Accepted Manuscript Title: Effect of intrahippocampal administration of anti-melanin-concentrating hormone on spatial food-seeking behavior in rats Author: Luciane Val´eria Sita Giovanne Baroni Diniz Newton Sabino Canteras Gilberto Fernando Xavier Jackson Cioni Bittencourt PII: DOI: Reference:

S0196-9781(15)30013-9 http://dx.doi.org/doi:10.1016/j.peptides.2015.12.007 PEP 69579

To appear in:

Peptides

Received date: Revised date: Accepted date:

28-7-2015 7-12-2015 23-12-2015

Please cite this article as: Sita Luciane Val´eria, Diniz Giovanne Baroni, Canteras Newton Sabino, Xavier Gilberto Fernando, Bittencourt Jackson Cioni.Effect of intrahippocampal administration of anti-melaninconcentrating hormone on spatial food-seeking behavior in rats.Peptides http://dx.doi.org/10.1016/j.peptides.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of intrahippocampal administration of anti-melanin-concentrating hormone on spatial food-seeking behavior in rats Luciane Valéria Sitaa, Giovanne Baroni Diniza, Newton Sabino Canterasa, c, Gilberto Fernando Xavierb, c, Jackson Cioni Bittencourta, c*

a

Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo - USP,

05508-000, Sao Paulo, Brazil. b

Department of Physiology, Institute of Biosciences, University of Sao Paulo – USP, 05508-090,

Sao Paulo, Brazil c

Center for Neuroscience and Behavior, Institute of Psychology, University of Sao Paulo, 05508-

030, Sao Paulo, Brazil

*Corresponding Author: Dr. Jackson C. Bittencourt, Laboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Science, University of São Paulo, Av. Prof. Lineu Prestes, 2415, Ed. B-III, São Paulo 05508-000 SP - Brazil. Tel: +55(11) 3091-7300 Fax: +55(11) 3091-8449 E-mail: [email protected]

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Highlights • Injection of an MCH antibody into the dorsal CA3 does not affect food-seeking behavior. • Injection of anti-MCH interferes with the hippocampal detection of a conflict situation. • Hippocampal MCH participates in the decision-making process related to feeding behavior.

ABSTRACT Melanin-concentrating hormone (MCH) is a hypothalamic peptide that plays a critical role in the regulation of food intake and energy metabolism. In this study, we investigated the potential role of dense hippocampal MCH innervation in the spatially oriented food-seeking component of feeding behavior. Rats were trained for eight sessions to seek food buried in an arena using the working memory version of the food-seeking behavior (FSB) task. The testing day involved a bilateral anti-MCH injection into the hippocampal formation followed by two trials. The anti-MCH injection did not interfere with the performance during the first trial on the testing day, which was similar to the training trials. However, during the second testing trial, when no food was presented in the arena, the control subjects exhibited a dramatic increase in the latency to initiate digging. Treatment with an anti-MCH antibody did not interfere with either the food-seeking behavior or the spatial orientation of the subjects, but the increase in the latency to start digging observed in the control subjects was prevented. These results are discussed in terms of a potential MCHmediated hippocampal role in the integration of the sensory information necessary for decision-making in the pre-ingestive component of feeding behavior.

Keywords: foraging, dry-land maze, spatial memory, working memory, hippocampus, food intake.

1. Introduction

Feeding behavior is a complex temporally organized process involving pre-ingestive, consummatory and post-ingestive mechanisms [28]. Distinct neuropeptide systems specifically contribute to the control of these different phases of feeding behavior [5]. The melanin-concentrating hormone (MCH) system plays a key role in the control of feeding behavior and energy metabolism; however, the feeding behavior phase in which this peptide 2

