Anorexia increases microglial density and cytokine expression in the hippocampus of young female rats

Behavioural Brain Research 363 (2019) 118–125

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Anorexia increases microglial density and cytokine expression in the hippocampus of young female rats

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Durairaj Ragu-Varman1, Mayra Macedo-Mendoza1, Francisco Emmanuel Labrada-Moncada, ⁎ Pamela Reyes-Ortega, Teresa Morales, Ataúlfo Martínez-Torres, Daniel Reyes-Haro Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Boulevard Juriquilla 3001, Juriquilla, Querétaro, CP76230, Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: Anorexia Hippocampus Microglia TNF-α IL-6 IL-1β

Anorexia by osmotic dehydration is an adaptive response to hypernatremia and hyperosmolaemia induced by ingestion of a hypertonic solution. Dehydration-induced anorexia (DIA) reproduces weight loss and avoidance of food, despite its availability. By using this model, we previously showed increased reactive astrocyte density in the rat dorsal hippocampus, suggesting a pro-inflammatory environment where microglia may play an important role. However, whether such anorexic condition increases a pro-inflammatory response is unknown. The aim of this study was to test if DIA increases microglial density in the dorsal hippocampus, as well as the expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL1β) in the hippocampus of young female rats. Our results showed that DIA significantly increased microglial density in CA2-CA3 and dentate gyrus (DG) but not in CA1. However, forced food restriction (FFR) only increased microglial density in the DG. Accordingly, the activated/resting microglia ratio was significantly increased in CA2-CA3 and DG, in DIA and FFR groups. Finally, western blot analysis showed increased expression of IBA1, TNF-α, IL-6 and IL-1β in the hippocampus of both experimental groups. We conclude that anorexia triggers increased reactive microglial density and expression of TNF-α, IL-6 and IL-1β; this environment may result in hippocampal neuroinflammation.

1. Introduction Appetite is regulated by neural networks that are responsible for stimulation and inhibition of feeding. The neurobiology of appetite is clinically relevant to reveal how pathological disruption of synaptic circuits contributes to eating disorders. Among them, anorexia is known to reduce appetite and, consequently, food intake [1]. Pathological anorexia includes anorexia nervosa and disease-associated anorexia; the latter may be linked to cachexia, characterized by a progressive wasting of adipose and muscle tissues observed in cancer, AIDS or chronic bacterial and parasitic diseases [1]. Although reduced appetite and food intake cannot be directly evaluated in animals, a pharmacologicallymediated increase of cytokines in the central nervous system (CNS) is used to induce what is known as inflammation-associated anorexia [2–4]. At cellular level, microglia activation is involved in this homeostatic feeding behavior [5]. Moreover, TLR2-induced activation of hypothalamic microglia targets POMC neurons, thereby leading to a negative energy balance via melanocortin pathway regulation [6]. In

contrast, adaptive anorexia occurs regularly at specific stages of the life cycle that are associated with hibernation, incubation and migration [7]. Another example of adaptive anorexia is the one induced by dehydration. Here, the reduction of food intake protects fluid balance by allowing water to move from the gut to the rest of the tissues; this reduces the intake of osmolytes from food and ameliorates hyperosmolaemia. Thus, dehydration-induced anorexia (DIA) is an alternative model that reproduces weight loss and reduced food intake despite its availability, and it has been extensively characterized [8–11]. This experimental model includes a pair-fed group with animals under forced food restriction (FFR). The FFR group allows to distinguish the effects of fasting over dehydration on the CNS. Briefly, negative energy balance promotes physiological adaptations, mainly through the hypothalamic-pituitary-thyroid (HPT) axis. During fasting the serum content of thyrotropin and thyroid hormones decreases, enhancing the survival rates of individuals. This adjustment is observed in the FFR group, whereas DIA rats fail to decrease their HPT axis function and present high thyrotropin blood concentration [10]. The hypothalamic

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Corresponding author. E-mail address: [email protected] (D. Reyes-Haro). 1 Equal Contribution. https://doi.org/10.1016/j.bbr.2019.01.042 Received 17 July 2018; Received in revised form 7 January 2019; Accepted 25 January 2019 Available online 25 January 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.

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paraventricular nucleus is involved in the HPT axis adjustment during food restriction via the inhibition of thyrotropin-releasing hormone mRNA expression as a result of an increased local T3 content [11]. Thyrotropin-releasing hormone expression is also modulated by orexigenic and anorexigenic peptides [9–11]. On the other hand, recent magnetic resonance imaging studies reported hippocampal volume reduction in patients with anorexia nervosa [12–15], suggesting that the hippocampus might be also affected by anorexia. In support of this hypothesis, alterations in cell proliferation and dendritic branching, reduced the expression of glial fibrillary acidic protein (GFAP) and increased the number of reactive astrocytes and related intermediate filaments (vimentin and nestin) in activity-based anorexia (ABA) and DIA murine models [16,17]. These alterations suggest an inflammatory process associated with anorexia, which may result from the action of cytokines released by microglia, the resident immune cells in the CNS [3,18,19]. Interestingly, pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) are involved in feeding inhibition [20–22]. Furthermore, mRNA expression of these cytokines is higher in serum samples of anorexic patients [23–25], and sickness behavior, including cytokinemediated anorexia, is triggered by microglial activation in the hypothalamic arcuate nucleus [6]. To date, whether anorexia can induce inflammatory responses in brain areas such as the hippocampus has not been explored. Thus, the aim of this study was to test if DIA increases microglial density and the expression of TNF-α, IL-6 and IL-1β in the hippocampus of young female rats. 2. Materials and methods 2.1. Animals and housing All the experimental protocols in this study were approved by the Ethics Committee of the Institute of Neurobiology at UNAM. Animals were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Briefly, female Wistar rats (180–200 g) were individually housed under a 12:12-h light/dark cycle and controlled temperature, with free access to food and water.

