cortical representations *p=0.002). The control and cKO forelimb representation area (±SEM), mapped at 90 µA and 300 µA, respectively, were found not significantly different (right cortical representation p=0.406 and left cortical representation p=0.223). (TIF 3837 KB)
429_2018_1647_MOESM5_ESM.tif (2.6 mb)
Online Source 5 Reeler and Dab1 cKO neuromuscular junction. A. The photographs show the neuromuscular junctions of an extensor muscle in reeler and WT, demonstrating no obvious deviation from one to the other. (Scale bar of the most left photograph =200 µm, scale bar of the three right photographs =50 µm) B. The photographs show the neuromuscular junctions of an extensor muscle in Dab1 cKO and Dab1 control, demonstrating no obvious deviation from one to the other. (Scale bar of the most left photograph =200 µm, scale bar of the three right photographs =50 µm) C. The bands were found at 70 kDa and 40 kDa, respectively for choline acetyltransferase and α-actin. The bar graph indicates the ratio of choline acetyltransferase expression per α-actin loading control. The samples consisted of homogenized extensor digitorum communis and carpi ulnaris of reeler mice and WT mice. The band quantification did not result in a significant difference between reeler and WT (p=0.827, quantified from n=3). D. The graph shows the comparison of the extensor digitorum weight per body weight (±SEM, p=0.851), comparing reeler, WT (quantified from n=7), Dab1 cKO, and Dab1 control (quantified from n=4). (TIF 2670 KB)

Online Source 6 Reeler video of somen stick handling task. The video shows a WT mouse and reeler mutant mouse performing the somen stick handling task. In each trial, a mouse ate an uncooked Japanese thin noodle (somen≤1 mm in diameter, cut in 2.8 cm length) in a Plexiglas test chamber or a home-cage environment. The forepaw adjustments were defined as a visible release and re-grasp on the stick, and extension-flexion, abduction-adduction movements of the digits. (MP4 37496 KB)

Online Source 7 Reeler video of sunflower seed handling task. The video shows a WT mouse and reeler mutant mouse performing the sunflower seed handling task. Time counting started from the pick-up of the sunflower seed (0.06-0.08 g) followed by the seed contact with the mouth and ended with letting go of the seed after eating was complete. The time counting was halted when the seed was dropped or the pick-up that was not followed by the mouth contact. Tests were done either in the home-cage environment or in a Plexiglas test chamber. (MP4 52759 KB)

Online Source 8 Dab1 cKO video of somen stick handling task. The video shows a Dab1 control mouse and Dab1 cKO mouse performing the somen stick (somen≤1 mm in diameter, cut in 2.8 cm length) handling task. Notice that Dab1 cKO required a longer time to consume the stick. (MP4 40184 KB)

Online Source 9 Dab1 cKO video of sunflower seed handling task. The video shows a Dab1 control mouse and Dab1 cKO mouse performing the sunflower seed (0.06-0.08 g) handling task. Notice that Dab1 cKO required a longer time to consume the seed. (MP4 130326 KB)


  1. Alaverdashvili M, Whishaw IQ (2008) Motor cortex stroke impairs individual digit movement in skilled reaching by the rat. Eur J Neurosci 28(2):311–322. CrossRefPubMedGoogle Scholar
  2. Alcantara S, Ruiz M, D’Arcangelo G, Ezan F, de Lecea L, Curran T, Sotelo C, Soriano E (1998) Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse. J Neurosci 18(19):7779–7799CrossRefPubMedGoogle Scholar
  3. Allred RP, Adkins DL, Woodlee MT, Husbands LC, Maldonado MA, Kane JR, Schallert T, Jones TA (2008) The vermicelli handling test: a simple quantitative measure of dexterous forepaw function in rats. J Neurosci Methods 170(2):229–244CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bidoia C, Misgeld T, Weinzierl E, Buffelli M, Feng G, Cangiano A, Lichtman JW, Sanes JR (2004) Comment on “Reelin promotes peripheral synapse elimination and maturation”. Science 303(5666):1977. (author reply 1977) CrossRefPubMedGoogle Scholar
  5. Blotnick E, Anglister L (2016) Exercise modulates synaptic acetylcholinesterase at neuromuscular junctions. Neuroscience 319:221–232. CrossRefPubMedGoogle Scholar
