Perinatal protein malnutrition modulates the immunoreactivity of 5-HT1B and density of 5-HT in the nucleus of the solitary tract (NTS) of young rats in response to a feeding stimulus / Desnutrição protéica perinatal modula a imunorreatividade de 5-HT1B e

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Braz. J. of Develop.,Curitiba, v. 6, n. 9, p.65497-65508 ,sep. 2020. ISSN 2525-8761

Perinatal protein malnutrition modulates the immunoreactivity of 5-HT1B and

density of 5-HT in the nucleus of the solitary tract (NTS) of young rats in

response to a feeding stimulus

Desnutrição protéica perinatal modula a imunorreatividade de 5-HT1B e a

densidade de 5-HT no núcleo do Trato Solitário (NTS) de ratos jovens em

resposta à estimulo alimentar

DOI:10.34117/bjdv6n9-107

Recebimento dos originais: 08/08/2020 Aceitação para publicação: 04/09/2020

Lívia de Almeida Lira Falcão

PhD in Neuropsychiatry and Behavioral Sciences

Graduate Program of Neuropsychiatry and Behavioral Sciences, Federal University of Pernambuco, Recife, Brazil.

Catholic University of Pernambuco – UNICAP, Recife, Brazil. Address: R. do Príncipe, 526 - Boa Vista, Recife - PE, 50050-900, Brasil

E-mail: ilalira@hotmail.com

Gabriel Araújo Tavares

Master in Nutrition

Graduate Program of Nutrition, Federal University of Pernambuco, Recife, Brazil. Nantes Université, INRAE, UMR 1280, PhAN, Nantes, France.

Address: Av. Prof. Moraes Rêgo, 1235, Cidade Universitária, Recife – PE. CEP 50670-901 E-mail: gabrieltavaresufpe@outlook.com

Larissa Cavalcanti do Amaral Almeida

PhD in Neuropsychiatry and Behavioral Sciences

Address: Av. Prof. Moraes Rêgo, 1235, Cidade Universitária, Recife – PE. CEP 50670-901 E-mail: ftlarisaalmeida@gmail.com

Lisiane dos Santos Oliveira

PhD in Nutrition

Nucleus of Nutrition – Academic Center of Vitória de Santo Antão, Federal University of Pernambuco

R. Alto do Reservatório - Alto José Leal, Vitória de Santo Antão - PE, Brasil E-mail: lisianenutricao@yahoo.com.br

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Sandra Lopes de Souza

PhD in Morphofunctional sciences

Address: Av. Prof. Moraes Rêgo, 1235, Cidade Universitária, Recife – PE. CEP 50670-901 E-mail: Sandra.lsouza2@ufpe.br

ABSTRACT

The nucleus of the solitary tract (NTS) has emerged as an important area in the regulation of eating behavior by the serotonergic system. Some evidence also points out that this area is susceptible to nutritional stimuli in the perinatal period. Thus, we aim to analyze the effects of perinatal protein malnutrition on the density of serotonin and the immunoreactivity (IR) of its 5-HT1B receptor in NTS. Female rats were submitted to the normoproteic (17% casein) (PN) or low protein diet (8% casein) (LP) during pregnancy and lactation. At 35 days of age, male offspring was subjected to a 4-hour food deprivation, followed by standard food offer for 90 minutes. Then they were euthanized by transcardiac perfusion and the brains were collected and processed for immunohistochemistry analyzes against serotonin and 5-HT1B. Malnutrition modified the density of serotonin mainly in the intermediate sub-nucleus (SOLIM) of the NTS, while the IR of 5-HT1B was increased in the ventral-lateral (SOLVL), intermediate (SOLIM) and medial (SOLM) sub-nuclei of the NTS. From this, we conclude that perinatal protein malnutrition modulates serotonergic activity in NTS in response to food stimulus.

