Activity‐Dependent Dynamics of Coexisting Brain‐Derived Neurotrophic Factor, Pro‐Opiomelanocortin and α‐Melanophore‐Stimulating Hormone in Melanotrope Cells ofXenopus laevis

Abstract

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…HPF has been previously combined with immunolabeling (Donohoe et al, 2007;McDonald, 1999;Mobius et al, 2002;Monaghan et al, 1998;Nicolas et al, 1997) and in particular has been applied to brain tissue focusing on myelin glycolipids (Kirschning et al, 1998), cytoskeletal tethers in rat hippocampal PSDs (Rostaing et al, 2006;Siksou et al, 2007), kainate receptors in the monkey striatum (Kieval et al, 2001), GABA receptors in the monkey subthalamic nucleus (Galvan et al, 2004) and neurohormonal/secretory activity in neuroendocrine melanotrope cells (Calle et al, 2005;Wang et al, 2004). In several of these studies (e.g.…”

“…Examples of tissue that fall into this category that have been successfully cryo-fixed with HPF include rodent skin (Al-Amoudi et al, 2005;Reipert et al, 2004), cartilage (Al-Amoudi et al, 2005;Studer et al, 1995), cultured pancreatic islet cells (Marsh et al, 2004), kidney (Vanhecke et al, 2006), Xenopus laevis pituitary gland (Wang et al, 2004) and liver (Hsieh et al, 2006).…”

Sortilin Controls Intracellular Sorting of Brain-Derived Neurotrophic Factor to the Regulated Secretory Pathway

“…Because BDNF has been shown to be targeted to secretory granules in neurons and PC12 cells Mowla et al, 1999;Wang et al, 2004), we assessed whether sortilin colocalized in the same compartment in differentiated PC12 cells (Fig. 4 A).…”

Plasticity in the Melanotrope Neuroendocrine Interface of <i>Xenopus laevis</i>

“…Moreover, quantitative RT-PCR has demonstrated an increased expression of this neurotropin in black-adapted animals. BDNF co-exists with α-MSH in melanotrope secretory granules [71]. This suggests that BDNF is co-secreted with α-MSH in animals on black background.…”