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acts remains unknown. The intracerebroventricular administration of MCH induces hyperphagia and increases body weight [21, 31, 48-50]. In addition, obese (ob/ob) mice and both fasted rats and mice exhibit MCH mRNA overexpression [25, 48, 63]. Furthermore, while MCH knock-out mice display a hypophagia-related decrease in body weight associated with increased energy expenditure [57], MCH receptor (MCH-1R) knock-out mice are lean, albeit hyperphagic and their leanness is a consequence of hyperactivity and increased metabolic rate [13, 32]. Moreover, the administration of MCH-1R-specific antagonists decreases body weight gain [9, 56, 62]. MCH-expressing neurons are primarily located in lateral hypothalamic (LHA) and incerto-hypothalamic areas (IHy), sending out widespread projections along the neuro-axis [8]. Projections from the arcuate nucleus to MCH neurons located in the LHA constitute an anatomical substrate underlying the function of MCH in food intake regulation [10, 17, 19, 53]. However, LHA and IHy are integrative centers that influence a variety of systems and functions but do not act as direct modulators of effector functions. The areas that are innervated by MCH neurons do not appear to be directly associated with hunger. Instead of promoting direct food ingestion, MCH neurons seem to affect arousal and locomotor activity, increasing the probability that an animal will find food [6, 52]. The distribution of the primary MCH immunoreactive fibers is widespread, including the hippocampal formation, prefrontal cortex, nucleus accumbens shell, medial septal nucleus, pars lateralis of the medial mammillary nucleus, median eminence and periaqueductal gray matter [8]. Several studies have suggested that this peptide is involved in a variety of functions, including arousal, exploratory behavior, depression, anxiety, reward, sleep homeostasis and memory processing [7-9, 12, 16, 18, 23, 33, 35, 36, 46, 65]. Within the hippocampal formation, the CA3 subfield is densely innervated with MCH immunoreactive fibers [8, 30]. In addition, the three hippocampal subfields, CA1, CA2 and CA3, are among the regions in the rat brain that most strongly express MCH-1R [51]. Maze learning is disrupted by dorsal, but not ventral, hippocampal damage [27, 58]. Moser and collaborators showed that dorsal hippocampal lesions, but not equally large ventral lesions, disrupt the acquisition of the spatial water maze task [39, 40]. In addition, dorsal CA1 pyramidal cell loss, which follows transient cerebral ischemia, disrupts spatial learning; interestingly, this deficit has been directly associated with the degree of cell loss [34, 44, 66]. More recently, several studies have demonstrated that the dorsal hippocampus plays a role in spatial working memory processes [14, 15]. Furthermore, the CA3 subfield is critical

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for the retention of spatial working memory [11, 20, 24, 26, 29, 60]. Interestingly, MCH modulates the activity of hippocampal cells, thus facilitating memory retention [36, 64]. Taken together, this evidence suggests that hippocampal MCH innervation might regulate the food-seeking component of feeding behavior in locations where food has been previously identified. To examine this hypothesis, the present study investigated the effects of bilateral topical micro-infusion of anti-MCH antiserum within the dorsal hippocampal formation on the performance of rats subjected to the food-seeking behavior (FSB) task to evaluate their spatial working memory [59].

2. Methods 2.1. Subjects For this study, 11 male Wistar rats, weighing 200-230 g at the beginning of the experiment, were maintained in individual cages under a 12 h light-dark cycle (lights on at 6 am) in a temperature-controlled and acoustically isolated room. All rats were food deprived until their weight was reduced to 85% of the ad-lib level and provided unlimited access to water. The food deprivation schedule was initiated at four days prior to the habituation sessions (see below), and the body weight of each animal was monitored daily. The animals were offered a single meal of chow two hours after daily training. All training and testing sessions were performed during the light phase of the cycle. All experiments were performed in accordance with the Guidelines for the Use of Animals in Neuroscience and Behavioral Research established by the US National Institutes of Health [42] and approved by the Institute of Biomedical Sciences, University of São Paulo Committee for Research and Animal Care (Approved Protocol 89/2010). We attempted to minimize the number of rats used, and every effort was made to ensure that no rat suffered unnecessarily.

2.2. Surgery At least 7 days before initiating the behavioral experiments, the animals were anesthetized via a subcutaneous injection of an aqueous solution containing ketamine (5 mg/100 g), xylazine (1 mg/100 g) and acepromazine (0.2 mg/100 g) and then placed in stereotaxic frames. Then, bilateral implants of guide cannula (22 gauge) aimed at the dorsal CA3 subfield of the hippocampus were introduced. The stereotaxic coordinates, based on the Paxinos & Watson’s rat brain atlas [45], were -3.6 (anteroposterior), ±3.3 (mediolateral), -3.1 (dorsoventral) mm. During the post-surgical time and before the beginning of the behavioral procedures, the rats were handled daily (once per day), and the surgical area was 4

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cleaned and disinfected with iodopovidine solution. The rats exposed to handling were later subjected to micro-infusion to habituate the animals to the procedure and prevent the implanted guide cannula from becoming obstructed, which involved the introduction of a needle with the same length as the guide cannula.