Keywords: Nucleus of the solitary tract, Serotonin, Undernutrition, Low-protein diet. RESUMO

O núcleo do trato solitário (NTS) surgiu como uma importante área na regulação do comportamento alimentar pelo sistema serotonérgico. Algumas evidências também apontam que essa área é suscetível a estímulos nutricionais no período perinatal. Assim, objetivamos analisar os efeitos da desnutrição protéica perinatal sobre a densidade da serotonina e a imunorreatividade (IR) do seu receptor 5-HT1B no NTS. Ratas foram submetidas à dieta normoprotéica (casina 17%) (NP) ou hipoprotéica (caseína 8%) (LP) durante a gestação e lactação. Aos 35 dias de idade, os filhos machos foram submetidos a uma privação alimentar de 4 horas, seguida de oferta alimentar padrão por 90 minutos. Em seguida, foram eutanasiados por perfusão transcardíaca e os cérebros foram coletados e processados para análises de imunohistoquímica contra serotonina e 5-HT1B. A desnutrição modificou a densidade da serotonina principalmente no subnúcleo intermediário (SOLIM) do NTS, enquanto o IR de 5-HT1B foi aumentado nos subnúcleos ventro-lateral (SOLVL), intermediário (SOLIM) e medial (SOLM) do NTS. A partir disso, concluímos que a desnutrição protéica perinatal modula a atividade serotonérgica no NTS em resposta ao estímulo alimentar.

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1 INTRODUCTION

The control of eating behavior is carried out by a highly complex system, dependent on the coordinated activity of the central nervous system (CNS) and the periphery of the body (1–3). Although many aspects and neural regions involved in the regulation of eating behavior are known, such as hypothalamic circuits, much remains to be clarified, especially with regard to pathologies associated with eating behavior (4,5). In addition to the hypothalamus, research suggests other brain areas acting together to generate more appropriate responses to the individual's needs (2).

Among these other brain regions, the nucleus of the solitary tract (NTS) has been directly involved in the control of eating behavior. In addition to integrating peripheral information, the NTS is responsive to taste stimuli and information related to motivation and reward (6–8). Additionally, it maintains direct and reciprocal connections with other brain structures involved in the control of food intake and energy balance such as the nucleus Accumbens (NAc), several hypothalamic nuclei, the amigdala, the ventral tegmental area (VTA), the dorsal raphe nucleus (DRN) and the posterior area (9–11). The serotonin seems to play a key role on NTS activity, as the presence of postsynaptic sites for the action of serotonin (12) and different subtypes of serotonin receptors, such as 5-HT1A / B (13,14), 5-HT2A / C (15), 5-HT3 (16), 5-HT5A (17), 5-HT6 (18) and 5-HT7 (19) have already been identified. Interestingly, the anti-obesity 5-HT2CR agonist lorcaserin targets both the POMC circuitry of the brainstem nucleus of the solitary tract to mediate appetite suppression (20).

In recent years, relevant epidemiological and experimental studies have highlighted the participation of nutritional stimuli during the early stages of development on the appearance of developmental disorders (21–23). It is well established that the inadequate nutritional supply during the perinatal period is associated with disruption of feeding behavior homeostasis (24–27). According to this, we believe that the NTS plays a key role on the serotonergic control of feeding behavior, which can be disrupted by the perinatal protein restriction. Thus, deepening the knowledge about the role of the NTS participation in this context is extremely important to understand a little more how perinatal stimuli can interfere in the ability of this region to control the energy balance, as well as the role of serotonin in this process.

2 MATERIALS AND METHODS

2.1 ANIMALS AND EXPERIMENTAL GROUPS

Wistar rats from the colony of the Department of Nutrition of the Federal University of

Pernambuco (UFPE) were used. 120-day-old females underwent a fifteen-days adaptation period under standard conditions of the vivarium: temperature of 23 ° C ± 1, 12/h12h inverted light-dark

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cycle hours (light on at 6 pm) and free access to water and standard laboratory chow (Presence ®). After adaptation, the rats in the estrous period were mated in the ratio of two females to one male, and pregnancy was detected by the presence of sperm in the vaginal smear. After pregnancy was confirmed, the females were placed in individual cages, and observed routinely to record the day of birth of the pups. Twenty-four hours after birth, the litters were culled to 8 puppies (4 males and 4 females) chosen randomly. Females were used only to compose the litters. The pups were weaned at 21 days, after which were offered standard laboratory chow. The experiments followed the ethical recommendation of COBEA (Brazilian College of Animal Experimentation). All procedures were approved in accordance with UFPE's Ethics Committee on Animal Experimentation (CEEA) (nº 23076.0374092011-64).

