Nöronostatin nöronları üzerinde glutamaterjik sistem etkilerinin immünohistokimyasal olarak araştırılması
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Date
2017-01-06
Authors
Koçoğlu, Sema Serter
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Yeni tanımlanan bir peptit olan nöronostatin, intraserebroventriküler yolla verildiğinde besin alımını baskıladığı için anoreksijenik bir peptit olarak kabul edilmektedir. Nöronostatinin etki mekanizmaları çeşitli çalışmalar ile gösterilmiş olmasına karşın, nöronostatin nöronlarını kontrol eden mekanizmaları açıklayan çalışmalara literatürde rastlanmamıştır. Çalışmamızda; hipotalamusta pekçok nöroendokrin nöronun aktivitesinin düzenlenmesinde rol oynayan ve eksitatör amino asit glutamatı nörotransmitter olarak kullanan glutamaterjik sistemin ve beslenme gibi periferik bir faktörün nöronostatin nöronları üzerindeki etkilerinin gösterilmesi amaçlanmıştır. Bu doğrultuda üç özgün hedef belirlenmiştir. Özgün hedef 1: Nöronostatin nöronlarının regülasyonunda glutamaterjik sistem etkilerinin ve aktivasyon yolaklarının araştırıldığı deneyde, deneklere (n=10; 5 dişi+5 erkek) glutamat reseptör agonistleri (kainik asit, AMPA ve NMDA) veya %0,9 NaCl intraperitoneal yolla verilerek nöronostatin nöronlarında c-Fos, fosforile-CREB ve fosforile-STAT proteinlerinin ekspresyonları değerlendirildi. Kainik asit uygulamasının sadece dişi deneklerde, AMPA ve NMDA uygulamalarının ise her iki cinsiyette de c-Fos eksprese eden nöronostatin nöronlarının sayısında istatistiksel anlamlı artışa neden olduğu gözlendi. Özgün hedef 2: Glutamaterjik sistemin nöronostatin nöronlarının fizyolojik uyarılması üzerindeki etkinliği araştırıldı. Bir grup denek 48 saat aç bırakılırken, ikinci grup 48 saat açlık sonrası ve üçüncü grup ise açlık süresinin bitiminde yapılan glutamat antagonisti (CNQX) uygulamasından 15 dakika sonra 2 saat süreyle beslendi. Beslenme ve açlık grupları arasında c-Fos eksprese eden nöronostatin nöronlarının sayısı açısından bir fark gözlenmezken, beslenmesinin her iki cinsiyette de nöronostatin-immünreaktif nöronlarda pSTAT5 ekspresyonunu arttırdığı ve CNQX uygulamasının bu artışı anlamlı olarak baskıladığı belirlendi. Özgün hedef 3 kapsamında ise nöronostatin nöronlarında glutamat reseptörlerinin varlığı ikili immünofloresans yöntemle araştırıldı. Nöronostatin nöronlarının NMDA ve non-NMDA reseptör alt birimlerini eksprese ettikleri gösterildi. Sonuç olarak, besin alımı ve glutamat agonistleri gibi periferik ya da merkezi sinyaller ile nöronostatin nöronlarının aktive olduğu ve bu aktivasyonda glutamaterjik sistemin etkin rol oynadığı düşünüldü.
Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when intracerebroventricularly given. Although the effect mechanism of neuronostatin have been shown in different studies, there is no reports in the literature describing the mechanisms controlling neuronostatin neurons. Therefore, the purpose of the present study was to examine, the effects of feeding as a peripheral factor on neuronostatin neurons as well as the effects of the glutamatergic system, which uses glutamate as an excitatory amino acid neurotransmitter and plays a role in regulating the activity of many neuroendocrine neurons in the hypothalamus. Three specific aims were proposed: Specific aim 1: Expression of c-Fos, pCREB and pSTAT proteins in neuronostatin neurons were assessed in the experiment which investigated the activation pathways and glutamatergic system effects in regulating the neuronostatin neurons where the animals received intraperitoneal injections of glutamate receptor agonists (kainic acid, AMPA and NMDA) or 0.9% NaCl. A statistically significant increase was observed in the number of c-Fos-expressing neuronostatin neurons in both genders following AMPA and NMDA injections, and an increase was detected only in female subjects after kainic acid injection. Specific aim 2: The the effectiveness of the glutamatergic system on the physiological stimulation of neuronostatin neurons was investigated. While one group of subjects was fasted for 48 hours, the second group was fed for 2 hours after 48 hours fasting and the third group was fed for 2 hours after 15 minutes of glutamate antagonist (CNQX) injection at the end of fasting period. While there was no difference in the number of c-Fos-expressing neuronostatin neurons between the feeding and fasting groups, it was found that feeding increased pSTAT5 expression in neuronostatin-immunoreactive neurons in both genders and that CNQX injection significantly suppressed this increase. Specific aim 3: The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunofluorescence method. The results showed that the neuronostatin neurons express most of the NMDA and non-NMDA receptor subunits. In conclusion, it was suggested that neuronostatin neurons were activated by peripheral or central signals including food intake and/or glutamatergic innervation, and the glutamate receptors play an important role in this activation.
Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when intracerebroventricularly given. Although the effect mechanism of neuronostatin have been shown in different studies, there is no reports in the literature describing the mechanisms controlling neuronostatin neurons. Therefore, the purpose of the present study was to examine, the effects of feeding as a peripheral factor on neuronostatin neurons as well as the effects of the glutamatergic system, which uses glutamate as an excitatory amino acid neurotransmitter and plays a role in regulating the activity of many neuroendocrine neurons in the hypothalamus. Three specific aims were proposed: Specific aim 1: Expression of c-Fos, pCREB and pSTAT proteins in neuronostatin neurons were assessed in the experiment which investigated the activation pathways and glutamatergic system effects in regulating the neuronostatin neurons where the animals received intraperitoneal injections of glutamate receptor agonists (kainic acid, AMPA and NMDA) or 0.9% NaCl. A statistically significant increase was observed in the number of c-Fos-expressing neuronostatin neurons in both genders following AMPA and NMDA injections, and an increase was detected only in female subjects after kainic acid injection. Specific aim 2: The the effectiveness of the glutamatergic system on the physiological stimulation of neuronostatin neurons was investigated. While one group of subjects was fasted for 48 hours, the second group was fed for 2 hours after 48 hours fasting and the third group was fed for 2 hours after 15 minutes of glutamate antagonist (CNQX) injection at the end of fasting period. While there was no difference in the number of c-Fos-expressing neuronostatin neurons between the feeding and fasting groups, it was found that feeding increased pSTAT5 expression in neuronostatin-immunoreactive neurons in both genders and that CNQX injection significantly suppressed this increase. Specific aim 3: The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunofluorescence method. The results showed that the neuronostatin neurons express most of the NMDA and non-NMDA receptor subunits. In conclusion, it was suggested that neuronostatin neurons were activated by peripheral or central signals including food intake and/or glutamatergic innervation, and the glutamate receptors play an important role in this activation.
Description
Keywords
Nöronostatin, Glutamat, Beslenme, Açlık, c-Fos, pSTAT5, Neuronostatin, Glutamate, Feeding, Fasting
Citation
Koçoğlu, S. S. (2017). Nöronostatin nöronları üzerinde glutamaterjik sistem etkilerinin immünohistokimyasal olarak araştırılması. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.