2.3. Apparatus The test apparatus [59] used in the FSB task was a round, 150 × 90 cm fiberglass arena, filled to a depth of 5 cm with washable dark blue granulated low-density polyethylene. To reduce the importance of olfactory cues associated with specific locations in the arena, 100 g of Froot LoopsTM cereal and 100 g of laboratory chow were crushed and mixed with the granulated polyethylene. A movable 20 × 20 × 20 cm opaque acrylic starting box, filled to a depth of 5 cm with granulated low-density polyethylene on the floor and a guillotine door in one of its walls, was used as a temporary cage in which the animal was housed from the beginning of the session until two hours after the daily task, when they received the daily amount of lab chow in their home cages. The same movable box was used to individually transfer the animals from their home cages into the arena, minimizing the stress of transfer and habituating the animals to the granulated polyethylene floor. The arena was located in a sound-attenuated room with high-contrast figures on the walls. A DVD camera (Sony Handycam Digital DCR-DVD203) equipped with a 0.6× wide-angle conversion lens was positioned approximately 280 cm above the center of the arena and used to register the sessions. In addition, the DVD camera was connected to a video monitor for the continuous monitoring of the session. Thus, the experimenter could intervene rapidly when the rat obtained food or when the time limit for finding food elapsed. For descriptive data analysis, the arena was imaginarily divided into four equal-area quadrants. The quadrants were separated along lines that intersected the edge of the arena at arbitrary cardinal starting locations, referred to as north, south, east and west. An imaginary area measuring 25 cm in diameter (the target counter) was located at the center of each quadrant, where Petri dishes containing food were offered (see below). These training counters provided more specific indices of the spatial location. Petri dish bottoms 60 mm in diameter, containing 1 g of food (6 pieces of lab chow) were covered with a single layer of ParafilmTM to avoid olfactory guidance. The external surface of the ParafilmTM did not contact the food.

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2.4. FSB task and topical micro-infusion of anti-MCH The FSB task has been fully described elsewhere [59] and involves a dry-land maze version of the Morris Water Maze [37, 38], modified to specifically examine food-seeking behavior and spatial working memory without olfactory cues. As the task aims to analyze the spatial memory component of the food-seeking, olfactory cues were removed because olfaction is a preponderant cue for rodents and could be used to locate the food. The subjects received 85% of the ad-lib chow level. Four days after the beginning of the food-deprivation, the animals were exposed to successive phases involving (1) two sessions of habituation to the apparatus, (2) two sessions of pretraining for the identification of Petri dishes containing food, (3) eight sessions of training in a working memory version of the task and (4) one session of testing involving the injection of anti-MCH (see below). A session was composed by daily trials. Before each session, the subjects were exposed to the micro-infusion handling procedure, as described above; for habituation, pre-training and training phases, no material was injected. The subjects were subsequently placed in an individual starting box for approximately 10 minutes before the beginning of the trials. The animals were transferred to the arena using the movable starting box, which was inserted in variable locations from trial to trial close to the arena wall, with the guillotine door oriented towards the arena center. Subsequently, the guillotine door was opened to release the animal. The habituation phase involved two sessions, with one session per day and two trials per session. In each trial, four Petri dishes were offered on the surface of the granulated polyethylene in the arena in the center of each quadrant. The animal was allowed to freely explore the arena for 3 minutes after its initial release from the starting box. If the subject either did not find or did not unseal any of the Petri dishes within 3 minutes, the experimenter manually guided the animal towards one of the Petri dishes and opened the ParafilmTM seal to allow the subject to obtain a pellet of lab chow. The animal was subsequently guided towards the starting box, where it remained for 20 minutes until the next trial. The pre-training phase involved two sessions, with one session per day and three trials per session. Each trial was similar to those of the habituation phase, except that the Petri dishes were progressively buried in the granulated polyethylene in the arena during all trials and sessions. The training phase involved eight sessions, with one session per day and three trials per session. Each trial was similar to that of the pre-training phase, except that only one Petri dish containing food was buried in the center of one of the quadrants. The quadrant containing the buried Petri dish varied randomly from session to session but was kept 6