The experimental groups were obtained according to the type of diet offered during the period of pregnancy and lactation. Pregnant females were randomly given either a normo-protein diet (17% casein) (NP) or a low-protein diet (8% casein) (LP), as described previously (28), throughout the gestation and lactation periods. From each litter, a maximum of 2 male animals were taken to compose the experimental groups to avoid the litter effect.

2.2 IMMUNOHISTOCHEMISTRY AGAINST SEROTONIN AND 5HT1B

The euthanizes for obtaining brain tissue were performed at age 35. Before this, the animals were submitted to a food stimulus, consisting of a 4-hour food deprivation period, followed by 90 minutes of standard food offer. After this, the animals were submitted to transcardiac perfusion. For this, the animals were deeply anesthetized with ketamine (50mg / kg) and xylazine (10mg / kg). After anesthesia, the thoracic cavity was dissected to obtain access to the heart, where a cannula attached to a peristaltic pump was inserted in the left ventricle. Initially, 150 ml of saline solution (Nacl, 0.9%) was infused at room temperature, followed by infusion of 400 mL of fixative solution (4% paraformaldehyde, pH 7.4, at 4º C). At the end of the fixator passage, the brains were removed from the skull and post-fixed in the same fixative solution plus sucrose (20%) for 4 hours. After this period, they were stored in cryoprotective solution (PBS sodium phosphate buffer - plus 20% sucrose). For the following immunohistochemical procedures, the brains were cryodissected in coronal sections of 40 µm.

For the immunohistochemistry procedure against serotonin or 5-HT1B, the coronal sections of a series (NP, n=5, LP, n=5) were washed in KPBS (3 x 10 min), then incubated in solution with primary antibody against serotonin (Protos Biotech Corporation, New York, NY, USA) or 5-HT1B (Santa Cruz Biotechnology, Texas USA) made in rabbit (Dilution 1: 5000). After the incubation the

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cuts were subjected to washes in PBS and subsequently incubated in solution with secondary antibody, made in goat against rabbit. Then, the cuts were again subjected to washes with PBS and another incubation with avidin-biotin-peroxidase 1% complex (Vectastain, Camon, Wiesbaden, Germany). After this, the cuts were subjected to washes with PB S and sodium acetate. The slices were colored with DAB (3,3′-Diaminobenzidine) staining kit.

The density and quantification were analyzed at the following sub nucleus of the NTS: ventrolateral (SOLVL), intermediate (SOLIM), medial (SOLM) and the commissural (SOLC) (13). The images were captured using a digital camera (Samsung SHC-410NAD) adapted to a microscope (Olympus BX50), and a computer. The acquisition of the images was performed using the 40X objective for 5-HT and 10XC objective for 5-TH1B, with the aid of the Motic Image Plus 2.0 software. The densities of the serotonergic terminals were obtained from the of bregma levels -12.96 and 13.96. The quantity of 5HT1BIR was obtained from the bregma levels 12.96, 13.96 and -14,40. For both, data were obtained considering only one cerebral hemisphere. For analysis of 5-HT, the images were processed using the Image Pro Plus software (version 6.0), identifing the serotonergic fibers density in relation to the area, represented in the optical density index (IOD) unit. Quantitative analysis of 5-HT1B neurons was performed using the ImageJ version 1.45 software. For the identification and delimitation of the studied areas, the Stereotactic Atlas of Rat was used as reference (29).