Fig. 1. Daily food intake and body weight. The food intake (A) and body weight (B) of young female rats in each experimental group was monitored daily for five days. DIA animals decreased their food intake from the first day, and this was consistently reflected in the body weight of the animals. The FFR group was pair-fed to DIA intake. (C) Representative example of IBA1 immunofluorescence in the dorsal hippocampus of a DIA rat. Selected regions for analysis were: Cornus Ammonis 1 and 2–3 (CA1 and CA2-CA3) and dentate gyrus (DG). Microglia and nuclei estimations were performed in the stratum oriens (s.o.) and stratum radiatum (s.r.) of CA1, CA2-CA3 and hilus region in the DG. The white arrow shows anatomical orientation: dorsal (D), ventral (V) and lateral (L). Scale bar = 200 μm. Data are shown as mean ± SEM. Significant differences were considered as + p < 0.05; ++,** p < 0.01; *** p < 0.0001.

2.2. Dehydration-induced anorexia procedure The experimental protocol of DIA was previously reported [8,17,26,27]. Briefly, three independent series of experiments were performed for five days; each one consisted of three groups of four animals randomly selected and placed in individual cages. The control group (N = 12) received water and food ad libitum. The DIA group (N = 12) received unrestricted access to food and a 2.5% NaCl solution as their sole drinking liquid. The FFR group (N = 12) received the same quantity of food consumed by DIA rats along with tap water ad libitum. The FFR group was a positive control to distinguish between reduced food intake and dehydration effects. Solid food intake and body weight were recorded daily at noon for five days, for all the experimental groups (Fig. 1A, B).

floating sections. The sections were rinsed three times in PBS buffer followed by treatment with 3% hydrogen peroxide (H2O2) for 10 min, rinsed another three times in PBS and incubated in 1.0% sodium borohydride for 6–8 min to reduce free aldehydes. The tissue was then placed in blocking solution (5% horse serum albumin/1% Triton X-100 in PBS) for 1 h. Sections were incubated overnight with polyclonal rabbit anti-IBA1 antibody (dilution 1:1000, Wako, Life Sciences). After washing, the primary antibody was detected with Alexa 594 (1:500, Invitrogen) coupled to goat anti-rabbit antibody. The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted with Vectashield H-1000 (Vector Laboratories, Burlingame, CA, USA). Coronal sections containing the hippocampus were mounted on slides and photographed with a digital camera (Photometrics Cool Snap FX, USA) attached to a Nikon microscope (Nikon Eclipse E600, Tokyo, Japan), and images were analyzed using IMAGE J version 1.41 (NIH, Bethesda, MD, USA). Zeiss LSM 780 Meta confocal microscope (Zeiss, Gottingen, Germany) was used for confocal images with Alexa 594 (excitation/emission wavelength 590/617 nm) and DAPI (excitation/

2.3. Histology and immunohistofluorescence Rats were euthanized with an overdose of pentobarbital (100 mg/ kg) and transcardially perfused with 100 ml of saline solution, followed by 250 ml of chilled paraformaldehyde 4% in phosphate-buffered saline (PBS), pH 7.4. Brains were removed and post-fixed overnight and were cryoprotected in graded sucrose solutions (10, 20 and 30% in PBS). Coronal sections (30 μm) along the dorsal hippocampus were obtained with a cryostat (Leica CM1850), and sections were collected and stored in cryoprotectant solution (30% ethylene glycol: 20% glycerol in PBS) at -20 °C [17,27,28]. The immunoreactivity for ionized calcium-binding adapter (IBA1), a specific marker for microglia [29], was performed on 119

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emission wavelength 350/460 nm) (Fig. 1C).

3. Results

2.4. Microglial cell counting

3.1. Body weight and food intake Food intake of the DIA group was reduced 23% from the first day of the experiment compared to the control group (100%, 19 ± 0.6 g), reaching a minimum intake on day 5, when rats consumed 82% less compared to control group ingestion (100%, 23.9 ± 0.5 g) (Fig. 1A). A two-way repeated measures ANOVA showed significant differences between groups (F2,4 = 457.0, p < 0.001), time (F5,10 = 76.30, p < 0.001) and the interaction between variables (F10,20 = 68.49, p < 0.001). DIA animals had a 14% reduction of body weight from day 2 vs control animals (Control = 100%; 187.3 ± 3 g), whereas the FFR group lost 15% of their body weight until day 3 vs the control group (Control = 100%; 195.3 ± 0.3 g), despite consuming the same amount of food as the DIA experimental group. Body weight loss was maintained until day 5, and was 30% and 22% less than control body weight (Control = 100%; 201.7 ± 1 g) in DIA and FFR groups, respectively (Fig. 1B). A two-way repeated measures ANOVA showed differences between groups (F2,4 = 27.55, p < 0.01), time (F5,10 = 11.20, p < 0.001) and in the interaction between variables (F10,20 = 14.25, p < 0.001).

