Figure 3 - uploaded by Fabio Bianco
Content may be subject to copyright.
Immunofluorescence staining on primary neuronal cultures. Immunofluorescence staining with GHS-R of either cortical (CN) or hippocampal (HN) neurons at different stages of development. Both neuronal cultures show correct maturation in vitro, as assayed by positive staining of vesicular marker for excitatory neurotransmitter transporter Vglut which shows punctuated expression along neuritis at mature stages of development. doi:10.1371/journal.pone.0064183.g003
Source publication
Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds...
Context in source publication
Context 1
... neuronal cultures showed correct maturation in vitro, given staining with vesicular marker for excitatory neurotransmitter transporter Vglut show punctuated expression along neuritis branches at mature stages of development. Interestingly, staining for GHS-R showed in both neuronal populations a transient positivity, with a stronger signal at earlier stages of development (up to 9 days) and a significant reduction at later stages (Fig.3). In order to evaluate at which stage of neuronal development there was a greater expression of GHS-R, qRT-PCR experiments were performed on the mRNA from primary rat hippocampal and cortical neurons; the expression of specific mRNA, assayed at 4, 9, 16 and 21 days ''in vitro'' and normalized on the expression at 4 div, showed increasing levels of expression until the reaching of mature stage (16 div) and subsequently a significant decrease at later stages in culture (21 div both in the cortex and in the hippocampus) (Fig. 4A). ...
Citations
... The brain protective function of ghrelin is based on its ability to penetrate the blood-brain barrier (BBB) (Banks et al. 2002). GHS-R1α is a G protein-coupled receptor, mediating the major effects of ghrelin (Abizaid and Hougland 2020), and is expressed in the hippocampal region (Lattuada et al. 2013). The hippocampal region is associated with memory, and ghrelin/GHS-R1α interaction enhanced memory directly (Buntwal et al. 2019) (Pereira et al. 2017). ...
This study is to observe the upregulation effect of astragaloside IV on ghrelin in diabetic cognitive impairment (DCI) rats and to investigate the pathway in prevention and treatment by reducing oxidative stress. The DCI model was induced with streptozotocin (STZ) in conjunction with a high-fat and high-sugar diet and divided into three groups: model, low-dose (40 mg/kg), and high-dose (80 mg/kg) astragaloside IV. After 30 days of gavage, the learning and memory abilities of rats, as well as their body weight and blood glucose levels, were tested using the Morris water maze and then detection of insulin resistance, SOD activity, and serum MDA levels. The whole brain of rats was sampled for hematoxylin–eosin and Nissl staining to observe pathological changes in the hippocampal CA1 region. Immunohistochemistry was used to detect ghrelin expression in the hippocampal CA1 region. A Western blot was used to determine changes in GHS-R1α/AMPK/PGC-1α/UCP2. RT-qPCR was used to determine the levels of ghrelin mRNA. Astragaloside IV reduced nerve damage, increased superoxide dismutase (SOD) activity, decreased MDA levels, and improved insulin resistance. Ghrelin levels and expression increased in serum and hippocampal tissues, and ghrelin mRNA levels increased in rat stomach tissues. According to Western blot, it increased the expression of the ghrelin receptor GHS-R1α and upregulated the mitochondrial function associated-protein AMPK-PGC-1α-UCP2. Astragaloside IV increases ghrelin expression in the brain to reduce oxidative stress and delay diabetes-induced cognitive impairment. It may be related to the promotion of ghrelin mRNA levels.
... Treatment: Pyramidal hippocampal neurons of 15 days in vitro (DIV) were treated with Gr 3 × 10 − 3 nM or 3 × 10 − 4 nM for 24hs Gr (SC1356, Polypeptide group) in order to evaluate the effect of the peptide on the number, differential distribution, density and morphology of the DS. We used cells of 15 days because it has been described that the expression of GHS-R in primary neurons depends on the maturation stage and day 16 shows the relatively highest levels of these receptors (Lattuada et al., 2013). Gr was dissolved in saline solution. ...
Ghrelin (Gr) is an orexigenic peptide that acts via its specific receptor, GHSR-1a distributed throughout the brain, being mainly enriched in pituitary, cortex and hippocampus (Hp) modulating a variety of brain functions. Behavioral, electrophysiological and biochemical evidence indicated that Gr modulates the excitability and the synaptic plasticity in Hp. The present experiments were designed in order to extend the knowledge about the Gr effect upon structural synaptic plasticity since morphological and quantitative changes in spine density after Gr administration were analyzed “in vitro” and “in vivo”. The results show that Gr administered to hippocampal cultures or stereotactically injected in vivo to Thy-1 mice increases the density of dendritic spines (DS) being the mushroom type highly increased in secondary and tertiary extensions. Spines classified as thin type were increased particularly in primary extensions. Furthermore, we show that Gr enhances selectively the expression of BDNF-mRNA species.
