Nesta análise a SCS foi avaliada 1 h após a aplicação de fluoxetina 1ml/100g

(10mg/kg, por via intraperitoneal) e imediatamente após a oferta de dieta padrão do biotério. Foi registrada, durante um período de 60 minutos, a duração dos seguintes comportamentos:

Quadro 1: Descrição dos Parâmetros Avaliados durante a Sequência Comportamental de Saciedade

Sequência Comportamental de Saciedade Parâmetro Avaliado Descrição

ALIMENTAÇÃO O registro desse comportamento foi iniciado imediatamente quando o rato foi observado junto ao comedouro iniciando a ingestão de ração. O mesmo foi finalizado no momento em que o rato abandonou o comedouro.

LIMPEZA O rato procedeu inicialmente o lamber de patas anteriores e movimentos dessas sobre a cabeça continuando-se com o lamber da região ventral, do dorso e das patas posteriores.

REPOUSO O rato foi observado em posição de descanso, apresentando o corpo repousado sobre o assoalho da gaiola.

ATIVIDADE Incluem outros comportamentos como: locomoção, cheirar, levantar as patas anteriores e explorar a área.

INGESTÃO ALIMENTAR

Foi obtida pela diferença do peso da ração antes e após a observação comportamental.

Avaliação da ingestão de alimento palatável sob estimulo de um inibidor seletivo de recaptação de serotonina durante a idade adulta

Nessa análise, foram utilizados animais previamente privados (por quatro horas) de alimento. Os animais receberam fluoxetina (10mg/kg de peso corporal) ou solução salina (Na cl 0,9%) no volume de 1ml/100g de peso corporal via intraperitoneal. Uma hora após as injeções, foi disponibilizado alimento palatável (Chocookies- chocolate Nabisco®) em quantidade conhecida. Após uma hora, o alimento foi removido e pesado para obtenção do consumo por diferença entre a quantidade oferecida e a rejeitada.

Análises estatísticas

Os dados foram apresentados em média e erro padrão sendo utilizado ANOVA, seguido de Bonferroni. O nível de significância foi considerado P igual ou menor que 0.05. Todos os dados foram analisados usando o programa GraphPad Prism 5, versão 7.

RESULTADOS

O artigo original deste estudo é intitulado “Efeito da desnutrição perinatal sobre

aspectos motivacionais da recompensa alimentar e da ação serotoninérgica sobre a ingestão de alimento palatável.” Será submetido como artigo original ao periodico The Journal of

Neuroscience- Behavioral/Systems/Cognitive, classificado como Qualis A1 pela CAPES.

Neste artigo foi verificado o papel da desnutrição perinatal sobre o comportamento motivacional, assim como a influência de ISRS sobre a ingestão de alimento palatável. Foi demonstrado que a desnutrição perinatal aumenta a motivação por recompensa alimentar, bem como o possível papel favorecedor da serotonina sobre a ingestão de alimento palatável.

Effect of perinatal malnutrition on the motivational aspects of food reward and serotonin action on the intake of palatable food

Authors:

Amanda Alves Marcelino da Silva1,Lisiane Santos Oliveira3, Tássia Karin Ferreira Borba2, Mayara Brasil de Sá Leitão1, Raul Manhães de Castro1, Sandra Lopes de Souza 2

1. Departamento de Nutrição – Universidade Federal de Pernambuco. – Recife- PE, Brazil. 2. Departamento de Anatomia – Universidade Federal de Pernambuco, Recife – PE, Brazil. 3 CAV-UFPE-Centro Acadêmico de Vitória UFPE, Vitória de Santo Antão-PE, Brazil.

Corresponding Author: Sandra Lopes de Souza

Corresponding author: Universidade Federal de Pernambuco- Departamento de Anatomia – UFPE Av. Prof. Moraes Rego, 1235- Cidade Universitária CEP:50670901-Recife-PE-Brasil

Fone: 55 81 2126 8567 /Fax: 55 81 21268554

E-mail: sanlopesufpe@gmail.com

Title: Effect of perinatal malnutrition on the motivational aspects of food reward and serotonin action on the intake of palatable food