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constant for the trials within a session to examine working memory. The trial ended when the subject either dug out and opened the Petri dish or carried the dish to the starting box. When the Petri dish was not found within 3 minutes after the initial release of the rat from the starting box, the subject was manually guided to the location of the dish, and the experimenter unburied the Petri dish to allow the subject to open it. If the animal did not then return to the starting box on its own, manual guidance was used to direct the animal back to the starting box, where it remained for 20 minutes until the next trial. Subsequently, a novel Petri dish containing food was buried in the same location for the next trial. The intertrial interval lasted 20 minutes. The testing phase involved one session with two trials. Each testing trial was similar to that of the training phase, except that in the second testing trial, no food was placed within the Petri dish buried in the arena (Probe test). Ten minutes before initiating the first testing trial, the subjects were individually placed in waiting cages (the movable starting boxes) and bilaterally micro-infused with either 1 l of the anti-MCH antibody (1:1, N=7, anti-MCH group) or 1 l of 0.9% saline (N = 4, control group), at an infusion rate of 1 l per minute. For microinjection, a 28-gauge infusion cannula that was 1 mm longer than the guide cannula was inserted. The infusion cannula was connected to a polyethylene tube for remote injection using a Hamilton microsyringe and was removed from the guide cannula 5 minutes after the microinjection. The subjects were then held for another 5 minutes prior to initiating the session. The anti-MCH used in this study was a generous gift from Dr. Wylie W. Vale (in memoriam) and Joan Vaughan from The Salk Institute (La Jolla, CA, USA). This antiserum (PBL #234) was raised in rabbit and it was prepared against synthetic peptide conjugated to human α-globulins via glutaraldehyde. Previous specificity tests showed that the staining for MCH was completely abolished by preincubation of the antiserum with micromolar concentrations of the peptide, and the staining with this antiserum was colocalized with in situ hybridization for the mRNA for the same protein [8, 41, 54].

2.5. Histology At the end of the behavioral procedures, the animals were anesthetized with a lethal dose of 35% chloral hydrate (1 ml, i.p.) and perfused via the ascending aorta with cold 0.9% saline (100 ml), followed by 4% formaldehyde fixative in borate buffer, pH 9.5, at 4°C (800– 1,000 ml). Immediately after perfusion, the brains of the animals were removed, postfixed overnight in the same fixative (supplemented with 20% sucrose) and then transferred to 20%

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sucrose in potassium phosphate-buffered saline (KPBS), pH 7.4. Series of 30-m coronal sections (1:5 series) were cut on a freezing microtome and stored at −30°C in antifreeze buffer. For reference purposes, one series from each animal was mounted on gelatin-coated slides, stained with thionin and coverslipped. The slides were examined under a light microscope to determine the placement of the cannula. Only those animals in which the tip of the injection cannula was positioned correctly were considered in the statistical evaluation of the results.

2.6. Analysis of the anti-MCH diffusion Series of sections of all anti-MCH and control animals were submitted to a standard peroxidase reaction in order to analyze the diffusion of the antiserum injected in the hippocampus (anti-MCH group) or its absence (control group). Sections were treated with 0.3% peroxide in KPBS for 15 min and then incubated in biotinylated secondary antibody made in donkey anti-rabbit (Jackson Labs, 1:800) and avidin-biotin complex (Vector, 1:333), both for 1h. Next, the sections were subjected to a peroxidase reaction using diaminobenzidine tetrahydrochloride (DAB, Sigma, 0,02%) as a chromogen solution, nickel sulfate 2,5% and 0,02% hydrogen peroxide dissolved in sodium acetate buffer. The reaction was terminated after 2-4 minutes with successive rinses of sodium acetate buffer and KPBS. Sections were mounted on gelatin-coated slides, dehydrated, cleared in xylene and coverslipped.

2.7. Data analysis For each trial, in both the training and testing sessions, we measured (1) the total amount of time the animal spent locating the Petri dish after release from the starting box (latency); (2) the total distance traveled (path length); (3) the latency until the first entry of the rat’s head into the quadrant where the Petri dish was buried (target quadrant); (4) the latency until the first entry of the rat’s head into the target counter after the first entry into the target quadrant; and (5) the latency to dig in the correct location after the first entry into the target counter. ANY-mazeTM behavioral analysis software (Stoelting, Wood Dale, IL, USA) was used to score the trial data. Only animals presented adequate cannula position were included in the statistical analysis. The training sessions were identical for all subjects, and until the end of this phase, there were no distinct group treatments. During the testing session, however, some of the subjects were injected with the anti-MCH antibody prior to testing, while other subjects were 8

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injected with saline, thus generating two groups. Nevertheless, the training data analysis was run retrospectively, considering the existence of these two groups. Hence, potential differences occurring during testing, but not during training, could reflect the drug treatment rather than pre-existing group differences. The behavioral scores were subjected to repeated measures analysis of variance (ANOVA), using the groups as the between-subject variables and days (sessions) and the trials as the within-subject variables (SAS Institute, Inc., Cary, NC). Separate ANOVAs for each measure were run for both the training and testing scores.

