Skip to main content
SpringerLink
Log in

Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat, with special reference to motoneurons and galanin

  • Published:
Experimental Brain Research Aims and scope Submit manuscript

Abstract

The extent to which the plasticity in peptide expression observed in developing spinal motoneurons occurs following proximal peripheral axotomy in the adult rat was examined using in situ hybridization and immunohistochemical techniques to visualize the changes. Transient upregulation of galanin, vasoactive intestinal polypeptide (VIP) and substance P messenger ribonucleic acids (mRNAs) was observed within subpopulations of motoneurons ipsilateral to lesion for periods lasting 2–3 weeks after injury. In contrast, the axotomy-induced heterogenous increases in somatostatin and neuropeptide tyrosine mRNA expression in ipsilateral motoneurons remained elevated, or, in the case of somatostatin, continued to increase for the time period studied (1 month). Immunohistochemical analysis agreed with the in situ hybridization results, showing some motoneurons within the injured ventral horn to contain galanin-, VIP-or somatostatin-like immunoreactivity. In some instances, galanin-immunoreactive motoneurons colocalized with calcitonin gene-related peptide immunoreactivity. Most of the neurons expressing the injury-induced peptides appeared large, presumably alpha-motoneurons, but there were also many small neurons expressing galanin in the ventral horn ipsilateral to lesion. This may represent evidence for peptide synthesis in gamma-motoneurons. The only peptide mRNA studied to be downregulated in response to axotomy was enkephalin. The results show that peptide expression in injured motoneurons is dramatically altered, the significance of which remains to be determined.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abelson L, Micevych PE (1991) Distribution of preprocholecystokinin mRNA in motoneurons of the rat brainstem and spinal cord. Mol Brain Res 10:327–335

    Google Scholar 

  • Agoston DV, Eiden LE, Brenneman DE, Gozes I (1991) Spontaneous electrical activity regulates vasoactive intestinal peptide expression in dissociated spinal cord cell cultures. Mol Brain Res 10:235–240

    Google Scholar 

  • Arvidsson U, Cullheim S, Ulfhake B, Hökfelt T, Terenius L (1989) Altered levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity of cat lumbar motoneurons after chronic spinal cord transection. Brain Res 489:387–391

    Google Scholar 

  • Arvidsson U, Johnson H, Piehl F, Cullheim S, Hökfelt T, Risling M, Terenius L, Ulfhake B (1990) Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons. Exp Brain Res 79:212–216

    Google Scholar 

  • Barron KD (1983) Comparative observations on the cytologic reactions of central and peripheral nerve cells to axotomy. In: Kao CC, Bunge RP, Reier PJ (eds) Spinal cord reconstruction. Raven, New York, pp 7–40

    Google Scholar 

  • Basi GS, Jacobson RD, Virag I, Schilling J, Skene JHP (1987) Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth. Cell 49:785–791

    Google Scholar 

  • Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I (1985) Calcitonin gene-related peptide is a potent vasodilator. Nature 313:54–57

    Google Scholar 

  • Brenneman DE, Nicol T, Warren D, Bowers LM (1990) Vasoactive intestinal peptide: a neurotrophic releasing agent and an astroglial mitogen. J Neurosci Res 25:386–394

    Google Scholar 

  • Buchan AMJ, Sikora LKJ, Lovy JG, McIntosh CHS, Dyck I, Brown JC (1985) An immunohistochemical investigation with monoclonal antibodies to somatostatin. Histochemistry 83:175–180

    Google Scholar 

  • Ch'ng JLC, Christofides ND, Anand P, Gibson SJ, Allen YS, Su HC, Tatemoto K, Morrison JFB, Polak JM, Bloom SR (1985) Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathway to injury. Neuroscience 16:343–354

    Google Scholar 

  • Coons AH (1958) Fluorescent antibody methods. In: Danielli JF (ed) Cytochemical methods. Academic, New York, pp 399–422

    Google Scholar 

  • Cortés R, Arvidsson U, Schalling M, Ceccatelli S, Hökfelt T (1990a) In situ hybridization studies on mRNAs for cholecystokinin, calcitonin gene-related peptide and choline acetyltransferase in the guinea pig with special reference to motoneurons. J Chem Neuroanat 3:467–485