  6. Bock HH, Herz J (2003) Reelin activates SRC family tyrosine kinases in neurons. Curr Biol 13(1):18–26CrossRefPubMedGoogle Scholar
  7. Caviness VS Jr, Rakic P (1978) Mechanisms of cortical development: a view from mutations in mice. Annu Rev Neurosci 1:297–326. CrossRefPubMedGoogle Scholar
  8. Chagnac-Amitai Y, Luhmann HJ, Prince DA (1990) Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features. J Comp Neurol 296(4):598–613. CrossRefPubMedGoogle Scholar
  9. Cheney PD (1985) Role of cerebral cortex in voluntary movements. A review. Phys Ther 65(5):624–635CrossRefPubMedGoogle Scholar
  10. Cheney PD, Fetz EE (1985) Comparable patterns of muscle facilitation evoked by individual corticomotoneuronal (CM) cells and by single intracortical microstimuli in primates: evidence for functional groups of CM cells. J neurophysiol 53(3):786–804CrossRefPubMedGoogle Scholar
  11. D’Arcangelo G, Miao GG, Chen SC, Soares HD, Morgan JI, Curran T (1995) A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374(6524):719–723. CrossRefPubMedGoogle Scholar
  12. Dekimoto H, Terashima T, Katsuyama Y (2010) Dispersion of the neurons expressing layer specific markers in the reeler brain. Dev Growth Differ 52(2):181–193. CrossRefPubMedGoogle Scholar
  13. Elston GN (2003) Cortex, cognition and the cell: new insights into the pyramidal neuron and prefrontal function. Cerebral cortex (New York, NY: 1991) 13(11):1124–1138Google Scholar
  14. Falconer DS (1951) Two new mutants, ‘trembler’ and ‘reeler’, with neurological actions in the house mouse (Mus musculus L.). J Genet 50(2):192–201CrossRefPubMedGoogle Scholar
  15. Frotscher M, Chai X, Bock HH, Haas CA, Forster E, Zhao S (2009) Role of Reelin in the development and maintenance of cortical lamination. J Neural Transm (Vienna, Austria: 1996) 116(11):1451–1455. CrossRefGoogle Scholar
  16. Geed S, McCurdy ML, van Kan PLE (2017) Neuronal correlates of functional coupling between reach- and grasp-related components of muscle activity. Front neural circ. CrossRefGoogle Scholar
  17. Goffinet AM (1984) Events governing organization of postmigratory neurons: studies on brain development in normal and reeler mice. Brain Res 319(3):261–296CrossRefPubMedGoogle Scholar
  18. Gomez CS-M, Ventura-Martinez J, Rodriguez RR (2006) The sunflower seed test: a simple procedure to evaluate forelimb motor dysfunction after brain ischemia. Drug Dev Res 67Google Scholar
  19. Gupta RC, Misulis KE, Dettbarn WD (1985) Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse. Exp Neurol 89(3):622–633CrossRefPubMedGoogle Scholar
  20. Guy J, Wagener RJ, Mock M, Staiger JF (2015) Persistence of functional sensory maps in the absence of cortical layers in the somsatosensory cortex of reeler mice. Cereb Cortex 25(9):2517–2528. CrossRefPubMedGoogle Scholar
  21. Guy J, Sachkova A, Mock M, Witte M, Wagener RJ, Staiger JF (2016) Intracortical network effects preserve thalamocortical input efficacy in a cortex without layers. Cereb Cort (New York, NY: 1991). CrossRefGoogle Scholar
  22. Harsan LA, David C, Reisert M, Schnell S, Hennig J, von Elverfeldt D, Staiger JF (2013) Mapping remodeling of thalamocortical projections in the living reeler mouse brain by diffusion tractography. Proc Natl Acad Sci USA 110(19):E1797–E1806. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hong SE, Shugart YY, Huang DT, Shahwan SA, Grant PE, Hourihane JO, Martin ND, Walsh CA (2000) Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nat Genet 26(1):93–96. CrossRefPubMedGoogle Scholar
  24. Hourihane JO, Bennett CP, Chaudhuri R, Robb SA, Martin ND (1993) A sibship with a neuronal migration defect, cerebellar hypoplasia and congenital lymphedema. Neuropediatrics 24(1):43–46. CrossRefPubMedGoogle Scholar
  25. Hussin AT, Boychuk JA, Brown AR, Pittman QJ, Teskey GC (2015) Intracortical microstimulation (ICMS) activates motor cortex layer 5 pyramidal neurons mainly transsynaptically. Brain Stimul 8(4):742–750. CrossRefPubMedGoogle Scholar
  26. Ikeda Y, Terashima T (1997) Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse. Dev Dyn 210(2):157–172.<157::aid-aja8>;2-fCrossRefPubMedGoogle Scholar
  27. Imai H, Shoji H, Ogata M, Kagawa Y, Owada Y, Miyakawa T, Sakimura K, Terashima T, Katsuyama Y (2016) Dorsal forebrain-specific deficiency of Reelin-Dab1 signal causes behavioral abnormalities related to psychiatric disorders. Cereb Cortex (New York, NY: 1991). CrossRefGoogle Scholar