3. Results 3.1. Histology Light microscopic evaluation of Nissl-stained sections from the rat brains indicated that the tip of the cannula was positioned correctly in the CA3 subfield of the hippocampus, bilaterally (Figure 1).

---------------------------------------------------------------------------Please insert Figure 1 about here -----------------------------------------------------------------------------

3.2. Antiserum diffusion Light microscopic analysis of the sections submitted to the peroxidase reaction to observe the diffusion of the antiserum around the cannula showed that the injected volume of anti-MCH was enough to diffuse to the CA3 region (Figure 2, A-F). CA2 may be received a limited amount of the antiserum, although most of the CA2 region was lesioned by the passage of the guide cannula in all animals. This analysis also suggested that the antiserum seems to be absorbed at different speeds among the animals, as revealed by different immunoreactivity areas. An anti-MCH animal showed weak labeling (Figure 2B) and other one a strong labeling (Figure 2C) making the establishment of diffusion boundaries difficult. The photomicrograph of an anti-MCH animal was omitted for aesthetical purposes. All control animals that were injected with saline presented absence of immunoreactivity.

---------------------------------------------------------------------------Please insert Figure 2 about here 9

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----------------------------------------------------------------------------3.3. FSB task training The acquisition of the food-seeking task was assessed by requiring the rats (n=11) to learn a novel food location every day, in a matching to place procedure with three trials per day and an inter-trial interval of 20 minutes. The animals exhibited a general improvement in performance during training, as revealed by significant Day effects for latency (F7,63 = 14.02, P < 0.0001), path length (F7,63 = 11.79, P < 0.0001), latency until the first entry into the target quadrant (F7,63 = 2.85, P < 0.02), latency until the first entry into the target counter after the first entry into the target quadrant (F7,63 = 2.60, P < 0.03) and latency to dig in the correct place after the first entry into the target counter (F7,63 = 7.10, P < 0.0001). Indeed, Figure 3 shows that there was a general decrement in these scores as the subjects learned to manage the basic requirements of the FSB task. Repeated measures ANOVA also revealed (1) significant Trial effects for the latency (F2,18 = 6.17, P < 0.01) and the latency until the first entry into the target counter (F2,18 = 6.66, P < 0.01); (2) slightly significant Trial effects for the path length (F2,18 = 3.51, P = 0.0518) and the latency to the first entry into the target quadrant (F2,18 = 3.51, P = 0.0516); and (3) significant Day × Trial interaction effects for the latency to the first entry into the target quadrant (F14,126 = 3.47, P < 0.001). Indeed, Figure 3 shows that performance, expressed as the latency, path length and latency to the first entry into the target counter, generally decreased during the trials, indicating that the animals are capable of storing information concerning the food location obtained from the first trial in their working memories for subsequent application in the remaining trials.

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As expected, ANOVA revealed no significant main Group, Group × Trial, Group × Day or Group × Trial × Day interaction effects for any of the training scores (Figure 3). This result suggests that the performance of the subjects in each of the groups did not differ prior to the administration of anti-MCH, ruling out the possibility that differences in the testing scores reflect pre-existing group differences.

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3.4. Testing In the testing session, the subjects received a bilateral intra-hippocampus injection of either an anti-MCH antibody or saline 10 minutes before the testing session. In the first trial, a sealed Petri dish containing food was buried in the center of one of the quadrants. In the second trial, the sealed Petri dish was empty (Probe test). The ANOVA revealed (1) a significant main Group effect for the latency (F1,9 = 7.48, P < 0.03) (Figure 3A) and path length (F1,9 = 7.83, p 0.5). As shown in Figure 3, the anti-MCH and control subjects were equally capable of finding the location where the food was buried in the first trial. However, in the second trial, when the sealed Petri dish was empty, although the subjects injected with anti-MCH within the hippocampus reached the target location at the same time as the control subjects (Figure 3C and 3D), these animals took longer to dig (Figure 3E), as reflected in the total latency (Figure 3A) and path length (Figure 3B).