    Google Scholar 

  • Cortés R, Ceccatelli S, Schalling M, Hökfelt T (1990b) Differential effects of intracerebroventricular colchicine administration on the expression of mRNAs for neuropeptide and neurotransmitter enzymes, with special emphasis on galanin: an in situ hybridization study. Synapse 6:369–391

    Google Scholar 

  • Cortés R, Villar MJ, Verhofstad A, Hökfelt T (1990c) Effects of central nervous system lesions on the expression of galanin: A comparative in situ hybridization and immunohistochemical study. Proc Natl Acad Sci USA 87:7742–7746

    Google Scholar 

  • Dagerlind Å, Friberg K, Bean A, Hökfelt T (1992) Sensitive mRNA detection using unfixed tissue: combined radioactive and nonradioactive in situ hybridization histochemistry. Histochemistry, 98:39–49

    Google Scholar 

  • Dumoulin FL, Raivich G, Streit WJ, Kreutzberg GW (1991) Differential regulation of calcitonin gene-related peptide (CGRP) in regenerating rat facial nucleus and dorsal root ganglion. Eur J Neurosci 3:338–342

    Google Scholar 

  • Ernfors P, Henschen A, Olson L, Persson H (1989) Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron 2:1605–1613

    Google Scholar 

  • Fahrenkrug J, Schaffalitzky de Muckadell OB (1977) Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in the plasma. J Lab Clin Med 89:1379–1388

    Google Scholar 

  • Fahrenkrug J, Schaffalitzky de Muckadell OB (1978) Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system. J Neurochem 31:1445–1451

    Google Scholar 

  • Fitzgerald M, Wall PD, Goedert M, Emson PC (1985) Nerve growth factor counteracts the neurophysiological and neurochemical effects of cronic sciatic nerve section. Brain Res 372:131–141

    Google Scholar 

  • Fontaine B, Klarsfeld A, Hökfelt T, Changeux JP (1986) Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci Lett 71:56–65

    Google Scholar 

  • Gibson SJ, Polak MJ, Bloom SR, Sabate IM, Mulderry PM, Ghatei MA, McGregor GP, Morrison JFB, Kelly JS, Evans RM, Rosenfeld MG (1984) Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J Neurosci 4:3101–3111

    Google Scholar 

  • Goedert M, Stoeckel K, Otten U (1981) Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons. Neurosciences 78:5895–5898

    Google Scholar 

  • Goodman RH, Aron DC, Ross BA (1989) Rat pre-prosomatostatin. J Biol Chem 258:5570–5573

    Google Scholar 

  • Graeber MB, Kreutzberg GW (1986) Astrocytes increase in glial fibrillary acidic protein during retrograde changes of facial motor neurons. J Neurocytol 15:363–373

    Google Scholar 

  • Haas CA, Streit WJ, Kreutzberg GW (1990) Rat facial motoneurons express increased levels of calcitonin gene-related peptide mRNA in response to axotomy. J Neurosci Res 27:270–275

    Google Scholar 

  • Hoffman PN (1989) Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons. J Neurosci 9:893–897

    Google Scholar 

  • Hökfelt T, Johansson O, Goldstein M (1984) Chemical anatomy of the brain. Science 225:1326–1334

    Google Scholar 

  • Hökfelt T, Wiesenfeld-Hallin Z, Villar MJ, Melander T (1987) Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett 83 217–220

    Google Scholar 

  • Hösli L, Hösli E, Heuss L, Rojas J (1990) Evidence for angiotensin and VIP receptors on astrocytes. In: Levi G (ed) Differentiation and functions of glial cells. Wiley-Liss, New York, pp 297–298

    Google Scholar 

  • Howells RD, Kilpatrick DL, Bhatt R, Monham JJ, Poonian M, Udenfriend S (1984) Molecular cloning and sequences determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissue. Proc Natl Acad Sci USA 81:7651–7655