Abstract

Malnutrition in early periods of life can promote permanent changes in brain structures responsible for control of food intake. Several mechanisms and cellular processes need to be clarified about the programming of feeding behavior. Thus, this study aimed to evaluate the effects of perinatal malnutrition on the serotonergic regulation of hedonic control of eating behavior. In this study, we used Wistar rats were divided into two groups according to the diet offered to mothers during the perinatal period: Control (C, diet with 17% casein) and Low protein (LP diet with 8% casein). We evaluated: a) Body weight during the lactation period until 35 days and 180 days of life. b) motivational behavior before food reward via the Runway. This test was conducted from 60 to 82 days of life, including 11 alternate sessions of training, where the animal was exposed to the reward for 5min. c) SCS with palatable food. 60min behaviors during feeding, cleaning, and resting were observed. d) Ingestion of palatable food under the effect of SSRIs. Animals were fasted for 4h. 1h assessment was used SSRIs (10mg/kg, pi). Perinatal malnutrition increases motivation for food reward, despite the

cognitive delay. The palatable food disrupts the behavioral sequence of satiety and the serotonergic system contributes to these events, increasing intake of palatable foods.

Keywords: perinatal malnutrition, programming, motivation, system reward, serotonin.

Introduction

Studies demonstrate that a relationship exists between this critical window of development and programming in an individual (1-3). Both factors are associated with nutritional status and fetal outcome, and this mother-son has aroused interest in the scientific world because of the relationship between these factors and the risk of diseases in adulthood (3). Nutritional manipulations have shown that according to the ontogeny of each phase (gestation, lactation or early childhood) and the species to be studied, the critical window of development can open up, and lack or excess of nutrients act by directing the body to remain metabolically about this condition (4-5).The process by which early insults at critical stages of development lead to permanent changes in tissue structure and function is known as intrauterine programming (4, 6).

Based on this concept, it is observed that the key point between perinatal diet and the emergence of the metabolic syndrome, is the maintenance of energy balance. This balance can be defined as the balance between supply and energy expenditure and is dependent on feeding behavior, which represents an adaptive response from the demand of the internal environment is modulated by the opportunities and limitations imposed by the external environment (7), through the complex interaction between peripheral and central mechanisms that control the processes of hunger and satiety (8). In general two types of systems are responsible for regulating eating behavior, a homeostatic and other hedonic (9). The hypothalamus is capable of integrating peripheral signals and central homeostatic control of this behavior (10). Within peripheral, the hypothalamus receives signals of hunger and satiety from the gastrointestinal tract, pancreas, liver and adipose tissue (11).While the cortical and limbic areas, such as the prefrontal cortex, nucleus accumbens and ventral tegmental area, participate in the hedonic aspects of food intake related to pleasure and sense of reward from eating. The access to palatable foods (which by definition are foods rich in fat or carbohydrates), and incorporate subjective values brings qualities to the food such as taste, texture. These properties are able to stimulate the motivational behavior of the individual.

The dopamine has a fundamental action in the motivation of appetite, this pathway consists of dopaminergic cell bodies located in the ventral tegmental area and projects to multiple nodes, including the nucleus accumbens, amygdala, prefrontal cortex and hippocampus (12). Another neurotransmitter involved with the ingestion of palatable food is serotonin (13) This action appears to be dependent on its effect on dopaminergic neurotransmission (14). The stimulation or inhibition of neurons of the nucleus raphe produces, respectively, increase and decrease in dopamine release in the nucleus accumbens, one of the key areas related to the hedonic control (15). Besides the direct administration of serotonin in the ventral tegmental area or nucleus accumbens increases extracellular dopamine levels (16-17)

The high prevalence of obesity today indicates that in the presence of palatable foods, the homeostatic control can be overwhelmed, experiencing excessive food intake. Our

hypothesis is that malnutrition increases the motivation for the reward and that changes in the serotonergic system are involved with the highest intake of palatable food. Therefore this study aimed to examine whether protein malnutrition can alter the hedonic control of eating behavior, through a possible serotonergic system programming.

