Elsevier

Neuroscience

Volume 97, Issue 2, April 2000, Pages 293-302
Neuroscience

Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors

https://doi.org/10.1016/S0306-4522(99)00597-7Get rights and content

Abstract

Recent evidence suggests that some types of neurotensin receptors may be expressed by astrocytes. In order to explore the function of neurotensin receptors in astrocytes, the effect of a neurotensin receptor agonist, neurotensin(8–13), on intracellular Ca2+ dynamics in mixed neuronal/glial cultures prepared from rat ventral tegmental area was examined. It was found that neurotensin(8–13) induces a long-lasting rise in intracellular Ca2+ concentration in a subset of glial fibrilary acidic protein-positive glial cells. This response displays extensive desensitization and appears to implicate both intracellular and extracellular Ca2+ sources. In the absence of extracellular Ca2+, neurotensin(8–13) evokes only a short-lasting rise in intracellular Ca2+. The neurotensin-evoked intracellular Ca2+ accumulation is blocked by the phospholipase C inhibitor U73122 and by thapsigargin, suggesting that it is initiated by release of Ca2+ from an inositol triphosphate-dependent store. The Ca2+-mobilizing action of neurotensin(8–13) in astrocytes is dependent on at least two receptors, because the response is blocked in part only by SR48692, a type 1 neurotensin receptor antagonist, and is blocked completely by SR142948A, a novel neurotensin receptor antagonist. The finding that the type 2 neurotensin receptor agonist levocabastine fails to mimic or alter the effects of neurotensin(8–13) on intracellular Ca2+ makes it unlikely that the type 2 neurotensin receptor is involved.

In summary, these results show that functional neurotensin receptors are present in cultured ventral tegmental area astrocytes and that their activation induces a highly desensitizing rise in intracellular Ca2+. The pharmacological profile of this response suggests that a type 1 neurotensin receptor is involved but that another, possibly novel, non-type 2 neurotensin receptor is also implicated. If present in vivo, such signalling could be involved in some of the physiological actions of neurotensin.

Section snippets

Cell culture

Primary cultures of rat VTA were prepared from neonatal animals (postnatal days 1–3). Two small blocks of tissue containing the left and right portions of the VTA were dissected out using a custom tissue micropunch from a coronal slice with a thickness of approximately 1.5 mm and localized rostrocaudally at the level of the midbrain flexure. The tissue was incubated in papaı̈n for 30 min and dissociated according to a protocol modified from Cardozo.12 Modifications to the protocol included the

Calcium-mobilizing action of neurotensin(8–13) on ventral tegmental area astrocytes

Experiments were performed on primary cultures of rat VTA. These preparations contained both neurons and astrocytes. Dopaminergic neurons comprised approximately 50% of the neuronal population, other neurons being mostly GABAergic. A glial cell monolayer covered most of the coverslip surface. These cells were exclusively astrocytes as suggested by their immunoreactivity to glial fibrillary acidic protein (not shown).

Intracellular Ca2+ concentration in astrocytes was monitored by fluorescence

Identity of neurotensin receptors on astrocytes

Although previous reports have already suggested that astrocytes can express NT receptors,34., 51., 52. the identity of the receptor(s) and the functional consequences of the activation of such receptors were unclear. The present work clarifies these issues by providing a detailed characterization of NT-evoked Ca2+ signalling in cultured VTA astrocytes. In line with the report of Hösli et al.,34 the present results are compatible with the idea that cultured VTA astrocytes express NT1 receptors.

Conclusions

This work provides clear evidence for the existence of functional NT receptors in astrocytes. These receptors cause a mobilization of intracellular Ca2+ and display pharmacological properties that are difficult to explain with the existence of a single type of NT receptor in astrocytes. Because the present work was performed in cultured cells, it remains unclear whether the reported observations have direct relevance for an understanding of the actions of NT in the intact brain. The first

Acknowledgements

This work was supported in part by the Medical Research Council of Canada, the EJLB Foundation, the Fonds de la Recherche en Santé du Québec and the Fonds pour les Chercheurs et l'Aide à la Recherche du Québec. Helpful comments on this manuscript were provided by Drs Philip Haydon, Pierre-Paul Rompré and Patrice Congar, as well as by François Michel. The assistance of Isabel Jutras and Marie-Josée Bourque in the preparation of cell cultures is acknowledged. The NT receptor antagonists SR48692

References (74)

  • G Grynkiewicz et al.

    A new generation of Ca2+ indicators with greatly improved fluorescence properties

    J. biol. Chem.

    (1985)
  • M Heaulme et al.

