Elsevier

Neuroscience

Volume 156, Issue 3, 15 October 2008, Pages 563-579
Neuroscience

Cellular neuroscience
Distribution and neuropeptide coexistence of nucleobindin-2 mRNA/nesfatin-like immunoreactivity in the rat CNS

https://doi.org/10.1016/j.neuroscience.2008.07.054Get rights and content

Abstract

The protein fragment nesfatin-1 was recently implicated in the control of food intake. Central administration of this fragment results in anorexia and reduced body weight gain, whereas antisense or immunological nesfatin-1 antagonism causes increased food intake and overweight. Nesfatin-1 is derived from the precursor nucleobindin-2 (NUCB2). To identify the neurocircuitry underpinning the catabolic effects of NUCB2/nesfatin-1, we have used in situ hybridization and immunohistochemistry to map the distribution of this protein and its mRNA in the rat CNS and performed double-labeling experiments to localize its expression to functionally defined neuronal populations. These experiments confirm previous observations but also present several novel NUCB2 cell populations. Both NUCB2 mRNA and nesfatin-like immunoreactivity was most concentrated in the hypothalamus, in the supraoptic, paraventricular, periventricular and arcuate nuclei and the lateral hypothalamic area/perifornical region. Additionally, outside of the hypothalamus, labeling was observed in the thalamic parafascicular nucleus, the Edinger-Westphal nucleus, locus coeruleus, ventral raphe system, nucleus of solitary tract and in the preganglionic sympathetic intermediolateral cell column of the spinal cord, and the pituitary anterior and intermediate lobes. In neurons, immunoreactivity was almost exclusively confined to perikarya and primary dendrites with virtually no labeling of axonal terminals. Double-labeling immunohistochemistry revealed colocalization of nesfatin with vasopressin and oxytocin in magnocellular neuroendocrine neurons, thyrotropin-releasing hormone, corticotropin-releasing hormone, somatostatin, neurotensin, and growth-hormone-releasing hormone in parvocellular neuroendocrine neurons, pro-opiomelanocortin (but not neuropeptide Y) in the arcuate nucleus and melanin-concentrating hormone (but not hypocretin) in the lateral hypothalamus. Furthermore, nesfatin was extensively colocalized with cocaine- and amphetamine-regulated transcript in almost all NUCB2-expressing brain regions. These data reveal a wider distribution of NUCB2/nesfatin-1 than previously known, suggesting that the metabolic actions of this protein may involve not only feeding behavior but also endocrine and autonomic effects on energy expenditure. In addition, the subcellular distribution of nesfatin-like immunoreactivity indicates that this protein may not be processed like a conventional secreted neuromodulator.

Section snippets

Animals

Male Sprague–Dawley rats (6–8 weeks; Scanlab, Stockholm, Sweden) were housed under 12-h dark/light conditions (lights on at 06:00 h and off at 18:00 h) in a temperature-controlled environment with free access to standard rat chow and tap water. All experiments had been approved by the local ethical committee (Stockholms Norra Djurförsöksetiska Nämnd) and the studies were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were

Results

The distribution of NUCB2 mRNA was investigated by in situ hybridization (Fig. 1). The four oligonucleotide probes, complementary to non-overlapping segments of the NUCB2 cDNA, tested separately, yielded qualitatively similar in situ hybridization patterns, albeit with varying signal strength. The highest signal-to-noise ratio resulted from a combination of probes against base pairs 438–486 and 1402–1450; in the results described below and figures this combination was used. Furthermore, the

Discussion

Here we have studied the rat CNS distribution of NUCB2 mRNA and its newly described protein fragment nesfatin-1 which has been ascribed anorexigenic properties. We show a wide distribution in the hypothalamus and a restricted set of other brain regions, as well as its colocalization with several neuropeptides implicated in the control of energy metabolism. The brain distribution is summarized schematically in Fig. 10 (NUCB2/nesfatin cells indicated on plates from Paxinos et al., 2007). Our

Acknowledgments

The authors gratefully acknowledge the donors of antisera used in this study (see Experimental Procedures). We also thank Drs. Staffan Cullheim and Tomas Hökfelt and their respective laboratories for sharing expertise and equipment, and Dr. David Lyons for helpful comments on the manuscript. Emanuela Santini is gratefully acknowledged for expert assistance with Western blot experiments. Financial support for this study and publication was provided by the European Neuroscience Institutes

References (63)

  • R. Poggioli et al.

    ACTH-(1–24) and alpha-MSH antagonize feeding behavior stimulated by kappa opiate agonists

    Peptides

    (1986)
  • R. Salin-Pascual et al.

    Hypothalamic regulation of sleep

    Neuropsychopharmacology

    (2001)
  • C.B. Saper et al.

    The need to feed: homeostatic and hedonic control of eating

    Neuron

    (2002)
  • C.B. Saper et al.

    Direct hypothalamo-autonomic connections

    Brain Res

    (1976)
  • L.W. Swanson et al.

    A direct projection from the ventromedial nucleus and retrochiasmatic area of the hypothalamus to the medulla and spinal cord of the rat

    Neurosci Lett

    (1980)
  • L.W. Swanson et al.

    Neural mechanisms for the functional coupling of autonomic, endocrine and somatomotor responses in adaptive behavior

    Brain Res

    (1981)
  • N. Taniguchi et al.

    The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm

    J Biol Chem

    (2000)
  • S. Barnikol-Watanabe et al.

    Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper proteinMolecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein

    Biol Chem Hoppe Seyler

    (1994)
  • G.C. Brailoiu et al.

    Nesfatin-1: distribution and interaction with a G protein-coupled receptor in the rat brain

    Endocrinology

    (2007)
  • C. Broberger

    Cocaine- and amphetamine-regulated transcript (CART) and food intake: Behavior in search of anatomy

    Drug Dev Res

    (2000)
  • C. Broberger

    Brain regulation of food intake and appetite: molecules and networks

    J Intern Med

    (2005)
  • C. Broberger et al.

    Hypocretin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems

    J Comp Neurol

    (1998)
  • C. Broberger et al.

    Neuropeptide Y innervation and neuropeptide-Y-Y1-receptor-expressing neurons in the paraventricular hypothalamic nucleus of the mouse

    Neuroendocrinology

    (1999)
  • A.A. Butler et al.

    Melanocortin-4 receptor is required for acute homeostatic responses to increased dietary fat

    Nat Neurosci

    (2001)
  • N. Chu et al.

    Norepinephrine-containing neurons: changes in spontaneous discharge patterns during sleeping and waking

    Science

    (1973)
  • R.D. Cone

    Anatomy and regulation of the central melanocortin system

    Nat Neurosci

    (2005)
  • Å. Dagerlind et al.

    Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

    Histochemistry

    (1992)
  • C.F. Elias et al.

    Characterization of CART neurons in the rat and human hypothalamus

    J Comp Neurol

    (2001)
  • W. Fan et al.

    Role of melanocortinergic neurons in feeding and the agouti obesity syndrome

    Nature

    (1997)
  • I.S. Farooqi et al.

    Dominant and recessive inheritance of morbid obesity associated with melanocortin 4 receptor deficiency

    J Clin Invest

    (2000)
  • K. Foo et al.

    Distribution and coexpression patterns of nucleobindin-2 (NUCB2) mRNA and nesfatin-1 peptide in the rat hypothalamus

    Soc Neurosci Abstr

    (2007)
  • Cited by (0)

    View full text