Loss of Pre-Inspiratory Neuron Synchroneity in Mice with DSCAM Deficiency

  • Kenji Amano
  • Morimitsu Fujii
  • Satoru Arata
  • Masaharu Ogawa
  • Kazuhiro Yamakawa
  • Akiko Arata
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 669)


Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam -/-) died within 24 hours after birth. Whole body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Further, a medulla-spinal cord preparation of Dscam -/- mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory (Pre-I) neurons located in the rostral ventrolateral medulla (RVLM) and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam -/- mice. Dscam +/− mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam -/- mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner. These results have been published (Amano et al. 2009).


Down Syndrome Facial Nerve Activity Facial Nucleus Rostral Ventrolateral Medulla Ventral Medulla 

1 Neonatal Death of Dscam-Deficient Mouse

Cell adhesion molecules of the neural Ig superfamily mediate a variety of cell-cell interactions that are important for neural development and function. We previously identified the DSCAM gene on human chromosome 21q22 and proposed it as an intriguing candidate gene for the etiology of Down syndrome (Yamakawa et al. 1998). To explore the physiological functions of DSCAM protein, we generated Dscam-deficient mice (Amano et al. 2009). The heterozygous knockout (Dscam +/−) mice were fertile and developed almost normally. Matings of heterozygotes (Dscam +/−) yielded homozygotes (Dscam -/-). Populations of pups for each genotype were in accordance with the expected Mendelian distribution. This indicates that deficiency of DSCAM does not lead to embryonic lethality. However, a majority of the null mutant mice (Dscam -/-) died within 24 hours after birth.

2 Dscam-/- Mice Show Irregular Central Respiratory Activities

Dscam -/- pups occasionally showed jerky or dyspnea-like movements. Whole body plethysmography showed irregular respiratory rhythm and frequent apneic episodes in Dscam -/- pups, whereas wild-type littermates showed regular and constant breathing (Fig. 1A). The heterozygotes (Dscam +/−) showed similar but less serious respiratory abnormalities. To investigate whether the abnormal respiratory rhythm in Dscam -/- pups was attributable to defects of the respiratory center in the brain, we recorded C4 ventral root activity in a medulla-spinal cord preparation as a measure of respiratory output (Arata et al. 1990). C4 ventral root recordings in Dscam -/- mice showed irregular activity (frequency per minute: \(4.1 \pm 0.6, n = 11\)) as compared to those seen in wild-type mice \((6.8 \pm 0.4, n = 7) (p < 0.05)\) (Fig. 1B). Continuous low-amplitude C4 activities representing apnea were often observed in Dscam -/- mice that well correlated with the lethality (Fig. 1B). The C4 activity of Dscam +/− mice (frequency per minute: \(7.3 \pm 0.8, n = 8\)) was also irregular but less severe than that of Dscam -/- mice (Fig. 1B). These results suggest that the abnormal respiration patterns in Dscam -/- and Dscam +/− mice were caused by defects of the central respiratory rhythm generator located in ventral medulla.
Fig. 1

Abnormal respiration in Dscam-deficient mice. Modified, with permission, from Amano et al. (2009) Society for Neuroscience

3 Pre-I Neuron Activity is Deficient in Dscam-/- Mouse

Dual respiratory rhythm generators have been suggested to exist in the rostral ventrolateral medulla (RVLM), one in the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) containing pre-inspiratory (Pre-I) neurons (Feldman et al. 2003; Onimaru and Homma 2003 ) and the other in the pre Bötzinger complex (preBötC) containing inspiratory (Insp) neurons (Smith et al. 1991; Rekling and Feldman 1998) (Fig. 2A, B). To visualize the spatio-temporal pattern of respiratory neuron activity in the ventral medulla, we employed an optical recording system that uses a brainstem-spinal cord preparation stained with a voltage-sensitive dye (Onimaru and Homma 2003; Onimaru et al. 2004). In order to reduce the input of inhibitory neuronal activity from the pons, the rostral limit of the medulla-spinal cord preparation was set at the middle of facial (VII) nucleus (Fig. 2A). Accumulated neuronal activities of 50 respiratory cycles, each of which was triggered by its C4 inspiratory activity on one side, were shown in images (Fig. 2C-H) and trajectories (Fig. 2I-K). In wild-type mice, we confirmed the patterns of two rhythm generator loci, Pre-I and Insp neurons, on each side (Fig. 2C, F), as reported previously (Onimaru et al. 2004). In contrast, the Pre-I neuron activity in the pFRG during the pre-inspiratory phase was reduced in Dscam +/− mice (Fig. 2D, J) and disappeared in Dscam -/- mice (Fig. 2E, K, L), although the Insp neuronal activities in preBötC during the inspiratory phase were largely retained in both the genotypes (Fig. 2G, H, J, K).
Fig. 2

