Neuropeptides 90 (2021) 102186 Available online 18 August 2021 0143-4179/© 2021 Elsevier Ltd. All rights reserved. The involvement of the central cholinergic system in the hyperventilation effect of centrally injected nesfatin-1 in rats Gokcen Guvenc-Bayram a,b, Murat Yalcin a,* a Department of Physiology, Faculty of Veterinary Medicine, Uludag University, Bursa 16059, Turkey b Department of Physiology, Faculty of Veterinary Medicine, Dokuz Eylul University, Kiraz, Izmir 35890, Turkey A R T I C L E I N F O Keywords: Nesfatin-1 Cholinergic muscarinic and nicotinic receptors Respiratory parameters Partial oxygen and carbon dioxide pressure Intracerebroventricular A B S T R A C T We recently demonstrated that peripheral and central administration of nesfatin-1 in fasting and satiety states generate hyperventilation activity by increasing tidal volume (TV), respiratory rate (RR), and respiratory minute ventilation (RVM). The present study aimed to investigate the mediation of central cholinergic receptors effective in respiratory control in the hyperventilation activity of nesfatin-1. Besides this, we intended to determine possible changes in blood gases due to hyperventilation activity caused by nesfatin-1 and investigate the mediation of central cholinergic receptors in these changes. Intracerebroventricular (ICV) administration of nesfatin-1 revealed a hyperventilation response with an in- crease in TV, RR, RMV, and pO2 and a decrease in pCO2 in saturated Sprague Dawley rats. ICV pretreatment with the muscarinic receptor antagonist atropine partially blocked the RR, RMV, pO2, and pCO2 responses produced by nesfatin-1 while completely blocking the TV response. However, central pretreatment with nicotinic receptor antagonist mecamylamine blocked the respiratory and blood gas responses induced by nesfatin-1. The study's conclusion demonstrated that nesfatin-1 had active hyperventilation effects resulting in an in- crease in pO2 and a decrease in pCO2. The critical finding of the study was that activation of central cholinergic receptors was involved in nesfatin-1-evoked hyperventilation and blood gas responses. 1. Introduction Nesfatin-1, having 82 amino acids, is one of the products of post- translational cleavage of prohormone NEFA/nucleobindin-2 (NUCB2) due to the action of the specific convertases. Nesfatin-1 was first described as an anorexigenic factor inducing satiety and inhibiting food and water intake (Oh-I et al., 2006). Subsequent studies revealed that nesfatin-1 acts as a very effective neuropeptide in the central nervous system (Palasz et al., 2012). As a neuropeptide, nesfatin-1 is released from numerous brain regions, primarily the hypothalamus, the center of autonomic regulation (Oh-I et al., 2006). Nesfatin-1, as a satiety neu- ropeptide, has been studied for its effects on the respiratory (Ciftci et al., 2019) and cardiovascular systems (Angelone et al., 2020; Schalla and Stengel, 2018) as well as on metabolism (Schalla et al., 2020; Blanco et al., 2018). Many neurotransmitters and neuromodulators involved in the con- trol and regulation of breathing have already been identified and described. It was reported that the neuropeptides such as leptin, bombesin, neuropeptide Y, and orexin etc., which effectively regulate food intake, like nesfatin-1, were also involved in respiratory regulation (Kaczynska et al., 2018). Moreover, orexin has a central interaction with the nesfatinergic (Gok-Yurtseven et al., 2019) and cholinergic (Ohno et al., 2008) neurons. We showed that centrally injected nesfatin-1 might cause a hyperventilation response by increasing both tidal vol- ume (TV) and respiratory rate (RR) (Ciftci et al., 2019). The central cholinergic system regulates respiration via nicotinic receptors (Shao and Feldman, 2009). We reported that CDP-choline as a cholinergic agent also produced a hyperventilation effect by activating central nicotinic receptors and central phospholipase to the thromboxane signaling pathway (Topuz et al., 2014). There is an interaction between central nesfatin-1 and the central cholinergic system. We showed that central cholinergic nicotinic and muscarinic receptors modulated car- diovascular responses evoked by nesfatin-1 (Aydin et al., 2018). Although nesfatin-1 induced hyperventilation effect is shown, the neurochemical and molecular background of nesfatin-1-induced hy- perventilation has not been fully understood