2.3 STATISTICAL ANALYSIS

The values of immunoreactive neurons to the 5-HT1B receptor or the density of serotonergic terminals in the NTS were evaluated by the t test to compare each group with its control. All data were analyzed using the software GraphPad Prism v8, and presented as mean ± SEM. Significance was considered when p ≤ 0.05.

3 RESULTS

3. 1 ANALYSIS OF THE DENSITY OF SEROTONERGIC TERMINALS AND 5-HT1B-IR IN NTS

On the bregma -12,96, LP group presented decreased density of 5-HT (t=5,072, df=4,132, p=0,0065), when compared to NP group (figure 1A). On the other hand, on the bregma -13,96, LP group presented increased 5-HT density in the whole NTS (t=2,516, df=7,120, p=0,0395), SOLIM (t=5,853, df=6,865, p=0,0007), and SOLM (t=2,389, df=7,967, p=0,0440) in comparison to NP

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group (Figure 1B). When summed the values of both bregmas, LP group showed increase in 5-HT density only in SOLIM (t=3,414, df=7,087, p=0,110), comparing to NP group (figure 1C).

Figure 1. Effects of perinatal protein malnutrition on 5-HT density in the NTS. Rats were submitted to normal protein (17%) or low protein (8%) diet during gestation and lactation. At day 35, brains of male rats (NP, n=5, LP, n=5) were collected and analysis of immunohistochemistry against 5-HT were performed. (A) analysis of 5-HT density on NTS on bregma -12,96, (B) analysis of 5-HT density on NTS on bregma -13,96, (C) analysis of 5-HT density on NTS on both bregmas -12,96 and -13,96 together. SOLVL, Sub-nucleus ventral-lateral; SOLIM, sub-nucleus intermediate; SOLM, sub-nucleus medial; SOLC, sub-nucleus commissural. Data are expressed as mean ± SEM, *p<0.05, **p<0.01. (□) NP, (■) LP.

3.2 QUANTITATIVE ANALYSIS OF 5-HT1B-IR NEURONS IN NTS.

On the bregma -12,96, the LP group showed increased 5-HT1B-IR in the whole NTS (t=3,573, df=7,940, p=0.0073), SOLIM (t=3,321, df=4,524, p=0,244) and SOLM (t=3,746, df=7,046, p=0.0071), when compared to NP group (figure 2A). In addition, on the bregma -13,96, the 5-HT1B-IR was increased in the whole NTS (t=5,989, df=7,233, p=0.0005), SOLVL (t=3,154, df=7,341, p=0.0151), SOLIM (t=7,690, df=5,563, p=0.0004) and SOLM (t=4,731, df=7,867, p=0.0015), in comparison to the NP group (figure 2B). On the bregma -14,40, only in the SOLM was observed increased 5-HT1B-IR in LP group (t=3,071, df=6,867, p=0.0185), compared to NP (figure 2C). On the other hand, when results of the three bregmas were summed, we observed increase in 5-HT1B-IR in the whole NTS (t=5,658, df=6,674, p=0.0009), SOLVL (t=3,696,

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df=6,503, p=0.0088), SOLIM (t=6,929, df=7,954, p=0.0001) and SOLM (t=5,754, df=7,989, p=0.0004), comparing to NP (figure 2D).

Figure 2. Effects of perinatal protein malnutrition on 5-HT1B immunoreactivity in the NTS. Rats were submitted to normal protein (17%) or low protein (8%) diet during gestation and lactation. At day 35, brains of male rats (NP, n=5, LP, n=5) were collected and analysis of immunohistochemistry against HT1B were performed. (A) analysis of IR on NTS on bregma -12,96, (B) analysis of 5-IR on NTS on bregma -13,96, (C) analysis of 5-HT1B-IR on NTS on bregma -14,40, (D) analysis of 5-HT1B-5-HT1B-IR on NTS on bregmas -12,96, -13,96 and -14,40 together. SOLVL, Sub-nucleus ventral-lateral; SOLIM, nucleus intermediate; SOLM, nucleus medial; SOLC, sub-nucleus commissural. Data are expressed as mean ± SEM, *p<0.05, **p<0.01. (□) NP, (■) LP.