Materials and Methods

Subjects

Virgin female Wistar rats (n=6) weighing 250-300g were obtained and maintained in the laboratory with an inverted light/dark cycle of 12 hours (lights on at 6:00 p.m.) for 15 days for adaptation, with water and a standard diet (Purina do Brasil S/A) ad libitum. The animals were maintained at a room temperature of 22° ±2C. After the adaptation period, females were assigned in a proportion of one female for one male. After confirmation of mating by visualization of spermatozoa in a vaginal smear, the females rats were housed individually and fed with either a control diet (17% casein) or low protein (8% casein). The day of the birth was considered day zero. Day one after birth, pups were divided into male and female groups and six male pups were assigned per dam. The experimental groups were classified in accordance with the diet consumed during the perinatal period, undernutrition (8% casein) (n=10, male) and control (17%casein) (n= 10, male). Female pups were discarded from the study to prevent variations due to sex-related differences in metabolic programming. After weaning at 21 days of age, both groups received a high fat diet until 35 days of postnatal life. From the 36th to 180th day of life, all animals were fed a standard diet. All experiments were performed in accordance with recommendations from the Brazilian Committee of Animal Experiments – COBEA, and were approved by the Ethics Committee on Animal Experimentation from Centre for Biological Sciences from the Federal University of Pernambuco.

Food Intake and body weight (experiment 1)

Body weight was measured daily during the 1st to the 35th day of life. We performed the analysis of dietary intake of standard diet for 150 days. The animals were isolated, suffered food deprivation for 4 hours, after which it was provided a standard diet (Labina - Purina) with known weight. Dietary intake was assessed for 1 hour. For measures of body weight and food intake was used electronic scale with a capacity of 4 kg and 0.1 g sensitivity (Marte, model S- 4000).

Runway Task Incentive (Experiment 2)

At 60 days all animals (control and low protein) were subjected to runway task incentive, after food deprivation for 4 hours. This test consists of a behavioral paradigm, which generates curves of acquisition of learning, as well as speed and trajectory traveled that express the motivation of the animal the stimulus of reward (PECIÑA et al., 2003). The test was performed between 12 to 14h. The runway apparatus consisted of three compartments: a start box (19x14x30), a central runway (150x14x30), and a goal box (19x14x30). The boxes were transparent acrylic and opaque polypropylene apparatus. Sliding doors separated the start and goal boxes from the runway alley. The images were captured by system of video camera positioned in the center of the runway in order to visualize the entire apparatus. The start box

could be moved anywhere along the alley to be close as 15cm from the goal box as far as 150cm from the goal box. A dish in the goal box contained 5 grams of cookies (Chocookies- chocolate- Nabisco). To extinguish any neophobia, rats were habituated to Chocookies- chocolate-Nabisco during the first 3 sessions. Runway training was conducted in 11 sessions on alternating days (22d training period) each session consisted of 5min. The animals were deprived of food for 4 hours. This was followed by one test trial per session in two further sessions (12 and 13). On the first three training sessions, rats were simply placed directly in the closed goal box and allowed to eat the reward that found there for 5 min. On training session 4, the start box was placed 15cm away from the reward. A rat was placed in the start box in the start box for 30 sec with door closed; then the door was elevated, and the rat was allowed to proceed into the runway. If a rat did not leave the start box within 3min, the rat was gently pushed toward the goal box. The start box was moved to 30 cm from the goal on session 5, to 60 cm on session 6, to 75cm on session 7, to 90 cm on session 8, 120 cm on session 9 and 150 cm 10-12. The task completion speed was completed for each session by dividing the latency to reach the goal box by the runway length on that day. Exit from the start box was recorded when all four limbs of the animal were outside the start box, and entry of the goal box was recorded when all four limbs of the animal were inside the goal box. Once the rat enter of the goal box and began eating, it was allowed to consume the reward for 30 sec before being retrieved. Incentive runway behavior analysis of (1) latency of leave the start box, (2) latency to reach the goal box, (3) number of pause in the runway, (4) number of reversals of direction in the runway en route to the goal (involving retracing o steps and usually accompanied by investigatory sniffing), (5) latency to being eating the reward.

