Cell and Tissue Research

, Volume 375, Issue 2, pp 345–357 | Cite as

Development of nNOS-positive preganglionic sympathetic neurons in the rat thoracic spinal cord

  • Konstantin Y. Moiseev
  • Irina V. Romanova
  • Andrey P. Masliukov
  • Petr M. MasliukovEmail author
Regular Article


To gain a better understanding of the neuroplasticity of sympathetic neurons during postnatal ontogenesis, the distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity was studied in sympathetic preganglionic neurons (SPN) in the spinal cord (Th2 segment) of female Wistar rats at different ages (newborn, 10-, 20-, 30-day-old; 2-, 6-month-old; 3-year-old). In all age groups, the majority of nNOS-immunoreactive (IR) neurons was observed in the nucleus intermediolateralis thoracolumbalis pars principalis. In the first month, the proportion of nNOS-IR neurons decreased significantly from 92 ± 3.4% in newborn to 55 ± 4.6% in 1-month-old, while the number of choline acetyltransferase (ChAT)-IR neurons increased from 74 ± 4.2% to 99 ± 0.3% respectively. Decreasing nNOS expression in the first 10 days of life was also confirmed by western blot analysis. Some nNOS-IR SPN also colocalized calbindin (CB) and cocaine and amphetamine-regulated transcript (CART). The percentage of NOS(+)/CB(−) SPN increased from 23 ± 3.6% in 10-day-old to 36 ± 4.2% in 2-month-old rats. Meanwhile, the proportion of NOS(+)/CART(−) neurons decreased from 82 ± 4.7% in newborn to 53 ± 6.1% in 1-month-old rats. The information provided here will also serve as a basis for future studies investigating the mechanisms of autonomic neuron development.


Spinal cord Sympathetic preganglionic neurons Nitric oxide synthase Development Immunohistochemistry 


Funding information

This work was supported by the RFBR (N 16-04-00538) grant.

Compliance with ethical standards

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Yaroslavl State Medical University and were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85–23, revised 1996) as well as the relevant Guidelines of the Russian Ministry of Health for scientific experimentation on animals.