... The ghrelin receptor GHS-R1a is expressed in hypothalamic neuropeptide Y-(NPY) and agouti-related peptide-(AgRP) expressing neurons (NPY/AgRP neurons), somatotropic cells and luteinising hormone (LH)-, thyroid-stimulating hormone (TSH)-, adrenocorticotropic hormone (ACTH)-and prolactin-(PRL) releasing cells in the anterior pituitary [26][27][28][29]. A lower expression of GHS-R1a is found in other brain regions such as the cingulate gyrus [30][31][32], amygdala, olfactory bulb and in hypothalamic ghrelin-receptive neurons adjoining the dorsal vagal complex (DVC) [33]. The expression of GHS-R1a is found across three distinct brain areas: pituitary gland (i.a. ...
Objectives: Ghrelin acts on a variety of central- and peripheral organs causing an orexigenic effect, conclusively followed by increased caloric intake. Recent studies have indicated that ghrelin's function as an orexigenic agent does not entirely reflect the full functional properties of the peptide. Specifically, ghrelin regulates stress-hormone synthesis and secretion therewith affecting the stress-axis. The role of stress in the development of obesity has been extensively studied. However, the orexigenic and underlying stress-regulatory effect of ghrelin has not yet been further considered in the development of stress-induced obesity.
Methods: Therefore, this review aims to accentuate the potential of ghrelin as a factor in the pathological development of stress-induced obesity.
Results: In this review we discuss (1) the ghrelin-mediated intracellular cascades and elucidate the overall bioactivation of the peptide, and (2) the mechanisms of ghrelin signalling and regulation within the central nervous system and the gastro-intestinal system.
Discussion: These biological processes will be ultimately discussed in relation to the pathogenesis of stress-induced obesity.
... Ghrelin, a 28-amino acid brain-gut peptide, activates the growth hormone secretagogue receptors which are expressed widely in the brain (Guan et al., 1997). Recent studies revealed that the receptor expression is developmental, with a stronger staining in the early stages and a weaker expression in the later stages of development (Lattuada et al., 2013). Ghrelin exerts critical roles in the regulation of energy homeostasis, neuroendocrine and neurodegenerative processes, especially in higher brain functions, such as learning and memory consolidation (Spitznagel et al., 2010;Rak-Mardyla, 2013;Murray et al., 2014;Panagopoulos and Ralevski, 2014;Jiao et al., 2017). ...
Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by cognitive deficits and neuronal loss. Deposition of beta-amyloid peptide (Aβ) causes neurotoxicity through the formation of plaques in brains of Alzheimer's disease. Numerous studies have indicated that the neuropeptides including ghrelin, neurotensin, pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y, substance P and orexin are closely related to the pathophysiology of Alzheimer's disease. The levels of neuropeptides and their receptors change in Alzheimer's disease. These neuropeptides exert neuroprotective roles mainly through preventing Aβ accumulation, increasing neuronal glucose transport, increasing the production of neurotrophins, inhibiting endoplasmic reticulum stress and autophagy, modulating potassium channel activity and hippocampal long-term potentiation. Therefore, the neuropeptides may function as potential drug targets in the prevention and cure of Alzheimer's disease.
... GHSR1a is extensively expressed in metabolically active tissues, like liver, pancreas, adipose tissue, pituitary glands, thyroid, kidneys, heart and gonads [50,51]. In the central nervous system, GHSR1a is expressed in cortex, ventral tegmental area, dorsal raphe nuclei, hypothalamus, substantia nigra and hippocampus [52][53][54][55]. This widespread expression of GHSR1a is accountable for the wide range of biological functions of ghrelin in glucose metabolism [56][57][58], lipid metabolism [59,60], in promoting appetite [61][62][63][64], gastric acid secretion [65], gut motility [65], regulation of growth hormone secretion [26,66,67], fertility [68][69][70], memory [57], learning and reward related pathways [62,71,72]. ...
Cachexia is a devastating complication of cancer and an important cause of morbidity and mortality and can have a great effect on quality of life, and sense of self-esteem. Unfortunately; there is no standard cure available for cancer cachexia. Ghrelin; a 28 amino acid orexigenic gut hormone and its mimetics have shown potential benefits in reversing the breakdown of protein and weight loss in catabolic states like cancer cachexia. Ghrelin has effects on several vital pathways in the regulation of appetite, and composition of the body. It increases the secretion of growth hormone and reduces energy expenditure. It plays an important role in regulation of processes associated with cancer and antagonizing protein breakdown in catabolic conditions such as cancer cachexia. Additionally, ghrelin has anti-inflammatory, anti-apoptotic and anxiolytic effects. Administration of ghrelin for short-term has been found to be well-tolerated and safe. These versatile actions of ghrelin and its safety can render it as a potentially useful novel therapy for patients with cancer cachexia. However; there is a need to generate more evidence to support the use of ghrelin in the management of cancer cachexia.