Behavioral Satiety Sequence (BSS) (Experiment 3)

The BSS study occurred on the 150th a day of life. The analysis of the behavioral satiety sequence was performed essentially as described by Halford et al (1998). Feeding and non- feeding behaviors during a 60 min test meal were continuously scored by a highly trained experimenter, blind to the nutritional status of the animals, and recorded on a videotape to be re-examined by a second skilled observer. Behaviors were categorized as: eating (ingesting food, gnawing, chewing or holding food in paws), grooming (body care movements with the mouth or forelimbs), and resting (sitting or lying in a resting position or sleeping). Other measure scored from the behavioral observation of feeding was rate (amount of food consumed (g)/ analysis of BSS duration (min). To promote feeding, food was removed from home cages 4h before the onset of the test and the presentation of food took place 1 h before the onset of the dark cycle. Food was weighed at the beginning and end of each session. The behavioral sequence of satiety was conducted in four stages, with all four experimental groups of animals, after 150 days of life: (a) In this analysis, BSS was assessed immediately after the provision of standard diet (Labina-Purina). (b) BSS was assessed immediately after the supply of palatable food (chocolate, Chocookies, Nabisco®). (c) BSS was measured 1h after application of saline1ml/100g body weight (0.9% NaCl, intraperitoneally) and immediately after the provision of standard diet. (d) BSS was assessed 1h after administration of fluoxetine 1ml/100g body weigth (10mg/kg, intraperitoneally) and immediately after the provision of standard diet.

After 48 hours of assessment BSS animals were first deprived of food for 4 hours. And one hour before the assessment was applied1ml/100g fluoxetine (10mg/kg/ intraperitoneally). Palatable food (chocolate Chocookies-Nabisco®) was available with known weight and observed for 1 h food intake of the animal.

Data analysis

Experimental results are expressed as means ± S.E.M. All data were analyzed using a GraphPad Prism 5 program. Body weight, runway performance and data from the BSS were analyzed using a two-way ANOVA followed by the Bonferroni test for multiple comparisons between groups. Statistical significance was set at P<0.05.

Results

Figure (1A-B) Body Weight

From the 6th day of life until the 35th day of life, the body weight the offspring undernutrition (10,41 ±4,19) (p<0,05) was lower significantly than control (15,71±5,46) (Figura 1A). This growth retardation persisted after 180 days of life, (370,3 ± 0,7) for the malnourished animals compared to the control group (435,7 ±0,7) (Figura 1B).

Runway performance

Evaluation of the number reverse direction, pauses, latency of leave the start box, latency to reach the goal box

Figure (2A-D)

Malnourished animals (0.70 ± 0.10, 0.40 ± 0.10) had fewer reverse direction compared to control (1.40 ± 0.13, 1.70 ± 0.13) during sessions 5 and 8 (Figure 2A). Between sessions 8 and 9 malnourished animals (0.4 ± 0.08, 0.3 ± 0.08) had fewer breaks compared to control animals (1.00 ± 0.10, 0.70 ± 0.09) (Figure2B).The evaluation of the response latency to target box showed that malnourished animals (20.90 ±0.30, 19.60±0.40) had higher latency only during sessions 5 and 7 in relation to its control (13.90 ±0.35, 13.20±0.24). During the 8 session malnourished animals (11.60± 0.27) had a lower latency than the control (32.20±0.56) (Figure 2C). The latency to leave the start box was greater for the malnourished animals (3.90±0.19, 3.0 ± 0.015) also during the sessions 5 and 7compared to control (2.50 ± 0.12, 2,00 ±0.08). During the 8 session malnourished animals (2.00 ±0.07) had a lower latency than the control (2.50±0.07) (Figure 2D).

Assessment of latency for consumption of food reward

During the adaptation sessions, the malnourished animals (247.0±1.00, 187.5 ± 0.90, 48.0±048) showed a higher latency for consumption of food reward compared to control (193.3±0,90; 145.40±0.90, 31.30±0.30) (Figure3A). During pre-exposure sessions (5, 6, 7) the malnourished animals (58.30 ± 0.75, 59.50 ±0.85, 57.20±0.60) had higher consumption of latency to reward ratio the control (48.80±0.45, 44.40±0.47, 42.60±0.55) .During the learning incentives the malnourished animals (48.80±0.68,36.90±0.50) showed lower latency for consumption of food reward compared to control (70.30 ± 0.80, 43,50 ± 0.41). During the last two sessions, 10 and 11 the malnourished animals (42.90±0.27, 38.80 ±0.29) showed longer latency to reward consumption compared to control (35.0±0.33; 33.60 ±0.31) (Figure 3B)

Speed

Figure 4

During the pre-exposure the malnourished animals (1.72 ± 0.09) had a lower speed (Figure 4) than control animals (3.75 ± 0.16) in session 5. At the stage of learning incentives, the malnourished animals (5.88 ± 0.17) had lower speed only in session 7 compared to control (6.86±0.18). And maintained higher speed during the sessions in 8 (9.66±0.20) and 9 (13.20±0.29) and stage-trained, 10 sessions (17.07 ± 0.27) and 11 (18.07±0.31) compared to control animals (6.07±0.23; 12.13±26; 15.18± 0.25; 16.10±0.27).

Palatable Food Intake and ISRS Figure (5A-B)

Malnourished animals (8.20 ± 0.14) showed a higher intake of dietary pattern in relation to control (6.13 ± 0.13). The intake of dietary pattern under the effect of SSRIs was greater in malnourished (6.13 ± 0.13) animals than control (4.10 ± 0.13). (Figure 5A). The intake of palatable food was greater in malnourished animals (16.40 ± 0.17) compared to control (13.53 ± 0.15). Under the effect of SSRI ingestion of palatable food was greater in malnourished animals (15.23 ± 0.20) compared to control (11.30 ± 0.18) (Figure 5B). By comparing the pattern of food intake (6.32 ± 0.11; 4.10 ± 0.13) and palatable (13.53± 0.15; 11.30 ± 0.18) under the effect of SSRIs in the control group was observed that the reduction in intake of both diets. A comparison of food intake pattern and palatable under the effect of SSRIs in the malnourished group, it was demonstrated that the reduction of standard diet (8.20 ± 0.14; 6.13 ± 0.13) but not palatable food (16.40 ± 0.17; 15.23 ± 0.20).

Behavioral Satiety Sequence (BSS) Figure s (6A-F, 7A-F, 8)

Each 5-min period was quantified duration of the behaviors of feeding, cleaning and rest and no statistical differences between experimental groups during the 12 evaluation periods (Figure 6). The point of satiety occurred at 37 minutos for control-diet standard (Figure 6A), and 40 minutes for the malnourished-diet standard (Figure 6B). The feeding rate (Figure 7) diet standard intake was not significantly different between the malnourished (1.2 ± 0.07) and control (1.2 ± 0.07) groups. When observed the effect of selective inhibitor of serotonin reuptake on the BSS, the point of satiety occurred at 27 minutes for the control-fluoxetine

(Figure 6C) and 28 minutes for the fluoxetine-malnourished (Figure 6D). The rate of feed intake of standard diet under the effect of SSRI was greater (p <0.01, two-way ANOVA followed by Bonferroni test) for the malnourished (1.2 ± 0.07) than for the control (0.74 ± 0.06). The point of satiety was not seen during the evaluation of BSS with palatable food (cookies) between control (Figure 6E) and malnutrition (Figure 6F). The feeding rate was higher (p <0.001, Two-way ANOVA followed by Bonferroni test) for the malnourished (1.4 ± 0.06) than for the control (1.09 ± 0.05).

Discussion

Malnutrition increases the perinatal behavior motivational before the food reward, translated by the higher speed to run the entire job, in other words is to follow through and respond to reward food. It promotes reducing the rate of distraction, observed by the lower number of breaks and reverse direction during the learning incentives. Although increase the latency to respond to food reward during the stages of adaptation and pre-exposure.

Protein malnutrition in the perinatal period causes a reduction in body weight that persists after nutritional recovery. Data on body weight in adulthood these individuals are still controversial. This fact is related to the period and type of malnutrition that occurs as well as the type of diet consumed during nutritional recovery. Thus we observe a higher body weight in rats malnourished during the perinatal period that were fed diets hypercaloric during the recovery phase (2), or for life (3), or persistent weight reduction when normocaloric diet was consumed (4 - 7).

During pre-exposure test conducted in this study motivation, the highest latencies to exit the box and respond to the initial target box can indicate delay of learning promoted by malnutrition. However, when activity was repeated by the training phase, learning incentives, this deficit was minimized. The malnutrition was related in mice and human learning and memory deficits (18-19). Nutritional restriction in early life affect the formation of the hippocampus, a structure that plays an important role in learning and memory (20). The prenatal malnutrition decreased 20% in the number of neurons in the CA1 region of hippocampus (21). These neurons are essential for the process of learning and memory. Transmitters important in learning and spatial memory are affected by malnutrition, such as reduction in the density of GABAergic neurons (22-23), the levels of acetylcholine and the