The quantification of IBA1+ cells was compared to the DAPI-labeled nuclei for each subfield of the dorsal hippocampus: CA1, CA2CA3 and dentate gyrus (DG). A total of 2–3 random fields in each of the three brain sections selected by similar or equivalent rostro-caudal extensions of the hippocampus of 12 rats per group was used for cell counting. Nine random fields from three tissue sections of each animal, in each experimental group, were used for estimations. A test square grid of 100 x 100 μm (0.01 mm2) was used to estimate, manually and with Cell Profiler, the number of DAPI-stained nuclei and IBA1+ cells [17,27,30]. Only process-bearing cells showing their soma in the plane of the analyzed area were counted, and their density was estimated (number of cells per mm2). Chosen fields located within each of the analyzed hippocampal subfields were counted by progressive displacement of a test square grid. The microglia/nuclei ratio was calculated by dividing the number of IBA1+ cells by the total number of DAPI-labeled nuclei. Resting microglia were identified as cells with thin and highly ramified processes that were longer than the diameter of the soma, whereas reactive microglia were defined by a pleomorphic cell body with shortened cellular processes that were smaller than the diameter of the soma [31,32]. The reactive/resting microglia ratio was estimated for all the experimental groups.

3.2. Anorexia increases microglial density in CA2-CA3 and DG but not in CA1 Nuclear density in the CA1 region of the dorsal hippocampus showed no significant changes between groups (F(2,33) = 0.02161, p = 0.97863) (Table 1). Interestingly, microglial density in CA1 showed significant differences between groups (F(2,33) = 11.15189, p < 0.001) for the DIA group (p < 0.001), but not for the FFR group (p = 1), when compared to the control (Table 1). Additionally, we determined the microglia/nuclei ratio by estimating the IBA1+ cells from the total nuclei, and there were no significant changes between control, DIA and FFR groups (F(2,33) = 3.1677, p = 0.05515) in CA1 (Fig. 2A and Table 1). We also analyzed the same parameters in the dorsal hippocampal CA2-CA3 region. One-way ANOVA showed significant differences between groups (F(2,33) = 5.56059, p = 0.00829) for nuclei density with a significant increase in DIA (p = 0.02542), but not for the FFR group (p = 1), when compared to the control. The estimated microglial density in CA2-CA3 also showed significant differences between groups (F(2,33) = 40.37377, p < 0.001) for DIA (p < 0.001), but not FFR group (p = 0.29), when compared to the control (Table 1). Additionally, we observed that the estimated microglia/nuclei ratio was significantly different between groups (F(2,33) = 15.09554, p < 0.001) (Fig. 2B) with a significant increase in DIA (p < 0.001), but not for the FFR group (p = 0.59207), when compared to the control. Lastly, we analyzed the dorsal hippocampal DG region. The nuclei density showed no significant changes in the DG region (F(2,33) = 0.35293, p = 0.70525) (Table 1). On the other hand, microglial density showed significant differences between groups (F(2,33) = 17.41930, p < 0.001). Surprisingly, microglial density increased significantly in the DIA group when compared to the control (p < 0.001), but no significance was observed between control and FFR groups (p = 0.15125). Finally, the estimated microglia/nuclei ratio was significantly increased in the DIA and FFR groups when compared to the control (F(2,33) = 27.43622, p < 0.001) (Fig. 2C, Table 1).

2.5. Western blot The whole hippocampus was dissected out as previously described [17]. Briefly, the hippocampi from three different rats for each experimental group (Control, DIA and FFR) were homogenized in ice-cold lysis buffer (200 mM glycine, 150 mM NaCl, 50 mM EGTA, 50 mM EDTA and 300 mM sucrose) along with a protease inhibitor (SigmaAldrich, USA). The total homogenate was centrifuged at 10,000 X g for 15 min at 4 °C; then the supernatants were stored at −80 °C. Concentration of protein from each sample was estimated by Bradford's method [33], and 30 μg of equal concentration of protein was resolved by 10% SDS-PAGE. Proteins were transferred electrophoretically to the PVDF membrane, which was then incubated in blocking solution (5% nonfat dry milk in TBS-T) for 3 h at room temperature. Membranes were incubated overnight at 4 °C with one of the following primary antibodies: (a) polyclonal rabbit anti-IBA1 (dilution 1:5000, Wako); (b) monoclonal mouse anti-TNF-α (dilution 1:1000, Santacruz, Dallas, TX, USA); (c) monoclonal mouse anti-IL-6 (dilution 1:1000, Santacruz, Dallas, TX, USA); (d) polyclonal rabbit anti-IL-1β (dilution 1:1000, Millipore) and (e) monoclonal mouse anti-GAPDH (dilution 1:1000, Santacruz, Dallas, TX, USA). The membranes were washed three times with TBS-T, and bound antibodies were detected by incubating for 3 h with either goat anti-mouse alkaline phosphatase conjugated antibody (dilution 1:2500; Santacruz, Dallas, TX, USA) or goat anti-rabbit alkaline phosphatase conjugated antibody (dilution 1:2500; Santacruz, Dallas, TX, USA). Alkaline phosphatase activity was detected by using BCIP/NBT AP-conjugate substrate reaction kit (Bio-Rad, USA). Images were acquired with a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Inc., USA) and optical density of trace quantity for each band was measured using Image Lab 3.0 software (Bio-Rad Laboratories Inc.). 2.6. Statistics

3.3. Anorexia increases reactive/resting microglia ratio in CA2-CA3 and DG due to reduced food intake

All the data corresponding to each treatment and hippocampal region were pooled and are presented as the mean ± standard error of the mean (S.E.M.). Statistical analysis of data was performed using a one-way ANOVA followed by a Bonferroni post-test with Origin 7.0 software; p < 0.05 was considered statistically significant.

The amount of reactive microglia was estimated for CA1, CA2-CA3 and DG regions. Reactive microglial cells were identified as IBA1+ with processes shorter than the diameter of the soma (Fig. 3). Reactive microglial density showed significant differences between groups in CA1 (F(2,15) = 6.100970, p = 0.01151). Thus, DIA significantly 120

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Table 1 Effect of dehydration induced anorexia (DIA) or forced food restriction (FFR) on microglial density. Hippocampal region Nuclei CA1 CA2-CA3 DG/SGZ Microglia CA1 CA2-CA3 DG/SGZ Microglia/nuclei ratio CA1 CA2-CA3 DG/SGZ Reactive microglia CA1 CA2-CA3 DG/SGZ Resting microglia CA1 CA2-CA3 DG/SGZ Reactive/Resting microglia ratio CA1 CA2-CA3 DG/SGZ

Control

DIA

FFR

733 ± 36 (N = 12) 1130 ± 64 (N = 12) 1192 ± 89 (N = 12)

758 ± 102 (N = 12) 1349 ± 61 (N = 12)* 1312 ± 112 (N = 12)

743 ± 100 (N = 12) 1117 ± 38 (N = 12) 1229 ± 110 (N = 12)

115 ± 11 (N = 12) 89 ± 8 (N = 12) 75 ± 3 (N = 12)

219 ± 27 (N = 12)*** 230 ± 14 (N = 12)*** 337 ± 52 (N = 12)***

111 ± 11 (N = 12) 117 ± 12 (N = 12) 167 ± 19 (N = 12)

0.19 ± 0.03 (N = 12) 0.09 ± 0.01 (N = 12) 0.07 ± 0.01 (N = 12)

0.43 ± 0.10 (N = 12) 0.19 ± 0.01 (N = 12)*** 0.26 ± 0.03 (N = 12)***

0.25 ± 0.06 (N = 12) 0.12 ± 0.02 (N = 12) 0.13 ± 0.01 (N = 12)*

51 ± 5 (N = 6) 48 ± 6 (N = 6) 32 ± 3 (N = 6)

96 ± 11 (N = 6)* 136 ± 13 (N = 6)*** 170 ± 21 (N = 6)***

69 ± 11 (N = 6) 79 ± 9 (N = 6) 80 ± 14 (N = 6)

61 ± 9 (N = 6) 73 ± 10 (N = 6) 37 ± 5 (N = 6)

64 ± 9 (N = 6) 66 ± 4 (N = 6) 49 ± 8 (N = 6)

34 ± 6 (N = 6) 37 ± 5 (N = 6)* 20 ± 2 (N = 6)

0.99 ± 0.13 (N = 6) 0.77 ± 0.09 (N = 6) 0.92 ± 0.11 (N = 6)

1.67 ± 0.31 (N = 6) 2.51 ± 0.27 (N = 6)** 4.54 ± 0.33 (N = 6)***

1.60 ± 0.26 (N = 6) 2.74 ± 0.41 (N = 6)*** 4.51 ± 0.59 (N = 6)***

Data are mean ± S.E.M. (* p < 0.05; ** p < 0.01; *** p < 0.001).

differences in DIA (F2,6 = 18.361, p < 0.005). Likewise, the expression of IL-6 increased significantly in DIA (66%) and FFR (65%) when compared to the control (Fig. 4). One-way ANOVA analysis showed significant differences in DIA (F2,6 = 25.812, p < 0.005). Finally, IL1β expression was significantly increased in DIA (105%) and FFR (102%) compared to the control (Fig. 4). One-way ANOVA analysis showed significant differences in DIA (F2,6 = 6.919, p < 0.05).

increased reactive microglial density (p = 0.0103), whereas the FFR group showed no changes (p = 0.56054) when compared to the control. In addition, reactive microglial density was estimated to be significantly different between groups for CA2-CA3 (F(2,15) = 21.62546, p < 0.001). Accordingly, reactive microglial density significantly increased in DIA (p < 0.0001), but not in FFR (p = 0.11153), when compared to the control (Table 1). Finally, reactive microglial density showed differences between groups in the DG (F(2,15) = 21.89490, p < 0.001), but only for the DIA group (p < 0.001) because no significant changes were observed for the FFR group (p = 0.11788), when compared to the control (Table 1). However, there were no significant changes in the density of resting microglia in CA1 and DG regions between the experimental groups and control, except for CA2-CA3 in the FFR group (F(2,15) = 8.084730, p = 0.00415) (see Table 1). Our next and final step was to estimate the reactive/resting microglia ratio. This ratio was not significantly modified in CA1 (F(2,15) = 2.322660, p = 0.1322) (Fig. 3, Table 1), whereas significant differences between groups were observed for CA2-CA3 (F(2,15) = 14.21470, p < 0.001). Thus, the reactive/resting microglia ratio increased in DIA (p = 0.0019) and FFR (p < 0.001) groups when compared to the control (Fig. 3, Table 1). Finally, the reactive/resting microglia ratio for the DG showed significant differences between groups (F(2,15) = 27.57863, p < 0.001). Thus, this ratio was significantly increased for DIA (p < 0.001) and FFR (p < 0.001) groups when compared to the control in the DG. (Fig. 3, Table 1).

4. Discussion In this study DIA, a murine model of adaptive anorexia, was tested on microglial density, IBA1 expression and pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. Our results showed that anorexia, but not dehydration, increased reactive microglial density in dorsal hippocampal CA2-CA3 and DG regions. Furthermore, the expression of IBA1, TNF-α, IL-6 and IL-1β also increased in the hippocampus. This environment may result in hippocampal neuroinflammation associated with anorexia. Thus, the DIA model may help to identify hippocampal regions involved in food avoidance, even when anorexigenic and orexigenic peptides inform the brain about energy reservoir depletion. Despite HPT axis dysregulation, DIA rats present metabolic changes similar to those of FFR rats; for example, decreased leptin, estradiol, insulin and thyroid hormones and increased corticosterone serum levels [9,10]. Moreover, increased neuropeptide Y (NPY) and decreased POMC expression patterns in hypothalamic ARC are similar for both experimental groups [10]. In this study female rats were not cycled but reduced levels of estradiol are associated with functional hypothalamic amenorrhea [34], this may explain the consistency of results observed in female rats under the DIA experimental protocol [9]. On the other hand, the role of microglia in short-term regulation of energy metabolism has been recently explored, and there is experimental evidence that microglia modulate synaptic plasticity of hypothalamic circuits involved in appetite [6]. Microglial behavior is highly dependent on cytokine environment, as cytokines are immune and neuro-modulatory messengers that can be released by reactive microglia. Moreover, cytokines can influence synaptic plasticity and disturb CNS functions when massively released. Cytokine-mediated physiological adaptations include endocrine axis adjustment via the hypothalamic nuclei involved in food intake [35]. In addition to hypothalamic nuclei, the

3.4. Anorexia increases hippocampal expression of IBA1, TNF-α, IL-6 and IL-1β due to food restriction We tested the effect of anorexia on IBA1, TNF-α, IL-6 and IL-1β expression by western blot from whole hippocampal samples at the end of the protocol (5th day) (Fig. 4). A significant increase of IBA1 expression was observed in DIA (51%) and FFR (54%) when compared to the control (Fig. 4). One-way ANOVA analysis showed significant differences in DIA (F2,6 = 6.091, p < 0.05). A similar effect was observed in the expression of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. Thereby, TNF-α expression increased significantly in DIA (48%) and FFR (47%) experimental groups when compared to the normalized control (Fig. 4). One-way ANOVA analysis showed significant 121

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increased only in DG. These results suggest that dehydration, and not reduced food intake, increases microglial density in CA2-CA3; however, reduced food intake is at least partially involved in the increase of microglial density observed in the DG. Next, we tested whether anorexia affected the activated/resting microglia ratio. Surprisingly, the activated/resting microglia ratio increased similarly in CA2-CA3 and DG for both experimental groups when compared to the control, indicating that reduced food intake and not dehydration is responsible for this increment. In support of these results, western blot studies showed that IBA1 expression was similarly increased by DIA and FFR when compared to the control group. Altogether our results indicate that reactive microglia density increases in the dorsal hippocampus due to anorexia and not dehydration. Accordingly, anorexia increases reactive astrocyte density and the expression of intermediate filaments, such as vimentin and nestin, in the dorsal hippocampus of young female rats [17]. Nevertheless, the differences observed in density versus reactivity can be attributed to the fact that the whole hippocampus (dorsal and ventral) was isolated for the western blot studies, whereas density studies were only performed for the dorsal hippocampus. This area is known to receive input from cognitive-related circuits; while the ventral hippocampus directly innervates neuro-endocrine-related medial hypothalamic areas and other forebrain structures to regulate homeostasis, metabolism and sexual behavior [42]. Although a direct relationship between ventral hippocampus and hypothalamus has been demonstrated, evidence about the involvement of the dorsal hippocampus in appetite and anorexia is limited [17]. Our study contributes in this direction. Thus, immunofluorescence studies were only performed in the dorsal hippocampus and reactive glial density was increased in CA2-CA3 and DG regions but not in CA1. This is in agreement with the anatomical and gene expression roadmap provided by the Hippocampus Gene Expression Atlas (HGEA), where unique gene expression was found in specific hippocampal regions (CA1 vs CA3) or in distinct combinations (DG and CA3 but not CA1) [42]. Furthermore, proteomic studies on CA1 and CA3 reported differential changes and dynamics in protein expression after object recognition or object location recognition [43]. Thus, it is reasonable to expect differences in hippocampal regions under pathological conditions. Accordingly, our studies reflect this major division of hippocampal regions, since increased reactive microglial density was observed in CA2-CA3 and DG regions but not in CA1. On the other hand, reactive glia may be responsible for decreased branching of hippocampal dendrites reported in the activity-based anorexia model [16], having important consequences on the synaptic plasticity of the hippocampus, which remain to be explored. Reactive glia, as found in most neuropathologies, is always associated with enhanced production of cytokines [32,35]. Thus, western blot studies showed a higher expression of TNF-α, IL-6 and IL-1β in the hippocampus of young female rats for both experimental groups (DIA and FFR). In this regard, TNF-α is considered a classical anorexigenic and pro-catabolic cytokine [2,44] and, in agreement with our results, previous studies showed that short-term caloric deprivation facilitates in vitro production of TNF-α [45]. Furthermore, this cytokine is increased in patients with anorexia nervosa [46]. A physiological link between increased TNF-α levels and cognitive alterations has been reported in both humans and animals [21,44,47]. A previous study in a model of multiple sclerosis reported a TNF-α-glial mediated signaling that impairs memory in the dentate gyrus–entorhinal cortex (DG-EC) circuit [48]. Here, microglia is hypothesized to increase TNF-α in the DG, whereas downstream activation of the receptor (TNFR1) expressed in astrocytes produces a persistent change in excitatory transmission associated with pathological states, resulting in alterations of the inputoutput relationship in the DG-EC circuit, which is critically involved in contextual memory processing [48]. Thus, the increment of TNF-α expression by anorexia may elicit a similar mechanism and influence a dorsal hippocampal memory to inhibit meal initiation and reduce the amount of food intake. On the other hand, IL-6 is known to play a central role in the regulation of appetite, energy expenditure and body

Fig. 2. Dehydration-induced anorexia (DIA) increases microglia density. Coronal sections of the dorsal hippocampus show nuclei labeled with DAPI and cells immunostained with IBA1 for CA1 (A), CA2-CA3 (B) and DG (C). The overlay of DAPI and IBA1 is shown for control (CTL), DIA and forced-food restricted (FFR) groups. Nuclei and microglia densities were estimated in the stratum oriens (s.o), stratum radiatum (s.r) and hilus; stratum pyramidale (s.p) and granular cell layer (GCL) are indicated as a reference. Scale bar = 50 μm. The IBA1 / DAPI ratio is plotted for each experimental group and each hippocampal region. No significant differences were observed among experimental groups for CA1. However, DIA significantly increased microglia density when compared to CTL and FFR (*** p < 0.001) in CA2-CA3. Finally, microglia density significantly increased for DIA (*** p < 0.001) and FFR (* p < 0.05) in dentate gyrus (DG). All values are summarized in Table 1 for comparison. Data are expressed as mean ± SEM.

hippocampus has also been recently linked to feeding behavior by regulating learned or motivational aspects of food intake through the action of endocrine hormones involved in appetite [36–38]. By contrast, there are few studies about how cognitive areas participate in the regulation of food intake. Previous studies reported an association between the dorsal hippocampus and the delay in meal initiation and the amount eaten during the next meal [39]. Knowing the synaptic circuits involved in meal initiation and frequency may advance our understanding of feeding behavior. In this regard, murine models of anorexia have also shown structural changes in the cellular organization of the hippocampus [16,17]. Nevertheless, few studies have linked microglia as a promoter of brain inflammation with anorexia [6], despite the fact that it is one of the main cytokine producers in the brain. Likewise, reduced food intake is observed when cytokines like IL-1, TNF and IL-6 are administered centrally; therefore, they may be involved in triggering anorexia [20–22,40,41]. Accordingly, our results showed that DIA significantly increased microglial density in CA2-CA3 and DG. However, microglial density in the FFR group was significantly 122

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Fig. 3. Anorexia increases reactive microglia. Resting microglia were identified morphologically as IBA1+ cells with processes longer than the diameter of the soma (see zoom of a cell in CTL as an example); while reactive microglia was defined as IBA1+ cells with processes smaller than the diameter of the soma (see zooms of representative cells in DIA and FFR). The reactive/resting microglia ratio increased significantly for DIA and FFR in CA2CA3 and DG (**p < 0.01; ***p < 0.001), but not in CA1. All values are summarized in Table 1 for comparison. Data are expressed as mean ± SEM.

with a concomitant increased expression of this cytokine in the hippocampus and hypothalamus [52]. Thereby, short-term memory studies showed that increased levels of IL-1β in the hippocampus during training in the context did not impair encoding (acquisition), but rather appeared to affect storage (memory consolidation) [53]. Thus, prolonged elevation of IL-1β, specifically in the hippocampus, may be responsible for hippocampal-dependent memory impairments [54]. In support of this hypothesis, a recent study with lentivirus-mediated IL1β knockdown in the hippocampus showed attenuation of memory deficits and anxiety- and depression-like behaviors induced by lipopolysaccharide in mice [55]. Furthermore, long-term potentiation studies showed impairment by IL-1β, indicating specific effects on hippocampal synaptic plasticity [56]. Thus, experimental evidence suggests that exposure to high levels of TNF-α, IL-6 or IL-1β can change basal synaptic function and interfere with critical behaviors that are known to involve the hippocampus, a region that is part of the limbic system and is involved in emotional responses, adult neurogenesis, neuroplasticity and cognitive function.

5. Conclusion We conclude that anorexia, independently of dehydration, increases reactive microglial density and the expression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in the hippocampus. These mechanisms might be extended to other brain regions; further studies will elucidate if reactive glia and pro-inflammatory cytokines are involved in triggering the onset of neuropsychiatric disorders such as anorexia nervosa. Thus, a better understanding of the effects of reduced food intake in this brain area is fundamental for developing new therapeutic approaches that may target neuroinflammation as a potential onset of anorexia.

Fig. 4. Effect of anorexia on IBA1, TNF-α, IL-6 and IL-1β expression after fifth day for control (CTL), DIA and FFR groups. (A) Representative western blot showing the level of expression level of IBA1, TNF-α, IL-6 and IL-1β for each experimental group (CTL, DIA and FFR). GAPDH was used as internal control. (B) The band intensity of DIA and FFR groups was normalized to their respective controls for IBA1, TNF-α, IL-6 and IL-1β. Data are shown as mean ± SEM. Significant differences were considered as * p < 0.05; *** p < 0.005.

Role of funding source Funding for this study was provided by grants to T.M. (IN202315), A.M.T. (IN201913) and D.R.H. (IN201915 and IN205718) from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) from Universidad Nacional Autónoma de México (UNAM). PAPIIT-UNAM had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the paper for publication.

weight. A previous study reported that serum levels of IL-6 were increased in women with anorexia [49]. Interestingly, in vivo exposure to elevated levels of IL-6 can result in enhanced hippocampal synaptic transmission and impair cognitive ability [50,51]. Lastly, central administration of IL-1β is known to induce anorexia, fever and lethargy 123

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Declaration of interest None

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Acknowledgements The authors are grateful to E. N. Hernández-Ríos, A. Castilla, L. Casanova, A. E. Espino, M. E. Ramos and M. García-Servín for their technical support. We are indebted to Dr. R. Arellano-Ostoa for allowing us to use his histological facilities. The authors thank LCC Jessica González Norris for proofreading the English version of this manuscript. DRV was supported by Dirección General de Asuntos del Personal Académico - Universidad Nacional Autónoma de México (DGAPA-UNAM) as a postdoctoral fellow. MMM received a fellowship from CONACYT (612313) and support from Programa de Apoyo a los Estudios de Posgrado (PAEP) as student from Programa de Maestría en Ciencias (Neurobiología) - UNAM.

[19] [20]

[21]

[22]

[23]

References

[24]

[1] A.G. Watts, D. Salter, Neural mechanisms of anorexia, in: E.M. Stricker, S.C. Woods (Eds.), Neurobiology of Food and Fluid Intake. Handbook of Behavioral Neurobiology, vol. 14, Springer, Boston, MA, 2004, , https://doi.org/10.1007/0306-48643-1_14. [2] T. Romanatto, M. Cesquini, M.E. Amaral, E.A. Roman, J.C. Morales, M.A. Torsoni, A.P. Cruz-Neto, L.A. Velloso, TNF-alpha acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient-effects on leptin and insulin signaling pathways, Peptides 28 (2007) 1050–1058, https://doi.org/10.1016/j.peptides. 2007.03.006. [3] L. Gautron, S. Layé, Neurobiology of inflammation-associated anorexia, Front. Neurosci. 3 (2009) 1–10, https://doi.org/10.3389/neuro.23.003.2009. [4] R. Shirazi, V. Palsdottir, J. Collander, F. Anesten, H. Vogel, F. Langlet, A. Jaschke, A. Schürmann, V. Prévot, R. Shao, J.O. Jansson, K.P. Skibicka, Glugagon-like peptide 1 receptor induced suppression of food intake, and body weight is mediated by central IL-1 and IL-6, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 16199–16204, https://doi.org/10.1073/pnas.1306799110. [5] C.X. Yi, M. Walter, Y. Gao, S. Pitra, B. Legutko, S. Kälin, C. Layritz, C. GarcíaCáceres, M. Bielohuby, M. Bidlingmaier, S.C. Woods, A. Ghanem, K.K. Conzelmann, J.E. Stern, M. Jastroch, M.H. Tschöp, TNFα drives mitochondrial stress in POMC neurons in obesity, Nat. Comm. 8 (2017) 15143, https://doi.org/10.1038/ ncomms15143. [6] S. Jin, J.G. Kim, J.W. Park, M. Koch, T.L. Horvath, B.J. Lee, Hypothalamic TLR2 triggers sickness behavior via microglia-neuronal axis, Sci. Rep. 6 (2016) 29424, https://doi.org/10.1038/srep29424. [7] N. Mrosovsky, D.F. Sherry, Animal anorexias, Science 207 (1980) 837–842, https:// doi.org/10.1126/science.6928327. [8] A.G. Watts, Dehydration-associated anorexia: development and rapid reversal, Physiol. Behav. 65 (1999) 871–878, https://doi.org/10.1016/S0031-9384(98) 00244-3. [9] L. Jaimes-Hoy, P. Joseph-Bravo, P. de Gortari, Differential response of TRHergic neurons of the hypothalamic paraventricular nucleus (PVN) in female animals submitted to food-restriction or dehydration-induced anorexia and cold exposure, Horm. Behav. 53 (2008) 366–377, https://doi.org/10.1016/j.yhbeh.2007.11.003. [10] C. García-Luna, M.I. Amaya, E. Alvarez-Salas, P. de Gortari, Prepro-orexin and feeding-related peptide receptor expression in dehydration-induced anorexia, Regul. Pept. 159 (2010) 54–60, https://doi.org/10.1016/j.regpep.2009.09.011. [11] C. García-Luna, P. Soberanes-Chávez, P. de Gortari, Impaired hypothalamic cocaine- and amphetamine regulated transcript expression in lateral hypothalamic área and paraventricular nuclei of dehydration-induced anorexic rats, J. Neuroendocrinol. 29 (2017) 11, https://doi.org/10.1111/jne.12541. [12] G.D. Giordano, P. Renzetti, R.C. Parodi, L. Foppiani, F. Zandrino, G. Giordano, F. Sardanelli, Volume measurement with magnetic resonance imaging of hippocampus-amygdala formation in patients with anorexia nervosa, J. Edoncrinol. Invest. 24 (2001) 510–514 07/BF03343884 https://doi.org/10.102015. [13] F. Connan, F. Murphy, S.E. Connor, P. Rich, T. Murphy, N. Bara-Carill, S. Landau, S. Krljes, V. Ng, S. Williams, Morris R.G, I.C. Campbell, J. Treasure, Hippocampal volumen and cognitive function in anorexia nervosa, Psychiatry Res. 146 (2006) 117–125, https://doi.org/10.1016/j.pscychresns.2005.10.006. [14] J.N. Beadle, S. Paradiso, M. Brumm, M. Voss, K. Halmi, L.M. McCormick, Larger hippocampus size in women with anorexia nervosa who exercise excessively than healthy women, Psychiatry Res. 232 (2015) 193–199, https://doi.org/10.1016/j. pscychresns.2014.10.013. [15] N.T. Burkert, K. Koschutnig, F. Ebner, W. Freidl, Structural hippocampal alterations, perceived stress, and coping deficiencies in patients with anorexia nervosa, Int. J. Eat. Disord. 48 (2015) 670–676, https://doi.org/10.1002/eat.22397. [16] T.G. Chowdhury, N.C. Barbarich-Marsteller, T.E. Chan, C. Aoki, Activity-based anorexia has differential effects on apical dendritic branching in dorsal and ventral hippocampal CA1, Brain Struct. Funct. 219 (2014) 1935–1945, https://doi.org/10. 1007/s00429-013-0612-9. [17] D. Reyes-Haro, F.E. Labrada-Moncada, D.R. Varman, J. Krüger, T. Morales,

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39] [40]

[41]

[42]

124

R. Miledi, A. Martínez-Torres, Anorexia reduces GFAP+ cell density in the rat Hippocampus, Neural Plast. 2016 (2016) 2426413, , https://doi.org/10.1155/ 2016/2426413. S.A. Liddelow, K.A. Guttenplan, L.E. Clarke, F.C. Bennett, C.J. Bohlen, L. Schirmer, M.L. Bennett, A.E. Münch, W.S. Chung, T.C. Peterson, D.K. Wilton, A. Frouin, B.A. Napier, N. Panicker, M. Kumar, M.S. Buckwalter, D.H. Rowitch, V.L. Dawson, T.M. Dawson, B. Stevens, B.A. Barres, Neurotoxic reactive astrocytes are induced by activated microglia, Nature 541 (2017) 481–487, https://doi.org/10.1038/ nature21029. H. Kettenmann, U.K. Hanisch, M. Noda, A. Verkhratsky, Physiology of microglia, Physiol. Rev. 91 (2011) 461–553, https://doi.org/10.1152/physrev.00011.2010. L. Kapás, J.M. Krueger, Tumor necrosis factor-beta induces sleep, fever, and anorexia, Am. J. Physiol. 263 (1992) R703–R707, https://doi.org/10.1152/ajpregu. 1992.263.3.R703. M. Fantino, L. Wieteska, Evidence for a direct central anorectic effect of tumornecrosis-factor-alpha in the rat, Physiol. Behav. 53 (1993) 477–483, https://doi. org/10.1016/0031-9384(93)90141-2. C.R. Plata-Salamán, G. Sonti, J.P. Borkoski, C.D. Wilson, J.M.B. French-Mullen, Anorexia induced by chronic central administration of cytokines at estimated pathophysiological concentrations, Physiol. Behav. 60 (1996) 867–875, https://doi. org/10.1016/0031-9384(96)00148-5. G. Sonti, S.E. Ilyn, C.R. Plata-Salamán, Anorexia induced by cytokine interactions at pathophysiological concentrations, Am. J. Physiol. 270 (1996) R1394–R1402, https://doi.org/10.1152/ajpregu.1996.270.6.R1394. C. Pomeroy, E. Eckert, S. Hu, B. Eiken, M. Mentink, R.D. Crosby, C.C. Chao, Role of interleukin-6 and transforming growth factor-beta in anorexia nervosa, Biol. Psychiatry 36 (1994) 836–839, https://doi.org/10.1016/0006-3223(94)90594-0. N.C. Raymond, M. Dysken, K. Bettin, E.D. Eckert, S.J. Crow, K. Markus, C. Pomeroy, Cytokine production in patients with anorexia nervosa, bulimia nervosa and obesity, Int. J. Eat. Disord. 28 (2000) 293–302, https://doi.org/10.1002/1098-108X (200011)28:3<293::AID-EAT6>3.0.CO;2-F. P. de Gortari, K. Mancera, A. Cote-Vélez, M.I. Amaya, A. Martínez, L. Jaimes-Hoy, P. Joseph-Bravo, Involvement of CRH-R2 receptor in eating behavior and in the response of the HPT axis in rats subjected to dehydration-induced anorexia, Psychoneuroendocrinology 34 (2009) 259–272, https://doi.org/10.1016/j. psyneuen.2008.09.010. D. Reyes-Haro, F.E. Labrada-Moncada, R. Miledi, A. Martínez-Torres, Dehydrationinduced anorexia reduces astrocyte density in the rat Corpus callosum, Neural Plast. (2015) 474917, , https://doi.org/10.1155/2015/474917. V. Cabrera, E. Ramos, A. González-Arenas, M. Cerbón, I. Camacho-Arroyo, T. Morales, Lactation reduces glial activation induced by excitotoxicity in the rat hippocampus, J. Neuroendocrinol. 25 (2013) 519–527, https://doi.org/10.1111/ jne.12028. K. Ohsawa, Y. Imai, Y. Sasaki, S. