Brain Structure and Function

, Volume 221, Issue 7, pp 3803–3823 | Cite as

Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord

  • Ming-Dong ZhangEmail author
  • Swapnali Barde
  • Edit Szodorai
  • Anna Josephson
  • Nicholas Mitsios
  • Masahiko Watanabe
  • Johannes Attems
  • Gert Lubec
  • Gábor G. Kovács
  • Mathias Uhlén
  • Jan Mulder
  • Tibor Harkany
  • Tomas Hökfelt
Original Article


Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent’s spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k+ neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2+ and/or calretinin+. Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.


Calbindin-D28k Ependymal cell PKCγ SST2A VGLUT1 VGLUT2 



Support for this study was provided by the Swedish Medical Research Council (T.Hö., T.Ha.), Karolinska Institutet, partial financing of graduate student funds (M.-D.Z., T.Ha., and T.Hö.); funding from the Karolinska Institutet (T.Hö., T.Ha.), the Novo Nordisk Foundation (T.Ha. and T.Hö.), the Swedish Brain Foundation (T.Ha. and T.Hö.), the Augusta and Petrus Hedlund Foundation (T.Ha. and T.Hö.) and the European Commission’s PAINCAGE 7th Framework Programme (T.Ha. and T.Hö.). Laser-scanning microscopy was made available by the Center for Live Imaging of Cells (CLICK) at Karolinska Institutet, an imaging core facility supported by the Knut and Alice Wallenberg Foundation.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

429_2016_1191_MOESM1_ESM.tif (2.7 mb)
Supplementary Figure S1 Distribution of NECAB1-LI in rat and human spinal dorsal horn after incubation with monoclonal or polyclonal antibodies. The polyclonal (a, c) and monoclonal (b, d) NECAB1 antibody show a similar distribution pattern in rat (a, b) and human (c, d) dorsal horn. Scale bars: 200 μm in a, b, 200 μm in c, d (TIFF 2775 kb)
429_2016_1191_MOESM2_ESM.tif (354 kb)
Supplementary Figure S2 Representative MS/MS fragmentation spectrum of a unique peptide identified from NECAB2 illustrating b and y fragment ions of GGTAVILDIFR sequence (Charge: +2 Monoisotopic m/z: 581.450 Da (±0.230 Da) MH+(mono): 1161.663 Da, RT: 79.84 min) (TIFF 354 kb)
429_2016_1191_MOESM3_ESM.tif (961 kb)
Supplementary Figure S3 Expression of PKCγ in mouse, rat and human spinal cord. a-c Low magnification pictures show overview of PKCγ expression in mouse, rat and human lumbar spinal cord, respectively. The heavily labeled area in lamina IIi by PKCγ is outlined with magenta dashed lines. The descending tracts of PKCγ in rodents’ dorsal funiculus and human’s lateral spinal cord are also labeled by magenta dashed lines. d, e The PKCγ expression pattern is highlighted in the spinal atlas from rodents and human. dCST: descending cortical spinal tract, LCST: lateral cortical spinal tract, Cc: central canal. Scale bars: 500 μm (TIFF 960 kb)
429_2016_1191_MOESM4_ESM.tif (2.1 mb)
Supplementary Figure S4 Expression of VGLUT1 and VGLUT2 in mouse, rat and human spinal cord. a-c Low magnification pictures show overview of VGLUT1 expression in mouse, rat and human lumbar spinal cord, respectively. d-f Low magnification pictures show overview of VGLUT2 expression in mouse, rat and human lumbar spinal cord, respectively. Scale bars: 200 μm in a, b, d, e 1 mm in c, f (TIFF 2156 kb)
429_2016_1191_MOESM5_ESM.tif (2.7 mb)
Supplementary Figure S5 Expression of calretinin and calbindin-D28k in mouse, rat and human spinal cord. a-c Low magnification pictures show overview of calretinin expression in mouse, rat and human lumbar spinal cord, respectively. d-f Low magnification pictures show overview of calbindin-D28k expression in mouse, rat and human lumbar spinal cord, respectively. Central canal is outlined by dashed line. Cc: central canal. Scale bars: 200 μm in a, b, d, e 1 mm in c, f (TIFF 2742 kb)
429_2016_1191_MOESM6_ESM.docx (44 kb)
Supplementary material 6 (DOCX 44 kb)
429_2016_1191_MOESM7_ESM.docx (66 kb)
Supplementary material 7 (DOCX 65 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ming-Dong Zhang
    • 1
    • 2
    Email author
  • Swapnali Barde
    • 1
  • Edit Szodorai
    • 3
    • 7
  • Anna Josephson
    • 1
  • Nicholas Mitsios
    • 4
  • Masahiko Watanabe
    • 5
  • Johannes Attems
    • 6
  • Gert Lubec
    • 7
  • Gábor G. Kovács
    • 8
  • Mathias Uhlén
    • 4
    • 9
  • Jan Mulder
    • 4
  • Tibor Harkany
    • 2
    • 3
  • Tomas Hökfelt
    • 1
  1. 1.Department of NeuroscienceKarolinska InstitutetStockholmSweden
  2. 2.Division of Molecular Neurobiology, Department of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
  3. 3.Department of Molecular Neurosciences, Center for Brain ResearchMedical University of ViennaViennaAustria
  4. 4.Science for Life Laboratory, Department of NeuroscienceKarolinska InstitutetStockholmSweden
  5. 5.Department of AnatomyHokkaido University School of MedicineSapporoJapan
  6. 6.Institute of Ageing and HealthNewcastle UniversityNewcastle upon TyneUK
  7. 7.Department of Pharmaceutical ChemistryMedical University of ViennaViennaAustria
  8. 8.Institute of NeurologyMedical University of ViennaViennaAustria
  9. 9.Science for Life Laboratory, Albanova University CenterRoyal Institute of TechnologyStockholmSweden

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