Defects of respiratory rhythm generation in medulla Pre-I neurons of Dscam-deficient mice. Reproduced, with permission, from Amano et al. (2009) Society for Neuroscience

Pre-I neuron activity has been reported to correlate with facial nerve activity in the newborn rat brainstem-spinal cord preparation (Onimaru et al. 2006). We therefore investigated the output of the facial nerve activity together with C4 inspiratory activity in Dscam -/- and wild-type mice using the preparation with more rostral anterior limit (open arrow in Fig. 2A) in order to keep the facial nucleus intact. In wild-type mice, the activities of the facial nerve were regular, constantly phasic, and roughly synchronized with their C4 inspiratory activities (n = 6), while that of Dscam -/- mice became tonic and lost their synchroneity to the C4 activities (n = 5) (Fig. 2 M).

4 Enlarged Medulla of Dscam-/- Mouse

Gross anatomical examination of Dscam -/- mice at E19 revealed a significant enlargement of the mediolateral width of the medulla compared to littermate controls, while that of cortex mostly remained unchanged (Fig. 3A, B). Light microscopic observation of Nissl-stained transverse sections confirmed the increased size of Dscam -/- medulla at E19 (Fig. 3C-G). The cell density remained the same, therefore suggesting that the total cell number had increased. The enlargement was prominent in Dscam -/- mice, especially at the area dorsolateral to the facial nucleus (Fig. 3H-K). Immunohistochemical analysis with SMI311 (monoclonal antibody to pan-neuronal non-phosphorylated neurofilament), a marker for neuronal soma and dendrites, revealed increases of immunosignals in this area dorsolateral to the facial nucleus, indicating that the number of neurons had increased (Fig. 3L-O). These abnormal neurons may possibly lead to the deficient output of the facial nerve activity in Dscam -/- mice (Fig. 3 M).
Fig. 3

Enlargement of medulla in Dscam-deficient mouse neonates. Reproduced, with permission, from Amano et al. (2009) Society for Neuroscience

5 Conclusion

In the present study, we found that Dscam deficiency in mice leads to death shortly after birth with central respiratory defects. Physiological analysis revealed that the synchroneity of Pre-I neurons was reduced in Dscam +/− mouse and lost in Dscam -/- mouse, whereas that of Insp neurons in Dscam +/− and Dscam -/- mice was essentially retained. These results indicate that Dscam deficiency in mice leads to specific defects in central respiratory regulation, and make DSCAM an interesting candidate gene for central respiratory disorders. Furthermore, it would be plausible that the overexpression of DSCAM on chromosome 21q22 could contribute to some of neurodevelopmental defects in patients with Down syndrome, such as their central nervous system-dependent respiratory defects (Ferri et al. 1997; Ferri et al. 1998) and/or mental retardation itself.



This work was partly supported by a grant from RIKEN Brain Science Institute, Grant-in-Aid for Scientific Research (KAKENHI) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We thank Dr. T. Takeuchi, N. Nishiyama, and Y. Onodera for technical support, Dr. K. Yamaguchi for helpful suggestions.


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Kenji Amano
    • 1
  • Morimitsu Fujii
    • 2
  • Satoru Arata
    • 3
  • Masaharu Ogawa
    • 4
  • Kazuhiro Yamakawa
    • 1
  • Akiko Arata
    • 2
    • 5
  1. 1.Laboratory for NeurogeneticsRIKEN Brain Science InstituteSaitamaJapan
  2. 2.Laboratory for Memory and LearningRIKEN Brain Science InstituteSaitamaJapan
  3. 3.Center for BiotechnologyShowa UniversityTokyoJapan
  4. 4.Laboratory for Cell Culture DevelopmentRIKEN Brain Science InstituteSaitamaJapan
  5. 5.Department of PhysiologyHyogo College of MedicineHyogoJapan

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