  1. Alkadhi KA, Alzoubi KH, Aleisa AM (2005) Plasticity of synaptic transmission in autonomic ganglia. Prog Neurobiol 75:83–108Google Scholar
  2. Anderson CR (1992) NADPH diaphorase-positive neurons in the rat spinal cord include a subpopulation of autonomic preganglionic neurons. Neurosci Lett 139:280–284Google Scholar
  3. Andressen C, Blumcke I, Celio MR (1993) Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271:181–208Google Scholar
  4. Barber RP, Phelps PE, Houser CR, Crawford GD, Salvaterra PM, Vaughn JE (1984) The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study. J Comp Neurol 229:329–346Google Scholar
  5. Cossenza M, Socodato R, Portugal CC, Domith IC, Gladulich LF, Encarnação TG, Calaza KC, Mendonça HR, Campello-Costa P, Paes-de-Carvalho R (2014) Nitric oxide in the nervous system: biochemical, developmental, and neurobiological aspects. Vitam Horm 96:79–125Google Scholar
  6. Dawson TM, Dawson VL (2018) Nitric oxide signaling in neurodegeneration and cell death. Adv Pharmacol 82:57–83Google Scholar
  7. Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Synder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci U S A 88:7797–7801Google Scholar
  8. Dun NJ, Dun SL, Kwok EH, Yang J, Chang J (2000) Cocaine- and amphetamine-regulated transcript immunoreactivity in the rat sympathoadrenal axis. Neurosci Lett 283:97–100Google Scholar
  9. Emanuilov AI, Korzina MB, Archakova LI, Novakovskaya SA, Nozdrachev AD, Masliukov PM (2008) Development of the NADPH-diaphorase-positive neurons in the sympathetic ganglia. Ann Anat 190:516–524Google Scholar
  10. Fenwick NM, Martin CL, Llewellyn-Smith IJ (2006) Immunoreactivity for cocaine- and amphetamine-regulated transcript in rat sympathetic preganglionic neurons projecting to sympathetic ganglia and the adrenal medulla. J Comp Neurol 495:422–433Google Scholar
  11. Gallo EF, Iadecola C (2011) Neuronal nitric oxide contributes to neuroplasticity-associated protein expression through cGMP, protein kinase G, and extracellular signal-regulated kinase. J Neurosci 31:6947–6955Google Scholar
  12. Gardette R, Listerud MD, Brussaard AB, Role LW (1991) Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro. Dev Biol 147:83–95Google Scholar
  13. Gibbs SM (2003) Regulation of neuronal proliferation and differentiation by nitric oxide. Mol Neurobiol 27:107–120Google Scholar
  14. Godfrey EW, Schwarte RC (2010) Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells. Exp Cell Res 316:1935–1945Google Scholar
  15. Gonsalvez DG, Kerman IA, McAllen RM, Anderson CR (2010) Chemical coding for cardiovascular sympathetic preganglionic neurons in rats. J Neurosci 30:11781–11191Google Scholar
  16. Grkovic I, Anderson CR (1997) Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat. J Comp Neurol 386:245–259Google Scholar
  17. Heiman MG, Shaham S (2010) Twigs into branches: how a filopodium becomes a dendrite. Curr Opin Neurobiol 20:86–91Google Scholar
  18. Judas M, Sestan N, Kostović I (1999) Nitrinergic neurons in the developing and adult human telencephalon: transient and permanent patterns of expression in comparison to other mammals. Microsc Res Tech 45:401–419Google Scholar
  19. Masliukov PM, Fateev MM, Nozdrachev AD (2000) Age-dependent changes of electrophysiologic characteristics of the stellate ganglion conducting pathways in kittens. Auton Neurosci 83:12–18Google Scholar
  20. Masliukov PM, Korobkin AA, Nozdrachev AD, Timmermans JP (2012) Calbindin-D28k immunoreactivity in sympathetic ganglionic neurons during development. Auton Neurosci 167:27–33Google Scholar
  21. Masliukov PM, Emanuilov AI, Madalieva LV, Moiseev KY, Bulibin AV, Korzina MB, Porseva VV, Korobkin AA, Smirnova VP (2014) Development of nNOS-positive neurons in the rat sensory and sympathetic ganglia. Neuroscience 256:271–281Google Scholar
  22. Masliukov PM, Emanuilov AI, Moiseev K, Nozdrachev AD, Dobrotvorskaya S, Timmermans JP (2015) Development of non-catecholaminergic sympathetic neurons in para- and prevertebral ganglia of cats. Int J Dev Neurosci 40:76–84Google Scholar
  23. Masliukov PM, Emanuilov AI, Nozdrachev AD (2016) Developmental changes of neurotransmitter properties in sympathetic neurons. Adv Gerontol 29:442–453Google Scholar
  24. Masliukov PM, Moiseev K, Budnik AF, Nozdrachev AD, Timmermans JP (2017) Development of calbindin- and calretinin-immunopositive neurons in the enteric ganglia of rats. Cell Mol Neurobiol 37:1257–1267Google Scholar
  25. Meller ST, Gebhart GF (1993) Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52:127–136Google Scholar
  26. Mukhutdinova KA, Kasimov MR, Giniatullin AR, Zakyrjanova GF, Petrov AM (2018) 24S-hydroxycholesterol suppresses neuromuscular transmission in SOD1(G93A) mice: a possible role of NO and lipid rafts. Mol Cell Neurosci 88:308–318Google Scholar
  27. Nikonenko I, Jourdain P, Muller D (2003) Presynaptic remodeling contributes to activity-dependent synaptogenesis. J Neurosci 23:8498–8505Google Scholar
  28. Patel BA, Dai X, Burda JE, Zhao H, Swain GM, Galligan JJ, Bian X (2010) Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs. Neurogastroenterol Motil 22:909–918Google Scholar
  29. Petho G, Reeh PW (2012) Sensory and signaling mechanisms of bradykinin eicosanoids platelet-activating factor and nitric oxide in peripheral nociceptors. Physiol Rev 92:1699–1775Google Scholar
  30. Phelps PE, Barber RP, Vaughn JE (1991) Embryonic development of choline acetyltransferase in thoracic spinal motor neurons: somatic and autonomic neurons may be derived from a common cellular group. J Comp Neurol 307:77–86Google Scholar
  31. Porseva VV, Shilkin VV, Strelkov AA, Masliukov PM (2014) Subpopulation of calbindin-immunoreactive interneurons in the dorsal horn of the mice spinal cord. Tsitologiia 56:612–618Google Scholar
  32. Porseva VV, Shilkin VV, Krasnov IB, Masliukov PM (2015) Calbindin-D28k immunoreactivity in the mice thoracic spinal cord after space flight. Int J Astrobiol 14:555–562Google Scholar
  33. Prast H, Philippu A (2001) Nitric oxide as modulator of neuronal function. Prog Neurobiol 64:51–68Google Scholar
  34. Pyner S, Coote JH (1994) A comparison between the adult rat and neonate rat of the architecture of sympathetic preganglionic neurones projecting to the superior cervical ganglion stellate ganglion and adrenal medulla. J Auton Nerv Syst 48:153–166Google Scholar
  35. Rubin E (1985) Development of the rat superior cervical ganglion: initial stages of synapse formation. J Neurosci 5:697–704Google Scholar
  36. Sanchez-Islas E, Leon-Olea M (2004) Nitric oxide synthase inhibition during synaptic maturation decreases synapsin I immunoreactivity in rat brain. Nitric Oxide 10:141–149Google Scholar
  37. Schmidt HHHW, Gagne GD, Nakane M, Pollock JS, Miller MF, Murad F (1992) Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclases and novel paraneural functions for nitrinergic signal transduction. J Histochem Cytochem 40:1439–1456Google Scholar
  38. Scruggs P, Lai CC, Scruggs JE, Dun NJ (2005) Cocaine- and amphetamine-regulated transcript peptide potentiates spinal glutamatergic sympathoexcitation in anesthetized rats. Regul Pept 127:79–85Google Scholar
  39. Schwaller B (2012) The use of transgenic mouse models to reveal the functions of Ca2+ buffer proteins in excitable cells. Biochim Biophys Acta 1820:1294–1303Google Scholar
  40. Siechen S, Yang S, Chiba A, Saif T (2009) Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals. Proc Natl Acad Sci U S A 106:12611–12616Google Scholar
  41. Smolen A, Raisman G (1980) Synapse formation in the rat superior cervical ganglion during normal development and after neonatal deafferentation. Brain Res 787:315–323Google Scholar
  42. Snyder SH (1992) Nitric oxide: first in a new class of neurotransmitters. Science 257:494–496Google Scholar
  43. Southam E, Charles SL, Garthwaite J (1996) The nitric oxide-cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion. Br J Pharmacol 119:527–532Google Scholar
  44. Wetts R, Vaughn JE (1994) Choline acetyltransferase and NADPH diaphorase are co-expressed in rat spinal cord neurons. Neuroscience 63:1117–1124Google Scholar
  45. Wetts R, Phelps PE, Vaughn JE (1995) Transient and continuous expression of NADPH diaphorase in different neuronal populations of developing rat spinal cord. Dev Dyn 202:215–228Google Scholar
  46. Yakovleva OV, Shafigullin MU, Sitdikova GF (2013) The role of nitric oxide in the regulation of neurotransmitter release and processes of exo- and endocytosis of synaptic vesicles in mouse motor nerve endings. Neurochem J 7:103–110Google Scholar
  47. Young HM, Cane KN, Anderson CR (2011) Development of the autonomic nervous system: a comparative view. Auton Neurosci 165:10–27Google Scholar
  48. Zhang P, Yu PC, Tsang AH, Chen Y, Fu AK, Fu WY, Chung KK, Ip NY (2010) S-nitrosylation of cyclin-dependent kinase 5 (cdk5) regulates its kinase activity and dendrite growth during neuronal development. J Neurosci 30:14366–14370Google Scholar

Copyright information

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

Authors and Affiliations

  • Konstantin Y. Moiseev
    • 1
  • Irina V. Romanova
    • 2
  • Andrey P. Masliukov
    • 3
  • Petr M. Masliukov
    • 1
    Email author
  1. 1.Department of Normal Physiology and BiophysicsYaroslavl State Medical UniversityYaroslavlRussia
  2. 2.Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of SciencesSt. PetersburgRussia
  3. 3.I.M. Sechenov First Moscow State Medical UniversityMoscowRussia

Personalised recommendations