yet. Therefore, in the light of previous evidence, we aimed to explain the central mediatory mechanism of nesfatin-1-evoked hyperventilation response in terms of * Corresponding author. E-mail address: muraty@uludag.edu.tr (M. Yalcin). Contents lists available at ScienceDirect Neuropeptides journal homepage: www.elsevier.com/locate/npep https://doi.org/10.1016/j.npep.2021.102186 Received 9 April 2021; Received in revised form 11 August 2021; Accepted 15 August 2021 mailto:muraty@uludag.edu.tr www.sciencedirect.com/science/journal/01434179 https://www.elsevier.com/locate/npep https://doi.org/10.1016/j.npep.2021.102186 https://doi.org/10.1016/j.npep.2021.102186 https://doi.org/10.1016/j.npep.2021.102186 http://crossmark.crossref.org/dialog/?doi=10.1016/j.npep.2021.102186&domain=pdf Neuropeptides 90 (2021) 102186 2 the central cholinergic receptors in the current study. Moreover, the present study also proposed the effect of centrally injected nesfatin-1 on partial O2 (pO2) and CO2 (pCO2) pressure and the mediation of the central cholinergic receptors in the nesfatin-1-produced blood gas responses. 2. Materials and methods 2.1. Animals and experimental procedure The experiments approved by Bursa Uludag University Laboratory Animal Study Ethical Committee were performed on the 80 rats (Spra- gue Dawley, male, 3–4 months old, 250–300 g body weight) obtained from Bursa Uludag University Laboratory Animal Center. The rats were placed as five animals in a cage with free access to food and water. The temperature (20–22 ◦C), humidity (60–70%), and light (12 h light/dark) of the room where the cages were located were adjusted to suit the living conditions of the rats. The preparation of rats for the experiments and the experiments were carried out under xylazine/ketamine (10/70 mg/kg; ip) mixture anes- thesia. Briefly, in the preparation of the rats, the tracheal catheterization to record respiratory parameters, the left femoral arterial catheteriza- tion to collect blood samples for blood gases, and right lateral cerebral ventricle cannulation to perform the ICV pretreatment and treatment was carried out. During the surgical preparation and the experiments, the rats were kept on a heated platform for stabilizing the 37 ◦C body temperature. Each rat was studied once in a single experimental group. The control and the experimental group animals were examined on the same day according to the experimental protocol. In the first set of the study, nesfatin-1 (200 pmol; n = 10) or saline (5 μl; n = 10) was injected ICV and then the respiratory parameters and blood gases change of the rats were monitored. In the second set of the study, the rats were pretreated with the cholinergic muscarinic receptor antagonist atropine (10 μg; ICV), cholinergic nicotinic receptor antag- onist mecamylamine (50 μg; ICV), or saline (5 μl; ICV). 5 min after the pretreatment, the rats were treated with nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV), and then the respiratory parameters and blood gases change of the rats were monitored. The respiratory parameters were recorded for the next 60 min after the treatment. The blood gases analysis was performed before as well as 20 min after the treatment. The blood sample collection time interval was chosen because nesfatin-1 was reported to have the most potential hyperventilation effect at the 20th min of the injection (Ciftci et al., 2019). The experimental protocol of the study was briefly summarized in Table 1. 2.2. Analysis of respiratory parameters and blood gases The TV, RR, and RMV parameters were recorded by using the MP36 system (BIOPAC Systems Inc., CA, USA) with the differential pressure transducer (SS40L) connected to the airflow head (RX137) through the tracheal catheter. The TV (ml) and RR (breath/min) parameters of the rats were obtained by monitoring the electronic airflow signal in Acq- Knowledge software (BIOPAC Systems Inc., CA, USA). The RMV (ml/ min) was calculated by multiplying the TV and the RR. The blood pO2 and pCO2 were measured from a 100 μl blood sample obtained from the arterial catheter 20 min before and after the treat- ments using the EPOC® blood gases reader and the EPOC BGEM™ test card (Epocal Inc., Ottawa, Canada). 2.3. Drugs and ICV injections Nesfatin-1, atropine, and mecamylamine purchased from Sigma- Aldrich Co. (Deisenhofen, Germany) were diluted freshly in 0.9% sa- line on the day of the experiment. The desired dose drug dilution was injected by using a 10 μl Hamilton syringe connected to polyethylene tubing and the injection cannula through the ICV cannulation at 5 μl volume within 60 s. The doses of nesfatin-1 (Ciftci et al., 2019), atropine, and mecamylamine (Aydin et al., 2018) were selected from our previous studies. 2.4. Data and statistical analysis The data are given as the mean and standard error. Bonferroni test following Repeated-measures analysis of variance (RM-ANOVA) was used for statistical analysis with p < 0.05 considered the level of sig- nificance. The data are given as the mean and standard error. 3. Results 3.1. Effects of ICV administrated nesfatin-1 on respiratory parameters The baseline TV, RR, and RMV of the anesthetized rats (n = 20) were 3.71 ± 0.05 ml, 71.1 ± 1.1 breaths/min and 263.7 ± 5.4 ml/min, respectively (Fig. 1). The central nesfatin-1 (200 pmol) treatment significantly (p < 0.05) increased TV (Fig. 1A), RR (Fig. 1B) and RMV (Fig. 1C) of the anesthetized animals. The maximum increase in respi- ratory parameters was detected at the 20th min after nesfatin-1 treat- ment and continued for almost 60 min (Fig. 1). Centrally injected nesfatin-1 (200 pmol) caused approximately a 12% increase in TV (Fig. 1C), 8% increase in RR (Fig. 1D), and 21% increase in RMV (Fig. 1E) in the anesthetized rats compared to the saline (5 μl; ICV) treated animals. Central injection of nesfatin-1 did not alter the breathing pattern of rats like apneas or episodes of breathing. Before nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) injection, the anesthetized rats (n = 14) had baseline pO2 and pCO2 levels of 94.94 and 35.58 mmHg, respectively (Fig. 2A, B). Central treatment with nesfatin- 1 led to significant (p < 0.05) increase in pO2 (Fig. 2A) and significant (p < 0.05) decrease in pCO2 levels (Fig. 2B) of the anesthetized rats as parallel to its respiratory effects. The treatment with nesfatin-1 gener- ated an almost 15% increase in pO2 level (Fig. 2A) and approximately a 16% decrease in pCO2 level (Fig. 2B) of the anesthetized rats contrasted to the saline-treated animals. 3.2. The mediation of central cholinergic receptors on the nesfatin-1- evoked respiratory effects Atropine (10 μg; ICV) or mecamylamine (50 μg; ICV) pretreatment did not change baseline respiratory values and breathing pattern of anesthetized rats at the end of the 10 min period. The pretreatment with atropine or mecamylamine significantly (p < 0.05) attenuated the nesfatin-1-evoked increase in TV (Fig. 3A, 5A), RR (Fig. 3B, 5B) and RMV (Fig. 3C, 5C). Atropine or mecamylamine pretreatment also caused partial inhibition in the nesfatin-1-induced increase in pO2 level (Fig. 4A, 6A) and decrease in pCO2 (Fig. 4B, 6B). Table 1 Experimental protocol. The central doses of the drugs were chosen from our previous study (Aydin et al., 2018). Pretreatment (ICV) Treatment (ICV) Experiment Saline (5μl) - n = 10 rats for each treatment. - After treatment, TV, RV, and RMV were recorded for 60 min. - The blood samples for blood gases were withdrawn 20th min of the treatment. - The treatments were performed 5 min after the pretreatments. Nesfatin-1 (200 pmol) Saline (5μl) Saline (5μl) Saline (5μl) Nesfatin-1 (200 pmol) Atropine (10 μg) Saline (5μl) Atropine (10 μg) Nesfatin-1 (200 pmol) Mecamylamine (50 μg) Saline (5μl) Mecamylamine (50 μg) Nesfatin-1 (200 pmol) G. Guvenc-Bayram and M. Yalcin Neuropeptides 90 (2021) 102186 3 4. Discussion The current data report that centrally injected nesfatin-1 elicited hyperventilation effects on the respiratory system, causing an increase in pO2 and a decrease in pCO2 in anesthetized rats. Moreover, the main findings of the present study revealed that the respiratory and blood gas effects induced by nesfatin-1 were mediated by central cholinergic muscarinic and nicotinic receptors. It has been reported that nesfatin-1, which can be released both centrally and peripherally and exhibit multi-directional autonomic ef- fects (Schalla and Stengel, 2018; Schalla et al., 2020), can also have important effects on the regulation of respiration (Ciftci et al., 2019). We recently showed that nesfatin-1, which is administered centrally and peripherally, can produce a hyperventilation effect, which varies in severity according to fasting and satiety conditions (Ciftci et al., 2019). This report observed that centrally administered nesfatin-1 