    Google Scholar 

  • Inaishi Y, Kashihara Y, Sakaguchi M, Nawa H, Kuno M (1992) Cooperative regulation of calcitonin gene-related peptide levels in rat sensory neurons via their central and peripheral processes. J Neurosci 12:518–524

    Google Scholar 

  • Ingram SM, Krause RG II, Baldino F Jr, Skeen LC, Lewis ME (1989) Neuronal localization of cholecystokinin mRNA in the rat brain by using in situ hybridization histochemistry. J Comp Neurol 287:260–272

    Google Scholar 

  • Johnson DG, De C Nogueria Araujo GM (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349–350

    Article  CAS  PubMed  Google Scholar 

  • Johnson H, Hökfelt T,Ulfhake B (1992) Galanin- and CGRP-like immunoreactivity coexist in rat spinal motoneurons. Neuro Report 3:303–306

    Google Scholar 

  • Ju G, Hökfelt T, Fischer JA, Frey P, Rehfeld JF, Dockray GJ (1986) Does cholecystokinin-like immunoreactivity in the rat primary sensory neurons represents calcitonin gene-related peptide? Neurosci Lett 68:305–310

    Google Scholar 

  • Karns LR, Ng SG, Freeman JA, Fishman MC (1987) Cloning of complementary DNA for GAP43, a neuronal growth-related protein. Science 236:597–600

    Google Scholar 

  • Kashiba H, Senba E, Kawai Y, Ueda Y, Tohyama M (1992) Axonal blockade induces the expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons. Brain Res 577:19–28

    Google Scholar 

  • Knyihár-Csillik E, Kreutzberg GW, Raivich G, Csillik B (1991) Vasoactive intestinal polypeptide in dorsal root terminals of the rat spinal cord is regulated by the axoplasmic transport in the peripheral nerve. Neurosci Lett 131:83–87

    Google Scholar 

  • Krause JE, Chirgwin JM, Carter MS, Xu ZS, Hershey D (1987) Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc Natl Acad Sci USA 84:881–885

    Google Scholar 

  • Kreutzberg GW (1982) Acute neuronal reaction to injury. In: Nicholls JG (ed) Repair and regeneration of the nervous system. Springer, Berlin Heidelberg New York, pp 57–69

    Google Scholar 

  • Larhammar D, Ericsson A, Persson H (1987) Structure and expression of the rat neuropeptide Y gene. Proc Natl Acad Sci USA 84:2068–2072

    Google Scholar 

  • Lazar P, Reddington M, Streit WJ, Raivich G, Kreutzberg GW (1991) Stimulation of adenosine 3′,5′-monophosphate accumulation by calcitonin gene-related peptide in astrocyte cultures from neonatal rat brain. Neurosci Lett 130:99–102

    Google Scholar 

  • Lieberman AR (1979) The axon reaction: a review of the principal features of perikaryal responses to axon injury. Int Rev Neurobiol 14:49–124

    Google Scholar 

  • Lindå H, Piehl F, Dagerlind Å, Verge VMK, Arvidsson U, Cullheim S, Risling, M, Ulfhake B, Hökfelt T (1992) Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons. Exp Brain Res. 91:284–295

    Google Scholar 

  • Magistretti PJ, Morrison GH, Shoemaker WJ, Sapin V, Bloom FE (1981) Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism. Proc Natl Acad Sci USA 78:6535–6539

    Google Scholar 

  • Magistretti PJ, Shorderet M (1984) VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex. Nature 308:438–440

    Google Scholar 

  • Marti E, Gibson SJ, Polak J, Facer, P, Springall DR, Van Aswegen G, Aitchison M, Koltzenburg M (1987) Ontogeny of peptideand amine-containing neurones in motor, sensory, and autonomie regions of rat and human spinal cord, dorsal root ganglia, and rat skin. J Comp Neurol 266:332–359

    Google Scholar 

  • Moore RY (1989) Cranial motor neurons contain either galanin- or calcitonin gene-related peptide-like immunoreactivity. J Comp Neurol 282:512–522

    Google Scholar 

  • New HV, Mudge AW (1986) Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. Nature 323:809–811