... GHSR1a is extensively expressed in metabolically active tissues, like liver, pancreas, adipose tissue, pituitary glands, thyroid, kidneys, heart and gonads [50,51]. In the central nervous system, GHSR1a is expressed in cortex, ventral tegmental area, dorsal raphe nuclei, hypothalamus, substantia nigra and hippocampus [52][53][54][55]. This widespread expression of GHSR1a is accountable for the wide range of biological functions of ghrelin in glucose metabolism [56][57][58], lipid metabolism [59,60], in promoting appetite [61][62][63][64], gastric acid secretion [65], gut motility [65], regulation of growth hormone secretion [26,66,67], fertility [68][69][70], memory [57], learning and reward related pathways [62,71,72]. ...
Cachexia is a devastating complication of cancer and an important cause of morbidity and mortality and can have a great effect on quality of life, and sense of self-esteem. Unfortunately; there is no standard cure available for cancer cachexia. Ghrelin; a 28 amino acid orexigenic
gut hormone and its mimetics have shown potential benefits in reversing the breakdown of protein and weight loss in catabolic states like cancer cachexia. Ghrelin has effects on several vital pathways in the regulation of appetite, and composition of the body. It increases the
secretion of growth hormone and reduces energy expenditure. It plays an important role in regulation of processes associated with cancer and antagonizing protein breakdown in catabolic conditions such as cancer cachexia. Additionally, ghrelin has anti-inflammatory, antiapoptotic
and anxiolytic effects. Administration of ghrelin for short-term has been found to be well-tolerated and safe. These versatile actions of ghrelin and its safety can render it as a potentially useful novel therapy for patients with cancer cachexia. However; there is a need to generate more evidence to support the use of ghrelin in the management of cancer cachexia
... This transient behavior in both cortical and hippocampal neurons indicates a timedependent manner modulation of this receptor that needs further investigation. (27) The wide distribution of the ghrelin receptor and the number of tissues and cell types known to respond to ghrelin through a yet unidentified mechanism suggest that a number of systems may be affected with the treatment with this hormone or its analogue. Thus, it is imperative that we understand the diverse functions of ghrelin and its mechanisms of action, including the receptors involved, to develop specific and safe agonists or antagonists targeting the mechanism of interest. ...
... These results confirmed our previous observations and indicate not only that GHSR expression in primary neurons is modulated in a time-dependent manner but also that there might be a selective regional distribution which might yield to functional modulatory effects of ghrelin in the brain areas. (27) ...
... Because there is abundant GHSR1a expression in different neuronal populations, (24) we then focused our attention on the brain tissue. We previous demonstrated (27) that there is a significantly higher expression of GHSR in hippocampal rather than cortical neurons, so an immunoprecipitation of rat hippocampus was carried out. In the Western blot analysis MAb 5C9H2 bound a 48 kDa protein band corresponding to the predicted size of GHSR, confirming its specific bind also in this tissue (Fig. 5). ...
Ghrelin is an orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. It has been shown to also play a role in numerous higher brain functions, such as the regulation of inflammation and cell proliferation. Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor highly expressed in brain and detectable in some peripheral tissues. The wide distribution of ghrelin receptor and the number of tissues and cell types known to respond to ghrelin suggest that a number of systems may be affected by treatment with this hormone or its analogues. In this study, we characterized a new GHSR specific monoclonal antibody recognizing specifically the ghrelin receptor. This could be a useful tool for immunoassays aimed at obtaining insights into the physiological and pathological significance of the GHSR/ghrelin system.
... However, it is also expressed in extrahypothalamic structures such as the hippocampus (hp), a brain structure related to learning and memory. The hp is one of the few brain regions that express high levels of GHS-R1a (Bennett et al. 1997;Guan et al. 1997;Lattuada et al. 2013). ...
Ghrelin (Ghr) is a peptide that participates in the modulation of several biological processes. Ghr administration into the hippocampus improves learning and memory in different memory tests. However, the possible mechanisms underlying this effect on memory have not yet been clarified.
The purpose of the present work is to add new insights about the mechanisms by which Ghr modulates long-term memory consolidation in the hippocampus. We examined Ghr effects upon processes related to increased synaptic efficacy as presynaptic glutamate release and changes in the expression of the NR2B-subunits containing n-methyl-d-aspartate receptors (NMDAR), which are critical for LTP induction. We also attempted to determine the temporal window in which Ghr administration induces memory facilitation and if the described effects depend on GHS-R1a stimulation.