4. Discussion In this study, we investigated the effect of interference with hippocampal MCH neurotransmission on spatially oriented food-seeking behavior. The effects of bilateral antiMCH microinjection into the dorsal hippocampal formation were investigated in rats exposed to an appetitive task (the FSB task), adapted to evaluate the contribution of the spatial memory to food-seeking behavior [59]. Animals previously trained in a working memory version of the FSB task were microinjected with anti-MCH 10 minutes before the testing session to evaluate the effects of anti-MCH on (1) foraging for food in one of four previously rewarded locations, i.e., the center of each of the quadrants of the arena; (2) the acquisition of information concerning the location of food in that specific testing session; and (3) the maintenance of this information in the working memory for at least 20 minutes for subsequent use in a second testing trial. 11

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In order to properly evaluate the effect of the antiserum into the hippocampus, cannulas were placed in the CA3 subfield of the dorsal hippocampus, since the CA3 subfield is denser innervated with MCH immunoreactive fibers than CA1 or CA2 subfields [8, 30]. We analyzed only animals with cannulas properly localized in this subfield. However, the postmortem analysis of the antiserum diffusion was unable to ensure that the diffusion was restricted to the CA3. We considered that the anti-MCH inactivated primarily the CA3 MCH innervation, but also promoted an uncertain inactivation in adjacent areas. It is also important to note that the guide cannula caused extensive damage in CA2 subfield. The histological and immunohistochemical analysis suggest that the behavioral effect described in this paper is a result of the disfunction of the dorsal hippocampal formation. Four different latency-related measures were determined to properly evaluate performance. In each trial, we measured the standard latency to locate the Petri dish after release from the starting box, the latency until the first entry into the target quadrant where the Petri dish was buried, the latency until the first entry into the target counter after the first entry into the target quadrant, and the latency to dig in the correct place after the first entry into the target counter. Decomposition of the general latency score into subcomponents facilitated a detailed evaluation of performance. That is, while the latencies until the first entry into the target quadrant and target counter represent specific indices of spatial orientation, reflecting the spatial memory of the subjects, the latency to dig in the correct location after the first entry into the target counter reflects either the motivation or the certainty of the subject relative to the location of food. The results obtained in the present study showed that injecting the MCH antibody into the dorsal hippocampus did not interfere with food foraging in previously rewarded locations based on an allocentric spatial cognitive map, as food seeking during the first testing trial was not disturbed in the anti-MCH-injected subjects compared to the control subjects. In the second testing trial, conducted 20 minutes after the first testing trial, an empty sealed Petri dish was present at the previously rewarded location. There were no significant differences between the anti-MCH and control subjects regarding the finding of the correct location, as reflected in both the latency to the first entry into the target quadrant and the latency to the first entry into the target counter. Surprisingly, however, the control subjects exhibited longer latencies to dig in the correct location after the initial entry into the target counter compared to anti-MCH subjects, which promptly dug in the correct place. As a consequence of the increase in the latency to dig, the control subjects also exhibited longer general latencies and path lengths than anti-MCH injected subjects. 12

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The decomposition of the latency measure applied in the present study revealed that the anti-MCH antibody did not affect spatial learning. The increase in latency to start digging in the correct location by the control subjects in the second testing trial relative to both (1) the corresponding scores by anti-MCH injected subjects and (2) their own latencies to start digging in the first testing trial is quite surprising and seems to be related to absence of food. ParafilmTM sealing seems to avoid the animals to use olfactory cues to locate the petri dish, but didn´t prevent them to recognize the presence or absence of food after locating the Petri dish. As revealed by the saline-injected group, food-originated olfactory cues are required to stimulate the digging behavior in this behavioral task. The injection of anti-MCH could have altered the attentional process necessary to the modulation of the motor behavior and the animals did not stop the behavior of searching for food, even in front of the absence of food, as expected. It has been suggested that the hippocampus plays a pivotal role in detecting conflict situations through interrupting ongoing behavior and increasing the level of arousal and attention to enhance the gathering of information [22]. The hippocampus functions as a context analyzer that modulates adaptive motor activity in response to particular sensory events in the environment [43]. MCH innervation of the hippocampus could be associated to this hippocampal role in detecting conflict situation, since the animals injected with antiMCH did not adapt their behavior in a test involving an unexpected absence of food. Adams et al. [3] showed that MCH knockout mice exhibit impaired food-seeking behavior associated with normal levels of exploratory activity and the authors concluded that the MCH played a role in regulating sensory integration from olfactory pathways. Besides, Adamantidis et al. [2] described that MCH-1R knockout mice present learning and memory impairments, probably secondary to their hyperactivity and their alterations of NMDA receptor function. According to Scharfman [55] the CA3 subfield receives inputs from a variety of sources, and may be an "entry point" to hippocampus that receives information which needs to be "broadcasted". Since hippocampus receives indirect olfactory afferents through the perforant path (entorhinal cortex projections to dentate gyrus and CA3 subfield), the antiMCH antibody could have prevented the integration of the olfactory information in the decision-making processes. This integratory function lies on the fact that the perforant path terminals in the CA3 subfield are mainly located in the stratum lacunosum-moleculare [4], where MCH terminals are also presented [8].