exerted a more effective hyperventilation effect in fed rats than fasting rats (Ciftci et al., 2019). In accordance with our previous study, which showed a more effective hyperventilation effect of nesfatin-1 in fed rats, the pre- sent findings were observed that centrally administered nesfatin-1 induced hyperventilation effect in fed rats. Moreover, the current observation showed, as a novel finding, that nesfatin-1 augmented the oxygenation of the organism by increasing pO2 and decreasing pCO2 due to the hyperventilation effect it produced. The hyperventilation and oxygenation response produced by nesfatin-1 may also be due to nesfatin-1 activating the sympatho-adrenergic system. Because the ICV injection of nesfatin-1 has stimulated renal sympathetic nerve activity (Tanida and Mori, 2011), has increased plasma catecholamine level (Yilmaz et al., 2015), and also has caused pressor effect by activating the peripheral α adrenergic receptors (Yosten and Samson, 2009). The present findings explained that nesfatin-1-induced hyperventi- lation and higher oxygenation responses are mediated by the central cholinergic nicotinic and muscarinic receptors. Similar to the current study's findings, we have shown in our previous report that central nicotinic and muscarinic receptors mediate the cardiovascular effects caused by centrally administered nesfatin-1 (Aydin et al., 2018). The parallel regulation of respiratory and cardiovascular control by the same brain regions and the expression of nesfatin-1 in cholinergic neurons of the brainstem (Brailoiu et al., 2007), which also plays an active role in the regulation of the respiratory and cardiovascular system, support the current findings. Moreover, nesfatin-1-produced a rise in hypothalamic extracellular choline and acetylcholine levels was also reported (Aydin et al., 2018). It has been shown that CDP-choline, as a cholinergic agent, activating central cholinergic nicotinic receptors caused the hyperventilation Time (minute) 0 10 20 30 40 50 60 TV (m l) 3,7 3,8 3,9 4,0 4,1 4,2 Saline Nesfatin-1 * * * * * * * A Time (minute) 0 10 20 30 40 50 60 R R (b re at h/ m in ) 72 74 76 78 Saline Nesfatin-1 * * * * B Time (minute) 0 10 20 30 40 50 60 R M V (m l/m in ) 260 270 280 290 300 310 320 Saline Nesfatin-1 * * * * * * * C Fig. 1. TV, RR, and RMV effects of centrally administrated nesfatin-1. After nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) injections, the TV (A), RR (B), and RMV (C) were recorded for 60 min. *p < 0.05 was vs saline group. pO 2 (m m H g) 80 85 90 95 100 105 110 115 pC O 2 (m m H g) 25 30 35 40 Saline Saline Nesfatin-1Nesfatin-1 * ** A B Fig. 2. pO2 and pCO2 effects of centrally administrated nesfatin-1. Before as well as 20 min after nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) injections, the pO2 (A) and pCO2 (B) were measured from arterial blood samples. *p < 0.05 was vs saline group. G. Guvenc-Bayram and M. Yalcin Neuropeptides 90 (2021) 102186 4 response (Topuz et al., 2014). The nucleus ambiguus neurons express nesfatin-1 (Goebel et al., 2009; Goebel-Stengel et al., 2011), and also nesfatin-1 could activate cardiac vagal neurons of nucleus ambiguus resulting in parasympathetic activation and so nesfatin-1 induced bradycardia (Brailoiu et al., 2013). The mediation of the vagus nerve and the nucleus ambiguus neurons in the essential physiological func- tions of breathing control is also well known (Chang et al., 2015; Ezure, 1990). The nucleus tractus solitarius, which is the first projection area of afferent fibers from cardiopulmonary receptors, is a region of the brainstem that has essential functions in controlling respiration (Zoccal et al., 2014). The presence of both nesfatin-1 (Oh-I et al., 2006; Brailoiu et al., 2007) and choline/acetylcholine (Shihara et al., 1999; Altinbas et al., 2016) has been reported in the nucleus tractus solitarus. Also, interestingly, central or peripheral injection of nesfatin-1 has been demonstrated to cause the activation of the nucleus tractus solitarius neurons (Maejima et al., 2009; Shimizu et al., 2009). Moreover, it is also known that both the central nesfatin-1 (Vas et al., 2013) and the central cholinergic system (Mu and Huang, 2019) have similar effects in regu- lating sleep. However, their interactions have not been demonstrated, Time (minute) 0 10 20 30 40 50 60 TV (m l) 3,7 3,8 3,9 4,0 4,1 4,2 Saline + Saline Saline + Nesfatin-1 