  28. Iwaniuk AN, Whishaw IQ (2000) On the origin of skilled forelimb movements. Trends Neurosci 23(8):372–376CrossRefPubMedGoogle Scholar
  29. Iwasato T, Nomura R, Ando R, Ikeda T, Tanaka M, Itohara S (2004) Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice. Genesis 38(3):130–138. CrossRefPubMedGoogle Scholar
  30. Jacquelin C, Strazielle C, Lalonde R (2012) Spontaneous alternation and spatial learning in Dab1scm (scrambler) mutant mice. Brain Res Bull 87(4–5):383–386. CrossRefPubMedGoogle Scholar
  31. Jankowska E, Padel Y, Tanaka R (1975) Projections of pyramidal tract cells to alpha-motoneurones innervating hind-limb muscles in the monkey. J Physiol 249(3):637–667CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kanagal SG, Muir GD (2009) Task-dependent compensation after pyramidal tract and dorsolateral spinal lesions in rats. Exp Neurol 216(1):193–206. CrossRefPubMedGoogle Scholar
  33. Lalonde R, Strazielle C (2001) Motor performance and regional brain metabolism of spontaneous murine mutations with cerebellar atrophy. Behav Brain Res 125(1–2):103–108CrossRefPubMedGoogle Scholar
  34. Lalonde R, Strazielle C (2003) Motor coordination, exploration, and spatial learning in a natural mouse mutation (nervous) with Purkinje cell degeneration. Behav Genet 33(1):59–66CrossRefPubMedGoogle Scholar
  35. Lalonde R, Hayzoun K, Derer M, Mariani J, Strazielle C (2004) Neurobehavioral evaluation of Reln-rl-orl mutant mice and correlations with cytochrome oxidase activity. Neurosci Res 49(3):297–305. CrossRefPubMedGoogle Scholar
  36. Liang F, Rouiller EM, Wiesendanger M (1993) Modulation of sustained electromyographic activity by single intracortical microstimuli: comparison of two forelimb motor cortical areas of the rat. Somatosens Motor Res 10(1):51–61CrossRefGoogle Scholar
  37. Lucas TH, Fetz EE (2013) Myo-cortical crossed feedback reorganizes primate motor cortex output. J Neurosci 33(12):5261–5274. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Morris R, Whishaw IQ (2016) A proposal for a rat model of spinal cord injury featuring the rubrospinal tract and its contributions to locomotion and skilled hand movement. Front Neurosci 10:5. PubMedPubMedCentralCrossRefGoogle Scholar
  39. Namikawa T, Kikkawa S, Inokuchi G, Terashima T (2015) Postnatal development of the corticospinal tract in the Reeler mouse. Kobe J Med Sci 61(3):E71–E81PubMedGoogle Scholar
  40. Nishibe M, Urban ET 3rd, Barbay S, Nudo RJ (2015) Rehabilitative training promotes rapid motor recovery but delayed motor map reorganization in a rat cortical ischemic infarct model. Neurorehabil Neural Repair 29(5):472–482. CrossRefPubMedGoogle Scholar
  41. Nitsche MA, Paulus W (2000) Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J Physiol 527 Pt 3:633–639CrossRefPubMedGoogle Scholar
  42. Ogawa M, Miyata T, Nakajima K, Yagyu K, Seike M, Ikenaka K, Yamamoto H, Mikoshiba K (1995) The reeler gene-associated antigen on Cajal–Retzius neurons is a crucial molecule for laminar organization of cortical neurons. Neuron 14(5):899–912CrossRefPubMedGoogle Scholar
  43. Phelps PE, Rich R, Dupuy-Davies S, Rios Y, Wong T (2002) Evidence for a cell-specific action of Reelin in the spinal cord. Dev Biol 244(1):180–198. CrossRefPubMedGoogle Scholar
  44. Quattrocchi CC, Huang C, Niu S, Sheldon M, Benhayon D, Cartwright J Jr, Mosier DR, Keller F, D’Arcangelo G (2004) Retraction. Science (New York, NY) 303(5666):1974. CrossRefGoogle Scholar
  45. Ramsay DA, Drachman DB, Drachman RJ, Stanley EF (1992) Stabilization of acetylcholine receptors at the neuromuscular synapse: the role of the nerve. Brain Res 581(2):198–207CrossRefPubMedGoogle Scholar
  46. Silva LR, Gutnick MJ, Connors BW (1991) Laminar distribution of neuronal membrane properties in neocortex of normal and reeler mouse. J Neurophysiol 66(6):2034–2040CrossRefPubMedGoogle Scholar
  47. Sleigh JN, Burgess RW, Gillingwater TH, Cader MZ (2014) Morphological analysis of neuromuscular junction development and degeneration in rodent lumbrical muscles. J Neurosci Methods 227:159–165. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Strazielle C, Lefevre A, Jacquelin C, Lalonde R (2012) Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice. Behav Brain Res 233(1):24–28. CrossRefPubMedGoogle Scholar