The present research demonstrated that Ghr increased glutamate release from hippocampal synaptosomes; intra-hippocampal Ghr administration increased NR2B-subunits expression in CA1 and DG subareas and also reversed the deleterious effects of the NR2B-subunit-specific antagonist, Ro 25-6981, upon memory consolidation and LTP generation in the hippocampus. These effects are likely to be the consequence of GHS-R1a activation.
According to the results above mentioned and previous findings, we can hypothesize some of the mechanisms by which Ghr modulates memory consolidation. At presynaptic level, Ghr stimulates glutamate release, probably by enhancing [Ca(2+)]i. At postsynaptic level, the glutamate released activates NMDAR while Ghr also mediates effects directly activating its specific receptors and increases NR2B-subunit expression.
... Although the highest concentration of it is found in the pituitary gland and hypothalamus, consistent with the known role of ghrelin in GH release and the regulation of body weight and metabolism [10,11], there is also abundant GHSR-1a expression in other, extra-hypothalamic areas including the cortex and brain stem [12][13][14][15][16][17] suggesting that ghrelin plays a much broader role [10,18,19]. A recent comparative study on hippocampal and cortical dissociated neurons showed that GHSR-1 mRNA expression is higher in the hippocampus than in the cortex, and depends on the maturation stage of the cultures [20]. In humans, GHSR-1 mRNA distribution is much more widespread than that of GHSR-1a, and varies spatially and quantitatively from that of the receptor [21,22]. ...
While ghrelin was initially related to appetite stimulation and growth hormone secretion, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of those processes is related to synaptic efficacy and plasticity. Previous studies have shown that ghrelin not only stimulates synapse formation in cultured cortical neurons and hippocampal slices, but also alters some of the electrophysiological properties of neurons in the hypothalamus, amygdala and other subcortical areas. However, direct evidence for ghrelin's ability to modulate the activity in cortical neurons is not available yet. In this study, we investigated the effect of acylated ghrelin on the development of the activity level and activity patterns in cortical neurons, in relation to its effect on synaptogenesis. Additionally, we quantitatively evaluated the expression of the receptor for acylated ghrelin - growth hormone secretagogue receptor-1a (GHSR-1a) during development.
We performed electrophysiology and immunohistochemistry on dissociated cortical cultures from neonates, treated chronically with acylated ghrelin. On average 76 +/- 4.6% of the cortical neurons expressed GHSR-1a. Synapse density was found to be much higher in ghrelin treated cultures than in controls across all age groups (1, 2 or 3 weeks). In all cultures (control and ghrelin treated), network activity gradually increased until it reached a maximum after approximately 3 weeks, followed by a slight decrease towards a plateau. During early developmental stages (1-2 weeks), the activity was much higher in ghrelin treated cultures and consequently, they reached the plateau value almost a week earlier than controls.
Acylated ghrelin leads to earlier network formation and activation in cultured cortical neuronal networks, the latter being a possibly consequence of accelerated synaptogenesis.
... In our study we found GHSR1 expression in dissociated cortical neurons already at 1 DIV, which gradually increased during the first two weeks. This is in consent with the finding of Lattuada et al. [42] who reported increasing level of GHSR1 expression in developing hippocampal and cortical neurons, followed by significant reduction in mature neurons at longer times in cultures. Therefore, it is possible that the effect of ghrelin on synaptogenesis in dissociated cortical neurons is mediated via GHSR1, as was shown in the ventral thalamic area and substantia nigra of mice and rat [43]. ...
Ghrelin was initially related to appetite stimulation and growth hormone secretion. However, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of these processes are related to synaptic efficacy and plasticity. Previous studies indicated that ghrelin has an excitatory effect on neuronal activity, and stimulates synaptic plasticity in vivo. Plasticity in the adult brain occurs in many different ways, including changes in synapse morphology and number. Therefore, we used in vitro neuronal cultures to investigate how ghrelin affects synaptogenesis. We used dissociated cortical cultures of newborn rats, chronically treated with different doses of ghrelin (0.5, 1, 1.5 and 2μM). After one-, two-, three- or four weeks cultures were immunostained for the presynaptic marker synaptophysin. In parallel, additional groups of non-treated cultures were immunostained for detection of ghrelin receptor (GHSR1). During development, GHSR1was increasingly expressed in all type of neurons, as well as the synaptophysin. Synaptic density depended on ghrelin concentration, and was much higher than in controls in all age groups. In conclusion, ghrelin leads to earlier network formation in dissociated cortical networks and an increase in number of synapses. The effect is probably mediated by GHSR1. These findings suggest that ghrelin may provide a novel therapeutic strategy for the treatment of disorders related to synaptic impairment.