4 DISCUSSION

In the present study, we observed that malnutrition was able to modulate 5-HT density and 5-HT1B-IR in NTS. This region shows an important role of 5-HT in regulating feeding behavior. In this context, the family of receptors 5-HT2, and the receptors 5-HT1A and 5-HT3 have been implicated in satiety signalization specifically in the NTS, through activation of GLP-1 producing neurons (20,30,31). Despite we know this, much remains to be elucidated, especially with regard to its responses to environmental stimuli.

In the context of malnutrition, we already know that it modulates several areas of the central nervous system, in association with changes in eating behavior (32–34). On the other hand, little is known about the effects of malnutrition on NTS. Some studies show that this region is susceptible to malnutrition. For example, perinatal malnutrition alter the morphology of neurons in the NTS

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(35) and the glutamatergic control of cardiovascular function (36). Here, depending on the bregma evaluated, the density of 5HT was higher or lower in each evaluated sub-nucleus, however, when both bregmas analyzed are placed together, the increase in serotonergic density can be highlighted in SOLIM. This result is interesting, and may suggest a programing for the animals to be hyperphagic in response to nutritional deprivation. According to our previously published data, malnourished animals show hyperphagia at 35 days of age, which is accompanied by an increase in the expression of c-fos in the NTS in response to feeding stimulus (28). Thus, the food stimulus in malnourished animals promotes an increase in c-fos and serotonin at the same time, but without a satiety effect, since the animals are hyperphagic.

Interestingly, when the 5-HT1B IR was evaluated, it was increased in several regions in all analyzed bregma. The 5-HT1B receptor has a known inhibitory action, either at the axonal terminal of the 5HT neuron or in postsynaptic neurons (37). In this case, since we observed, in general, an increase in serotonergic density, the increase in IR of 5-HT1B here probably occurs in postsynaptic neurons. Thus, an increase in 5-HT1B in postsynaptic neurons added to an increase in serotonin may be the cause of inhibition of postsynaptic neurons that act in satiety signaling. This suggestion makes sense when compared to the fact that malnourished animals are in fact hyperphagic at 35 days of age (Lira et al., 2014). In addition, when adults, these same animals show a delay in satiety point and an increase in the meal size, associated with increased c-fos IR in NTS (Lira et al., 2014). This suggests that these changes in the serotonin activity pattern may have long-term effects, promoting persistent hyperphagic behavior in response to perinatal malnutrition.

In view of the results obtained, we can see that the NTS is a region susceptible to the influences of perinatal malnutrition. Likewise, the impact of malnutrition on the serotonergic neurotransmission system in this nucleus is evident. However, despite this being the first report about the effects of this early manipulation on the serotonergic system in NTS, further investigations need to be carried out to clarify the altered mechanisms.

FUNDING STATEMENT

This work was supported by the Universidade Federal de Pernambuco – UFPE and by the

Fundação de Amparo a Ciência de Tecnologia de Pernambuco (FACEPE). Additionally, this study

was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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CONFLICT OF INTERESTS DISCLOSURE

The authors declare that there is no conflict of interests.

ETHICS APPROVAL STATEMENT

The experiments followed the ethical recommendation of COBEA (Brazilian College of Animal Experimentation). All procedures were approved in accordance with UFPE's Ethics Committee on Animal Experimentation (CEEA) (nº 23076.0374092011-64).

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTION STATEMENT

LF and SS conceptualized the work. LF and LA performed the immunohistochemical analysis. LF and GT wrote the manuscript. LO and SS edited the manuscript. All authors contributed

to and have approved the final manuscript.

AKNOWLEDGMENTS

The authors would like to thank the Neuroplasticity and Behavior Research Group for the personal and technical support. Additionally, the authors would like to thank Dr. Bertrand Kaeffer for the comments on the manuscript.

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