  49. Tennant KA, Asay AL, Allred RP, Ozburn AR, Kleim JA, Jones TA (2010) The vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice. J Vis Exp. PubMedPubMedCentralCrossRefGoogle Scholar
  50. Terashima T (1995a) Anatomy, development and lesion-induced plasticity of rodent corticospinal tract. Neurosci Res 22(2):139–161CrossRefPubMedGoogle Scholar
  51. Terashima T (1995b) Course and collaterals of corticospinal fibers arising from the sensorimotor cortex of the reeler mouse. Dev Neurosci 17(1):8–19CrossRefPubMedGoogle Scholar
  52. Terashima T, Inoue K, Inoue Y, Mikoshiba K, Tsukada Y (1983) Distribution and morphology of corticospinal tract neurons in reeler mouse cortex by the retrograde HRP method. J Comp Neurol 218(3):314–326. CrossRefPubMedGoogle Scholar
  53. Terashima T, Takayama C, Ichikawa R, Inoue Y (1992) Dendritic arbolization of large pyramidal neurons in the motor cortex of normal and reeler mutant mouse. Okajimas Folia Anat Jpn 68(6):351–363CrossRefPubMedGoogle Scholar
  54. Trotter J, Lee GH, Kazdoba TM, Crowell B, Domogauer J, Mahoney HM, Franco SJ, Muller U, Weeber EJ, D'Arcangelo G (2013) Dab1 Is Required for Synaptic Plasticity and Associative Learning. J Neurosci 33(39):15652–15668. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Viaro R, Bonazzi L, Maggiolini E, Franchi G (2017) Cerebellar modulation of cortically evoked complex movements in rats. Cereb Cortex (New York, NY: 1991) 27(7):3525–3541. CrossRefGoogle Scholar
  56. Voigt MB, Hubka P, Kral A (2017) Intracortical microstimulation differentially activates cortical layers based on stimulation depth. Brain Stimul 10(3):684–694. CrossRefPubMedGoogle Scholar
  57. Whishaw IQ, Coles BL (1996) Varieties of paw and digit movement during spontaneous food handling in rats: postures, bimanual coordination, preferences, and the effect of forelimb cortex lesions. Behav Brain Res 77(1–2):135–148CrossRefPubMedGoogle Scholar
  58. Whishaw IQ, Pellis SM, Gorny B, Kolb B, Tetzlaff W (1993) Proximal and distal impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions. Behav Brain Res 56(1):59–76CrossRefPubMedGoogle Scholar
  59. Whishaw IQ, Piecharka DM, Drever FR (2003) Complete and partial lesions of the pyramidal tract in the rat affect qualitative measures of skilled movements: impairment in fixations as a model for clumsy behavior. Neural Plast 10(1–2):77–92. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Whishaw IQ, Piecharka DM, Zeeb F, Stein DG (2004) Unilateral frontal lobe contusion and forelimb function: chronic quantitative and qualitative impairments in reflexive and skilled forelimb movements in rats. J Neurotrauma 21(11):1584–1600. CrossRefPubMedGoogle Scholar
  61. Yamamoto T, Sakakibara S, Mikoshiba K, Terashima T (2003) Ectopic corticospinal tract and corticothalamic tract neurons in the cerebral cortex of yotari and reeler mice. J Comp Neurol 461(1):61–75. CrossRefPubMedGoogle Scholar
  62. Yamamoto T, Setsu T, Okuyama-Yamamoto A, Terashima T (2009) Histological study in the brain of the reelin/Dab1-compound mutant mouse. Anat Sci Int 84(3):200–209. CrossRefPubMedGoogle Scholar
  63. Yazdan-Shahmorad A, Lehmkuhle MJ, Gage GJ, Marzullo TC, Parikh H, Miriani RM, Kipke DR (2011) Estimation of electrode location in a rat motor cortex by laminar analysis of electrophysiology and intracortical electrical stimulation. J Neural Eng 8(4):046018. CrossRefPubMedGoogle Scholar
  64. Young NA, Vuong J, Flynn C, Teskey GC (2011) Optimal parameters for microstimulation derived forelimb movement thresholds and motor maps in rats and mice. J Neurosci Methods 196(1):60–69. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular Neuroscience, Graduate School of MedicineOsaka UniversitySuitaJapan
  2. 2.The Institute of Academic InitiativesOsaka UniversitySuitaJapan
  3. 3.WPI Immunology Frontier Research CenterOsaka UniversitySuitaJapan
  4. 4.Department of AnatomyShiga University of Medical ScienceOtsuJapan
  5. 5.Department of Frontier ScienceOsaka UniversitySuitaJapan

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