Atropine + Saline Atropine + Nesfatin-1 * * * * * * * A Time (minute) 0 10 20 30 40 50 60 R R (b re at h/ m in ) 70 72 74 76 78 Saline + Saline Saline + Nesfatin-1 Atropine + Saline Atropine + Nesfatin-1 * * * * * * * * * * ** * + + + + B Time (minute) 0 10 20 30 40 50 60 R M V (m l/m in ) 260 270 280 290 300 310 320 Saline + Saline Saline + Nesfatin-1 Atropine + Saline Atropine + Nesfatin-1 ** * * * * + + C Fig. 3. Role of atropine pretreatment on hyperventilation response evoked by nesfatin-1. Pretreatment with atropine (10 μg; ICV) or saline (5 μl; ICV) was performed 5 min before treatment with nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) (time point “0”). The TV (A), RR (B), and RMV (C) were recorded for the following 60 min after the treatments. *p < 0.05 was vs. “Saline + Saline” or “Atropine + Saline” group. +p < 0.05 was vs. “Atropine+ Nesfatin-1” group. p O 2 (m m H g) 80 85 90 95 100 105 110 115 Before treatment After treatment Saline + Saline Saline + Nesfatin-1 Atropine + Saline Atropine + Nesfatin-1 * * A # pC O 2 (m m H g) 25 30 35 40 Before treatment After treatment Saline + Saline Saline + Nesfatin-1 Atropine + Saline Atropine + Nesfatin-1 * * B # Fig. 4. Role of atropine pretreatment on oxygenation-enhancing effect evoked by nesfatin-1. Pretreatment with atropine (10 μg; ICV) or saline (5 μl; ICV) was performed 5 min before treatment with nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV). Before as well as 20 min after treatments, the pO2 (A) and pCO2 (B) were measured from arterial blood samples. *p < 0.05 was vs. “Saline + Saline” or “Atropine + Saline” group. #p < 0.05 was vs. “Atropine+ Nesfatin-1” group. G. Guvenc-Bayram and M. Yalcin Neuropeptides 90 (2021) 102186 5 yet. Given the reports, we suggest that centrally injected nesfatin-1 may stimulate the release of extracellular acetylcholine and choline by innervating central cholinergic neurons, so nesfatin-1 showed hyper- ventilation higher oxygenation responses indirectly by activating both types of cholinergic receptors. The current finding demonstareted that nesfatin-1-evoked hyper- ventilation and higher oxygenation responses are mediated by both the central cholinergic nicotinic and muscarinic receptors, at least in part. In this case, the other neurotransmitters' or neuromodulators' receptors, which nesfatin-1 interact, could be involved in nesfatin-1-produced respiratory effects. Moreover, the hyperventilation effect produced by nesfatin-1 also appeared to be compatible in terms of the interaction of nesfatin-1 with other neuromodulators. It was reported that central cyclooxygenase-thromboxane (Erkan et al., 2016) -prostaglandin E, –D, or -F2α (Erkan et al., 2017) signaling pathways and the central lip- oxygenase pathway (Guvenc-Bayram et al., 2020a) were involved in the arachidonic acid-induced hyperventilation resulting in an increase in pO2. We recently reported that centrally administered nesfatin-1 Time (minute) 0 10 20 30 40 50 60 TV (m l) 3,7 3,8 3,9 4,0 4,1 4,2 Saline + Saline Saline + Nesfatin-1 Mecamylamine + Saline Mecamylamine + Nesfatin-1 * * * * * * + A Time (minute) 0 10 20 30 40 50 60 R R (b re at h/ m in ) 72 74 76 78 Saline + Saline Saline + Nesfatin-1 Mecamylamine + Saline Mecamylamine + Nesfatin-1 * * * * * * * + + + * B Time (minute) 0 10 20 30 40 50 60 R M V (m l/m in ) 260 270 280 290 300 310 320 Saline + Saline Saline + Nesfatin-1 Mecamylamine + Saline Mecamylamine + Nesfatin-1 * * * * * * * * + + C Fig. 5. Role of mecamylamine pretreatment on hyperventilation response induced by nesfatin-1. The rats were treated with nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) (time point “0”) 5 min after the pretreatment with meca- mylamine (10 μg; ICV) or saline (5 μl; ICV). The TV (A), RR (B), and RMV (C) were recorded for the following 60 min after the treatments. *p < 0.05 was vs. “Saline + Saline” or “Mecamylamine + Saline” group. +p < 0.05 was vs. “Mecamylamine + Nesfatin-1” group. pO 2 (m m H g) 80 85 90 95 100 105 110 115 Before treatment After treatment Saline + Saline Saline + Nesfatin-1 * A Mecamylamine + Saline Mecamylamine + Nesfatin-1 # * pC O 2 m m H g) 25 30 35 40 Before treatment After treatment Saline + Saline Saline + Nesfatin-1 Mecamylamine + Saline Mecamylamine + Nesfatin-1 * B # * Fig. 6. Role of mecamylamine pretreatment on oxygenation-enhancing effect induced by nesfatin-1. The rats were treated with nesfatin-1 (200 