    Google Scholar 

  • Nishizawa M, Hayakawa Y, Yanaihara N, Okamoto H (1985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett 183:55–59

    Google Scholar 

  • Noguchi K, Senba E, Morita Y, Sato M, Tohyama M (1990) Alpha-CGRP and beta-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglia. Mol Brain Res 7:299–304

    Google Scholar 

  • Pease PC (1962) Buffered formaldehyde as a killing agent and primary fixative for electron microscopy. Anat Rec 142:342

    Google Scholar 

  • Piehl F, Arvidsson U, Johnson H, Cullheim S, Villar M, Dagerlind Å, Terenius L, Hökfelt T, Ulfhake B (1991) Calcitonin gene-related peptide (CGRP)-like immunoreactivity and CGRP mRNA in rat spinal cord motoneurons after different types of lesions. Eur J Neurosci 3:737–757

    Google Scholar 

  • Platt JL, Michael AF (1983) Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J Histochem Cytochem 31:840–842

    Google Scholar 

  • Rosenfeld MG, Mermod J-J, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WV, Evans RM (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129–135

    Google Scholar 

  • Said SI, Mutt V (1972) Polypeptide with broad biological activity: isolation from small intestine. Science 169:1217–1218

    Google Scholar 

  • Saika T, Senba E, Noguchi K, Sato M, Kubo T, Matsunaga T, Tohyama, M (1991) Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection. Mol Br Res 11:187–196

    Google Scholar 

  • Schalling M, Dagerlind Å, Brene S, Hallman H, Djurfeldt M, Persson H, Terenius L, Goldstein M, Schlessinger D, Hökfelt T (1988) Coexistence and gene expression of phenylethanoamine N-ethyltransferase, tyrosine hydroxylase and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine. Proc Natl Acad Sci USA 85:8306–8310

    Google Scholar 

  • Schalling M (1990) In situ hybridization studies on regulatory molecules in neural and endocrine tissues with special reference to expression of coexisting peptides. PhD thesis. Karolinska Institute, Stockholm (ISBN 91–628–0036–1)

    Google Scholar 

  • Schiffman SN, Teugels E, Halleux P, Menu R, Vanderhaeghen JJ (1991) Cholecystokinin mRNA detection in rat spinal cord motoneurons but not in dorsal root ganglia neurons. Neurosci Lett 123:123–126

    Google Scholar 

  • Schrøder HD (1983) Localization of cholecystokinin-like immunoreactivity in the rat spinal cord, with particular reference to the autonomie innervation of the pelvic organs. J Comp Neurol 217:176–186

    Google Scholar 

  • Schrøder HD (1984) Somatostatin in the caudal spinal cord: an immunocytochemical study of the spinal centers involved in the innervation of pelvic organs. J Comp Neurol 223:400–414

    Google Scholar 

  • Schwartz JP, Pearson J, Johnson EM (1982) Effect of exposure to anti-NGF on sensory neurons of adult rats and guinea pigs. Brain Res 244:378–381

    Google Scholar 

  • Shu S, Ju G, Fan L (1988) The glucoseoxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169–171

    Google Scholar 

  • Streit WJ, Dumoulin FL, Raivich G, Kreutzberg GW (1989) Calcitonin gene-related peptide increases in rat facial motoneurons after peripheral nerve transection. Neurosci Lett 101:143–148

    Google Scholar 

  • Suburo AM, Gibson SJ, Moscoso G, Terenghi G, Polak JM (1992) Transient expression of neuropeptide Y and its C-flanking peptide immunoreactivities in the spinal cord and ganglia of human embryos and fetuses. Neuroscience 46:71–584

    Google Scholar 

  • Sutin EL, Jacobowitz DM (1990) Detection of CCK mRNA in the motor nucleus of the rat trigeminal nerve with in situ hybridization histochemistry. Mol Brain Res 8:63–68

    Google Scholar 

  • Tetzlaff W, Alexander SA, Miller FD, Bisby MA (1991) Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP43. J Neurosci 11(8): 2528–2544

    Google Scholar 

  • Tsui BJ, Cassar SL, Tetzlaff W (1991) Changes in mRNA levels for GAP43, tubulin and neurofilament-M in rat spinal motoneurons after proximal versus distal axotomy. Soc Neurosci Abstr 17:47

    Google Scholar 

  • Verge VMK, Richardson PM, Wiesenfeld-Hallin Z, Hökfelt T (1991) Differential influence of nerve growth factor on neuropeptide expression in intact and injured adult sensory neurons. Eur J Neurosci [Suppl] 4:288

    Google Scholar 

  • Villar MJ, Huchet M, Hökfelt T, Changeux JP, Fahrenkrug J, Brown JC (1988) Existence and coexistence of calcitonin generelated peptide, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivities in spinal cord motoneurons of developing embryos and post-hatch chicks. Neurosci Lett 86:114–118

    Google Scholar 

  • Villar MJ, Cortés R, Theodorsson E, Wiesenfeld-Hallin Z, Schalling M, Fahrenkrug J, Emson PC, Hökfelt T (1989a) Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33:587–604

    Google Scholar 

  • Villar MJ, Roa M, Huchet M, Hökfelt T, Changeux JP, Fahrenkrug J, Brown JC, Epstein M, Hersh L (1989b) Immunoreactive calcitonin gene-related peptide, vasoactive intestinal polypeptide, and somatostatin in developing chicken spinal cord motoneurons. Distribution and role in regulation of cAMP in cultured muscle cells. Eur J Neurosci 1:269–287

    Google Scholar 

  • Villar MJ, Meister B, Cortés R, Schalling M, Morris M, Hökfelt T (1990) Neuropeptide gene expression in hypothalamic magnocellular neurons of normal and hypophysectomized rats: a combined immunohistochemical and in situ hybridization study. Neuroscience 36:181–199

    Google Scholar 

  • Villar MJ, Roa M, Huchet M, Changeux JP, Valentin KL, Hökfelt T (1991) Occurrence of neuropeptide K-like immunoreactivity in ventral horn cells of the chicken spinal cord during development. Brain Res 541:149–153

    Google Scholar 

  • Vrontakis ME, Peden LM, Duckworth ML, Friesen HG (1987) Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J Biol Chem 262:16755–16758

    Google Scholar 

  • Wiesenfeld-Hallin Z, Bartfai T, Hökfelt T (1992) Galanin in sensory neurons and spinal cord. Front Neuroendocrinol 13:319–343

    Google Scholar 

  • Wilkin GP, Reid JC, Marriott DR, Cholewinski AJ (1990) Peptide receptors on astrocytes: Characterization and regional heterogeneity. In: Levi G (ed) Differentiation and functions of glial cells. Wiley-Liss, New York, pp 265–274

    Google Scholar 

  • Young WS III (1990) In situ hybridization histochemistry. In: Björklund A, Hökfelt T, Wouterlood FG, van den Pool AN (eds) Handbook of chemical neuroanatomy, vol 8. Analysis of neuronal microcircuits and synaptic interactions. Elsevier, Amsterdam, pp 481–512

    Google Scholar 

  • Zamboni I, De Martino C (1967) Buffered picric acid formaldehyde. A new rapid fixative for electron microscopy (abstract). J Cell Biol 35:148

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Histology and Neurobiology, Karolinska Institutet, Box 60400, S-10401, Stockholm, Sweden

    X. Zhang, V. M. K. Verge & T. Hökfelt

  2. Department of Clinical Physiology, Section of Clinical Neurophysiology, Huddinge Hospital, Karolinska Institutet, Box 60400, S-10401, Stockholm, Sweden

    Z. Wiesenfeld-Hallin

  3. Department of Anatomy, Karolinska Institutet, Box 60400, S-10401, Stockholm, Sweden

    F. Piehl

Authors
  1. X. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. M. K. Verge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Z. Wiesenfeld-Hallin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Piehl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. Hökfelt
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, X., Verge, V.M.K., Wiesenfeld-Hallin, Z. et al. Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat, with special reference to motoneurons and galanin. Exp Brain Res 93, 450–461 (1993). https://doi.org/10.1007/BF00229360

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00229360

Key words

Search

Navigation