    Involvement of potentially distinct neurotensin receptors in neurotensin-induced stimulation of striatal [3H]dopamine release evoked by KCl versus electrical depolarization

    Neuropharmacology

    (1997)
  • E Hermans et al.

    Rapid desensitization of agonist-induced calcium mobilization in transfected PC12 cells expressing the rat neurotensin receptor

    Biochem. biophys. Res. Commun.

    (1994)
  • E Hermans et al.

    Mechanisms of regulation of neurotensin receptors

    Pharmac. Ther.

    (1998)
  • E Hermans et al.

    Phospholipase C activation by neurotensin and neuromedin N in Chinese hamster ovary cells expressing the rat neurotensin receptor

    Brain Res. molec. Brain Res.

    (1992)
  • L.J Holmes et al.

    Dopamine-dependent contralateral circling induced by neurotensin applied unilaterally to the ventral tegmental area in rats

    Brain Res. Bull.

    (1985)
  • E Hosli et al.

    Autoradiographic and electrophysiological evidence for the existence of neurotensin receptors on cultured astrocytes

    Neuroscience

    (1995)
  • F.B Jolicoeur et al.

    Relationships between structure and duration of neurotensin's central action: emergence of long acting analogs

    Neuropeptides

    (1984)
  • Y Masuo et al.

    Regulation of neurotensin-containing neurons in the rat striatum. Effects of unilateral striatal lesions with quinolinic acid and ibotenic acid on neurotensin content and its binding site density

    Brain Res.

    (1990)
  • J Mazella et al.

    The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor

    J. biol. Chem.

    (1998)
  • E Moyse et al.

    Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [125I]Tyr3-neurotensin

    Neuroscience

    (1987)
  • E Nalivaiko et al.

    Electrophysiological evidence for putative subtypes of neurotensin receptors in guinea-pig mesencephalic dopaminergic neurons

    Neuroscience

    (1998)
  • D Nouel et al.

    Centrally administered [d-Trp11]neurotensin, as well as neurotensin protected from inactivation by thiorphan, modifies locomotion in rats in a biphasic manner

    Peptides

    (1990)
  • D Nouel et al.

    Pharmacological, molecular and functional characterization of glial neurotensin receptors

    Neuroscience

    (1999)
  • R.D Pinnock

    Neurotensin depolarizes substantia nigra dopamine neurones

    Brain Res.

    (1985)
  • R Quirion

    Interactions between neurotensin and dopamine in the brain: an overview

    Peptides

    (1983)
  • R Quirion et al.

    Autoradiographic distribution of [3H]neurotensin receptors in rat brain: visualization by tritium-sensitive film

    Peptides

    (1982)
  • R Robitaille

    Modulation of synaptic efficacy and synaptic depression by glial cells at the frog neuromuscular junction

    Neuron

    (1998)
  • P.P Rompre et al.

    Facilitation of brain stimulation reward by mesencephalic injections of neurotensin(1–13)

    Eur. J. Pharmac.

    (1992)
  • M Sato et al.

    Neurotensin and neuromedin N elevate the cytosolic calcium concentration via transiently appearing neurotensin binding sites in cultured rat cortex cells

    Brain Res. devl Brain Res.

    (1991)
  • P Schaeffer et al.

    Human umbilical vein endothelial cells express high affinity neurotensin receptors coupled to intracellular calcium release

    J. biol. Chem.

    (1995)
  • V Seutin et al.

    Electrophysiological effects of neurotensin on dopaminergic neurones of the ventral tegmental area of the rat in vitro

    Neuropharmacology

    (1989)
  • J Singh et al.

    Effects of microinjections of cholecystokinin and neurotensin into lateral hypothalamus and ventral mesencephalon on intracranial self-stimulation

    Pharmac. Biochem. Behav.

    (1997)
  • F Sotty et al.

    Differential effects of neurotensin on dopamine release in the caudal and rostral nucleus accumbens: a combined in vivo electrochemical and electrophysiological study

    Neuroscience

    (1998)
  • R Steinberg et al.

    SR 48692, a non-peptide neurotensin receptor antagonist, differentially affects neurotensin-induced behaviour and changes in dopaminergic transmission

    Neuroscience

    (1994)
  • R Steinberg et al.

    Neurochemical and behavioural effects of neurotensin vs [d-Tyr11]neurotensin on mesolimbic dopaminergic function

    Neuropeptides

    (1995)
  • K Tanaka et al.

    Structure and functional expression of the cloned rat neurotensin receptor

    Neuron

    (1990)
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