Skip to main content
SpringerLink
Log in
Book cover

Stimulation and Inhibition of Neurons pp 107–120Cite as

In Vivo Manipulation of Intracellular Signalling Pathways

  • Protocol
  • First Online:

  • 1276 Accesses

Part of the book series: Neuromethods ((NM,volume 78))

Abstract

Manipulation of intracellular signalling pathways and ligand protein interactions can lead to new therapeutic strategies in many cardiovascular diseases. One of the most commonly used methods, performed on different disease models of rat, is the microinjection of various agonist and antagonists of ligand and proteins into specific regions of the brain that are implicated in a variety of cardiovascular diseases. This chapter will describe a detailed step-by-step method for manipulation of intracellular signalling pathways in specific regions of the rat brainstem. In this method, the rostral ventrolateral medulla, an area of the brainstem which is essential for sympathetic activity and blood pressure, is studied with regard to the cyclic adenosine 3′-5′-monophosphate pathway. With this method, a more detailed interpretation of the physiological state of the rat in vivo can be achieved.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Dampney RA (1994) Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74:323–364

    Article  PubMed  CAS  Google Scholar 

  2. Guyenet PG (2006) The sympathetic control of blood pressure. Nat Rev Neurosci 7:335–346

    Article  PubMed  CAS  Google Scholar 

  3. Sved AF, Ito S, Sved JC (2003) Brainstem mechanisms of hypertension: role of the rostral ventrolateral medulla. Curr Hypertens Rep 5:262–268

    Article  PubMed  Google Scholar 

  4. Oshima N, Kumagai H, Onimaru H, Kawai A, Pilowsky PM, Iigaya K, Takimoto C, Hayashi K, Saruta T, Itoh H (2008) Monosynaptic excitatory connection from the rostral ventrolateral medulla to sympathetic preganglionic neurons revealed by simultaneous recordings. Hypertens Res 31:1445–1454

    Article  PubMed  Google Scholar 

  5. Oshima N, McMullan S, Goodchild AK, Pilowsky PM (2006) A monosynaptic connection between baroinhibited neurons in the RVLM and IML in Sprague-Dawley rats. Brain Res 1089:153–161

    Article  PubMed  CAS  Google Scholar 

  6. Lipski J, Kanjhan R, Kruszewska B, Smith M (1995) Barosensitive neurons in the rostral ventrolateral medulla of the rat in vivo: morphological properties and relationship to C1 adrenergic neurons. Neuroscience 69:601–618

    Article  PubMed  CAS  Google Scholar 

  7. Brown DL, Guyenet PG (1985) Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ Res 56:359–369

    Article  PubMed  CAS  Google Scholar 

  8. Farnham MM, Li Q, Goodchild AK, Pilowsky PM (2008) PACAP is expressed in sympathoexcitatory bulbospinal C1 neurons of the brain stem and increases sympathetic nerve activity in vivo. Am J Physiol Regul Integr Comp Physiol 294:R1304–1311

    Article  PubMed  CAS  Google Scholar 

  9. Krieger MH, Moreira ED, Oliveira EM, Oliveira VL, Krieger EM, Krieger JE (2006) Dissociation of blood pressure and sympathetic activation of renin release in sinoaortic-denervated rats. Clin Exp Pharmacol Physiol 33:471–476

    Article  PubMed  CAS  Google Scholar 

  10. Miyawaki T, Goodchild AK, Pilowsky PM (2002) Activation of mu-opioid receptors in rat ventrolateral medulla selectively blocks baroreceptor reflexes while activation of delta opioid receptors blocks somato-sympathetic reflexes. Neuroscience 109:133–144

    Article  PubMed  CAS  Google Scholar 

  11. Gaede AH, Pilowsky PM (2010) Catestatin in rat RVLM is sympathoexcitatory, increases barosensitivity, and attenuates chemosensitivity and the somatosympathetic reflex. Am J Physiol Regul Integr Comp Physiol 299:R1538–R1545