pmol; ICV) or saline (5 μl; ICV) 5 min after the pretreatment with mecamylamine (10 μg; ICV) or saline (5 μl; ICV). Before as well as 20 min after treatments, the pO2 (A) and pCO2 (B) were measured from arterial blood samples. *p < 0.05 was vs. “Saline + Saline” or “Mecamylamine + Saline” group. #p < 0.05 was vs. “Mecamyl- amine + Nesfatin-1” group. G. Guvenc-Bayram and M. Yalcin Neuropeptides 90 (2021) 102186 6 increased hypothalamic extracellular total prostaglandin concentration by activating the hypothalamic cyclooxygenase and lipoxygenase en- zymes (Guvenc-Bayram et al., 2020b). These reports likewise might also suggest that the hyperventilation effect evoked by nesfatin-1 may be an indirect effect of nesfatin-1 by activating the central arachidonic acid cascade. The relationship between respiration and energy metabolism plays a curicial role in regulating the respiratory system according to the O2 and CO2 needs of the body. Nesfatin-1 regulates the energy metabolism of the body by restricting food intake, decreasing blood glucose levels due to rise in insulin release (Nakata and Manaka, 2011), and also increasing the release of adrenocorticotropin, cortisone (Konczol et al., 2010), and thyrotropin-releasing hormone (Gotoh et al., 2013). Also, the cholin- ergic system is related to energy metabolism by increasing plasma in- sulin levels (Ilcol et al., 2008), declining appetite ratings (Killgore et al., 2010), and also stimulating adrenocorticotropin and thyroid- stimulating hormone release (Cavun and Savci, 2004). The cholinergic system has a potential role at the plasma nesfatin-1 level, depending on food intake (Usta et al., 2019). Central and peripheral injected cholin- ergic agent caused an increase in plasma nesfatin-1 levels in the satiated rats but a decrease in 12 h fasted rats (Usta et al., 2019). In the satiated rats, the higher plasma nesfatin-1 level with cholinergic agent admin- istration and the more potential hyperventilation effect with nesfatin-1 administration suggest the interaction between nesfatin-1 and the cholinergic system, considering their similar effects on energy metabolism. The present findings clearly show that nesfatin-1 has hyperventila- tion and oxygenation-enhancing effects in parallel with its impact on energy metabolism. The current results suggest that the central cholin- ergic nicotinic and muscarinic receptors mediate the hyperventilation and oxygenation-enhancing effects of nesfatin-1. Nesfatin-1 may also positively affect respiratory system pathologies such as sleep apnea by creating hyperventilation and oxygenation-enhancing effects. Support- ing this interpretation, it has been reported that there is a negative correlation between the severity of sleep apnea and nesfatin-1 levels (Araz et al., 2015). 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Yalcin http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0180 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0180 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0185 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0185 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0185 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0190 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0190 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0190 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0195 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0195 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0195 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0195 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0200 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0200 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0200 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0205 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0205 http://refhub.elsevier.com/S0143-4179(21)00072-X/rf0205 The involvement of the central cholinergic system in the hyperventilation effect of centrally injected nesfatin-1 in rats 1 Introduction 2 Materials and methods 2.1 Animals and experimental procedure 2.2 Analysis of respiratory parameters and blood gases 2.3 Drugs and ICV injections 2.4 Data and statistical analysis 3 Results 3.1 Effects of ICV administrated nesfatin-1 on respiratory parameters 3.2 The mediation of central cholinergic receptors on the nesfatin-1-evoked respiratory effects 4 Discussion Acknowledgment References