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Taking together, our data suggest that MCH hippocampal innervation is associated to the hippocampal role in detecting potential conflicts that might occur during normal exploratory activity, which is based on sensory integration. Antagonizing this effect through the anti-MCH local injection disturbed the integration of olfactory information and prevented the animals to realize the absence of food as control animals did. Concluding, lateral hypothalamic area MCH-containing cells and the broadly distribution of such terminals are in the position to subserve the control of motivated behaviors, memory and learning [1, 61]. The widespread distribution of MCH suggests a role for MCH in mammals, i.e., a function in generalized arousal and sensorimotor integration [8], which can, in turn, play a role in information gathering. Although our current knowledge regarding MCH suggests that this peptide is a critical hypothalamic regulator of energy homeostasis, affecting both feeding behavior and energy expenditure [47], it is also reasonable to consider that somatosensory information might converge on MCH cells that mediate diffuse activational functions, such as feeding [53]. Taking into account that feeding behavior includes different categories of temporally distinct behaviors, i.e., pre-ingestive (food-seeking) and consummatory mechanisms [5], the results obtained in the present study suggest that MCH terminals in the dorsal hippocampus might play a role in the integration of the sensory information that is necessary for the decision making involved in the preingestive component of feeding behavior.

Disclosure Policy The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements: The authors would like to thank Dr. Wylie Vale (in memoriam) and Dr. Joan Vaughan for the generous gift of anti-MCH antiserum and Amanda Ribeiro de Oliveira for expert technical assistance. Grants and fellowships were obtained from CNPq (521.799/96-1), CAPES and FAPESP (#04/13849-5, #04/13850-3, #11/09816-8). J.C.B., N.S.C and G.F.X. are CNPq Investigators.

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FIGURE LEGENDS

Figure 1. A representative brightfield photomicrograph of a Nissl-stained section obtained from a rat showing gliosis of the cannula implants in the hippocampus. Abbreviations: CA1, CA1 subfield of the hippocampus; CA2, CA2 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; cc, corpus callosum; DG, dentate gyrus. Bar: 500µm. Figure 2. Antiserum diffusion in the hippocampus. Brightfield unilateral photomicrograph of sections from bilateral injection of anti-MCH (A-F) and saline injected animals (G-J) submitted to peroxidase protocol showing an immunostained anti-MCH injection (antiMCH animals, arrows) or the absence of immunoreactivity in control animals injected with saline. Abbreviations: CA1, CA1 subfield of the hippocampus; CA2, CA2 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; DG, dentate gyrus; fi, fimbria of the hippocampus. Bar: 500µm.

Figure 3. Performance of the rats during the 8 days of training in a working memory version of the food-seeking behavior (FSB) task, with 3 trials per day, and along with 2 trials on a single testing day before which the rats received a single bilateral injection of either antiMCH (open squares) or saline (black circles). The treatments were implemented only before the testing session. Therefore, for purposes of representation, the analysis of the training data in the separate groups is only retrospective. See main text for detailed explanations. (A) Latency to locate the food. (B) Mean distance traveled (path length) to locate the food. (C) Latency until first entry into the target quadrant. (D) Latency until the first entry into the target counter after the first entry into the target quadrant. (E) Latency to dig in the correct place after the first entry into the target counter. The data are reported as mean scores ±SEM.

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Fig.2

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