    Article  PubMed  CAS  Google Scholar 

  12. Seyedabadi M, Goodchild AK, Pilowsky PM (2001) Differential role of kinases in brain stem of hypertensive and normotensive rats. Hypertension 38:1087–1092

    Article  PubMed  CAS  Google Scholar 

  13. Chan SH, Tai MH, Li CY, Chan JY (2006) Reduction in molecular synthesis or enzyme activity of superoxide dismutases and catalase contributes to oxidative stress and neurogenic hypertension in spontaneously hypertensive rats. Free Radic Biol Med 40:2028–2039

    Article  PubMed  CAS  Google Scholar 

  14. Miyawaki T, Suzuki S, Minson J, Arnolda L, Chalmers J, Llewellyn-Smith I, Pilowsky P (1997) Role of AMPA/kainate receptors in transmission of the sympathetic baroreflex in rat CVLM. Am J Physiol 272:R800–R812

    PubMed  CAS  Google Scholar 

  15. Miyawaki T, Goodchild AK, Pilowsky PM (2003) Maintenance of sympathetic tone by a nickel chloride-sensitive mechanism in the rostral ventrolateral medulla of the adult rat. Neuroscience 116:455–464

    Article  PubMed  CAS  Google Scholar 

  16. White HS, Driver PS, Isom GE (1979) Studies on the central pressor activity of dibutyryl cyclic AMP. Eur J Pharmacol 57:107–113

    Article  PubMed  CAS  Google Scholar 

  17. Clipsham PJ, Hamilton TC, Hunt AA, Poyser RH (1980) Cyclic nucleotides and central cardiovascular control in the conscious cat. Eur J Pharmacol 65:193–200

    Article  PubMed  CAS  Google Scholar 

  18. Walland A (1975) cAMP as a second messenger in central blood pressure control. Naunyn Schmiedebergs Arch Pharmacol 290:419–423

    Article  PubMed  CAS  Google Scholar 

  19. Johnson JD, White HS, Isom GE (1983) Local application of cyclic AMP in the rat brain: characterization of the cardiovascular response. Eur J Pharmacol 91:343–351

    Article  PubMed  CAS  Google Scholar 

  20. Xu Y, Krukoff TL (2006) Adrenomedullin in the rostral ventrolateral medulla inhibits baroreflex control of heart rate: a role for protein kinase A. Br J Pharmacol 148:70–77

    Article  PubMed  CAS  Google Scholar 

  21. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272

    PubMed  CAS  Google Scholar 

  22. Johannes FJ, Prestle J, Dieterich S, Oberhagemann P, Link G, Pfizenmaier K (1995) Characterization of activators and inhibitors of protein kinase C mu. Eur J Biochem 227:303–307

    Article  PubMed  CAS  Google Scholar 

  23. Van Haastert PJ, Van Driel R, Jastorff B, Baraniak J, Stec WJ, De Wit RJ (1984) Competitive cAMP antagonists for cAMP-receptor proteins. J Biol Chem 259:10020–10024

    PubMed  Google Scholar 

  24. Yokozaki H, Tortora G, Pepe S, Maronde E, Genieser HG, Jastorff B, Cho-Chung YS (1992) Unhydrolyzable analogues of adenosine 3′:5′-monophosphate demonstrating growth inhibition and differentiation in human cancer cells. Cancer Res 52:2504–2508

    PubMed  CAS  Google Scholar 

  25. Mei FC, Qiao J, Tsygankova OM, Meinkoth JL, Quilliam LA, Cheng X (2002) Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation. J Biol Chem 277:11497–11504

    Article  PubMed  CAS  Google Scholar 

  26. Honegger KJ, Capuano P, Winter C, Bacic D, Stange G, Wagner CA, Biber J, Murer H, Hernando N (2006) Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC). Proc Natl Acad Sci USA 103:803–808

    Article  PubMed  CAS  Google Scholar 

  27. Vliem MJ, Ponsioen B, Schwede F, Pannekoek WJ, Riedl J, Kooistra MR, Jalink K, Genieser HG, Bos JL, Rehmann H (2008) 8-pCPT-2′-O-Me-cAMP-AM: an improved Epac-selective cAMP analogue. Chembiochem 9:2052–2054

    Article  PubMed  CAS  Google Scholar 

  28. Christensen AE, Selheim F, de Rooij J, Dremier S, Schwede F, Dao KK, Martinez A, Maenhaut C, Bos JL, Genieser HG, Doskeland SO (2003) cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension. J Biol Chem 278:35394–35402

    Article  PubMed  CAS  Google Scholar 

  29. NHMRC AG (2004) Australian code of practise for the care and use of animals for scientific purposes (7th edn). This is a small booklet on the web put by the Australian Gov. The web URL is: http://www.nhrmc.gov.au/publications/eahome.htm

    Google Scholar 

  30. Morrison SF, Reis DJ (1989) Reticulospinal vasomotor neurons in the RVL mediate the somatosympathetic reflex. Am J Physiol 256:R1084–R1097

    PubMed  CAS  Google Scholar 

  31. Gootman PM, Cohen MI (1970) Efferent splanchnic activity and systemic arterial pressure. Am J Physiol 219:897–903

    PubMed  CAS  Google Scholar 

  32. Makeham JM, Goodchild AK, Pilowsky PM (2001) NK1 receptor and the ventral medulla of the rat: bulbospinal and catecholaminergic neurons. Neuroreport 12:3663–3667

    Article  PubMed  CAS  Google Scholar 

  33. Paine TA, Neve RL, Carlezon WA Jr (2009) Attention deficits and hyperactivity following inhibition of cAMP-dependent protein kinase within the medial prefrontal cortex of rats. Neuropsychopharmacology 34:2143–2155

    Article  PubMed  CAS  Google Scholar 

  34. Holz GG, Kang G, Harbeck M, Roe MW, Chepurny OG (2006) Cell physiology of cAMP sensor Epac. J Physiol 577:5–15

    Article  PubMed  CAS  Google Scholar 

  35. Bos JL (2003) Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4:733–738

    Article  PubMed  CAS  Google Scholar 

  36. Kaupp UB, Seifert R (2002) Cyclic nucleotide-gated ion channels. Physiol Rev 82:769–824

    PubMed  CAS  Google Scholar 

  37. Ster J, de Bock F, Bertaso F, Abitbol K, Daniel H, Bockaert J, Fagni L (2009) Epac mediates PACAP-dependent long-term depression in the hippocampus. J Physiol 587:101–113

    Article  PubMed  CAS  Google Scholar 

  38. Zhong N, Zucker RS (2005) cAMP acts on exchange protein activated by cAMP/cAMP-regulated guanine nucleotide exchange protein to regulate transmitter release at the crayfish neuromuscular junction. J Neurosci 25:208–214

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Australian School of Advanced Medicine, Macquarie University, North Ryde, NSW, Australia

    V. J. Tallapragada

Authors
  1. V. J. Tallapragada
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to V. J. Tallapragada .

Editor information

Editors and Affiliations

  1. , Australian School of Advanced Medicine, Macquarie University, Technology Place 2, North Ryde, 2113, New South Wales, Australia

    Paul M. Pilowsky

  2. , Australian School of Advanced Medicine, Macquarie University, Technology Place 2, North Ryde, 2113, New South Wales, Australia

    Melissa M.J. Farnham

  3. , Australian School of Advanced Medicine, Macquarie University, Technology Place 2, North Ryde, 2113, New South Wales, Australia

    Angelina Y. Fong

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Tallapragada, V.J. (2013). In Vivo Manipulation of Intracellular Signalling Pathways. In: Pilowsky, P., Farnham, M., Fong, A. (eds) Stimulation and Inhibition of Neurons. Neuromethods, vol 78. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-233-9_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-233-9_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-232-2

  • Online ISBN: 978-1-62703-233-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation