Advertisement

Experimental Brain Research

, Volume 106, Issue 2, pp 205–214 | Cite as

Electrophysiological characterization of dopaminergic and non-dopaminergic neurones in organotypic slice cultures of the rat ventral mesencephalon

  • B. H. Steensen
  • S. Nedergaard
  • K. Østergaard
  • J. D. C. Lambert
Research Article

Abstract

The aim of the present study was to characterize electrophysiologically neurones in organotypic cultures of the rat ventral mesencephalon and to compare these results with results published for the same neurones in other types of preparation. Intracellular recordings were obtained in 3- to 8-week-old organotypic slice cultures of the ventral mesencephalon prepared from newborn rats. Dopaminergic neurones were distinguished from non-dopaminergic neurones by staining with the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and briefly viewing the preparation with short exposures to ultraviolet (UV) light (365 nm). Short exposures to UV light did not affect the electrophysiological properties. There were no significant differences between dopaminergic and non-dopaminergic neurones with regard to resting membrane potential or action potential threshold and amplitude, and in both types of neurone spontaneous burst activity and glutamatergic excitatory postsynaptic potentials were seen. There were differences in the following parameters, which can be used to distinguish between the two types of neurone. Dopaminergic neurones had broad action potentials (2–9 ms), high input resistance (mean 81 MΩ), were silent or fired spontaneously at a low frequency (0–9 Hz), and no spontaneous GABAA-ergic inhibitory postsynaptic potentials or inward rectification were present. In contrast, non-dopaminergic neurones had fast action potentials (0.6–3.2 ms), low input resistance (mean 32 MΩ), were silent or fired spontaneously at relatively high firing frequency (0–28 Hz), and sometimes inhibitory postsynaptic potentials and inward rectification were seen. In the presence of 1 μM tetrodotoxin and 10 mM tetraethylammonium, Ca2+ spikes could be evoked in both dopaminergic and non-dopaminergic neurones. Dopaminergic neurones in 3- to 8-week-old organotypic slice cultures have a number of distinguishing electrophysiological characteristics similar to those recorded in other types of acute or cultured preparations. However, some intrinsic regulatory mechanisms, namely the slow oscillatory potentials, inward rectification and the K+ current, IA, seem to be missing in the cultured neurones.

Key words

Organotypic slice culture 5,7-Dihydroxytryptamine Dopaminergic neurones Ventral mesencephalon Substantia nigra Rat 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baumgarten HG, Klemm HP, Lachenmayer L, Björklund A, Lovenberg W, Schlossberger HG (1978) Mode and mechanism of action of neurotoxic indoleamines: a review and progress report. Ann NY Acad Sci 305: 3–24Google Scholar
  2. Caeser M, Bonhoeffer T, Bolz J (1989) Cellular organization and development of slice cultures from rat visual cortex. Exp Brain Res 77: 234–244Google Scholar
  3. Cardozo DL (1993) Midbrain dopaminergic neurons from postnatal rat in long-term primary culture. Neuroscience 56: 409–421Google Scholar
  4. Chiodo LA, Kapatos G (1992) Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture. Synapse 11: 294–309Google Scholar
  5. Chiodo LA, Antelman SM, Caggiula AR, Lineberry CG (1980) Sensory stimuli alter the discharge rate of dopamine (DA) neurons: evidence for two functional types of DA cells in the substantia nigra. Brain Res 189: 544–549Google Scholar
  6. Fujimura K, Matsuda Y (1989) Autogenous oscillatory potentials in neurons of the guinea pig substantia nigra pars compacta in vitro. Neurosci Lett 104: 53–57Google Scholar
  7. Gähwiler BH (1984) Slice cultures of cerebellar, hippocampal and hypothalamic tissue. Experientia 40: 235–308Google Scholar
  8. Gähwiler BH (1988) Organotypic cultures of neural tissue. Trends Neurosci 11: 484–489Google Scholar
  9. Grace AA (1988) In vivo and in vitro intracellular recordings from rat midbrain dopamine neurons. Ann NY Acad Sci 537: 51–76Google Scholar
  10. Grace AA (1991) Regulation of spontaneous activity and oscillatory spike firing in rat midbrain dopamine neurons recorded in vitro. Synapse 7: 221–234Google Scholar
  11. Grace AA, Bunney BS (1984a) The control of firing pattern in nigral dopamine neurons: single spike firing. J Neurosci 4: 2866–2876Google Scholar
  12. Grace AA, Bunney BS (1984b) The control of firing pattern in nigral neurons: burst firing. J Neurosci 4: 2877–2890Google Scholar
  13. Grace AA, Onn S-P (1989) Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci 9: 3463–3481Google Scholar
  14. Guyenet PG, Aghajanian GK (1978) Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra. Brain Res 150: 69–84Google Scholar
  15. Hainsworth AH, Röper J, Kapoor R, Ashcroft FM (1991) Identification and electrophysiology of isolated pars compacta neurons from guinea-pig substantia nigra. Neuroscience 43: 81–93Google Scholar
  16. Harris NC, Webb C, Greenfield SA (1989) A possible pacemaker mechanism in pars compacta neurons of the guinea-pig substantia nigra revealed by various ion channel blocking agents. Neuroscience 31: 355–362Google Scholar
  17. Jaeger C, Ruiz AG, Llinás R (1989) Organotypic slice cultures of dopaminergic neurons of substantia nigra. Brain Res Bull 22: 981–991Google Scholar
  18. Kang Y, Kitai ST (1993) Calcium spike underlying rhythmic firing in dopaminergic neurons of the rat substantia nigra. Neurosci Res 18: 195–207Google Scholar
  19. Kita T, Kita H, Kitai ST (1986) Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation. Brain Res 372: 21–30Google Scholar
  20. Lacey MG, North RA (1988) An inward current activated by hyperpolarization (Ih) in rat substantia nigra zona compacta neurones in vitro (abstract). J Physiol (Lond) 406:18PGoogle Scholar
  21. Lacey MG, Mercuri NB, North RA (1987) Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol (Lond) 392: 397–416Google Scholar
  22. Lacey MG, Mercuri NB, North RA (1989) Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. J Neurosci 9: 1233–1241Google Scholar
  23. Lacey MG, Calabresi P, North RA (1990) Muscarine depolarizes rat substantia nigra zona compacta and ventral tegmental neurons in vitro through M1-like receptors. J Pharmacol Exp Ther 253: 395–400PubMedGoogle Scholar
  24. Levine MS, Fisher RS, Hull CD, Buchwald NA (1982) Development of spontaneous neuronal activity in the caudate nucleus, globus pallidus-entopeduncular nucleus, and substantia nigra of the cat. Dev Brain Res 3: 429–441Google Scholar
  25. Masuko S, Nakajima S, Nakajima Y (1992) Dissociated high-purity dopaminergic neuron cultures from the substantia nigra and the ventral tegmental area of the postnatal rat. Neuroscience 49: 347–364Google Scholar
  26. Nedergaard S, Greenfield SA (1992) Sub-populations of pars compacta neurons in the substantia nigra: the significance of qualitatively and quantitatively distinct conductances. Neuroscience 48: 423–437Google Scholar
  27. Nedergaard S, Flatman JA, Engberg I (1991) Excitation of substantia nigra pars compacta neurones by 5-hydroxy-tryptamine in vitro. Neuro Report 2: 329–332Google Scholar
  28. Nedergaard S, Flatman JA, Engberg I (1993) Nifedipine- and omega-conotoxin-sensitive Ca2+ conductances in guinea-pig substantia nigra pars compacta neurones. J Physiol (Lond) 466: 727–747Google Scholar
  29. Ort CA, Futamachi KJ, Peacock JH (1988) Morphology and electrophysiology of ventral mesencephalon nerve cell cultures. Dev Brain Res 39: 205–215Google Scholar
  30. Østergaard K, Schou JP, Zimmer J (1990) Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum, hippocampus, and cerebellum. Exp Brain Res 82: 547–565Google Scholar
  31. Østergaard K, Schou JP, Gähwiler BH, Zimmer J (1991) Tyrosine hydroxylase immunoreactive neurons in organotypic slice culures of the rat striatum and neocortex. Exp Brain Res 83: 357–365Google Scholar
  32. Pitts DK, Freeman AS, Chiodo LA (1990) Dopamine neuron ontogeny: electrophysiological studies. Synapse 6: 309–320Google Scholar
  33. Segal M (1986) 5,7-Dihydroxytryptamine modifies excitability of rat dorsal raphe serotonergic neurons in vitro. Neurosci Lett 71: 306–310Google Scholar
  34. Shepard PD, German DC (1988) Electrophysiological and pharmacological evidence for the existence of distinct subpopulations of nigrostriatal neurons in the rat. Neuroscience 27: 537–546Google Scholar
  35. Silva NL, Mariani AP, Harrison NL, Barker JL (1988) 5,7-dihydroxytryptamine identifies living dopaminergic neurons in mesencephalic cultures. Proc Natl Acad Sci USA 85: 7346–7350Google Scholar
  36. Tepper JM, Trent F, Nakamura S (1990) Postnatal development of the electrical activity of rat nigrostriatal dopaminergic neurons. Exp Brain Res 54: 21–33Google Scholar
  37. Walsh JP, Cepeda C, Buchwald NA, Levine MS (1991) Neurophysiological maturation of cat substantia nigra neurons: evidence from in vitro studies. Synapse 7: 291–300Google Scholar
  38. Wang RY (1981) Dopaminergic neurons in the rat ventral tegmental area. I. Identification and characterization. Brain Res Rev 3: 123–140Google Scholar
  39. Wuttke W, Hancke JL, Höhn KG, Baumgarten HG (1978) Effect of intraventricular injection of 5,7-dihydroxytryptamine on serum gonadotropins and prolactin. Ann NY Acad Sci 305: 423–436Google Scholar
  40. Yung WH, Häusser MA, Jack JJB (1991) Electrophysiology of dopaminergic and non-dopaminergic neurones of the guinea-pig substantia nigra pars compacta in vitro. J Physiol (Lond) 436: 643–667Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • B. H. Steensen
    • 1
  • S. Nedergaard
    • 1
  • K. Østergaard
    • 2
  • J. D. C. Lambert
    • 1
  1. 1.Institute of Physiology, University of AarhusÅrhus CDenmark
  2. 2.Department of Anatomy and Cell BiologyUniversity of OdenseOdense CDenmark

Personalised recommendations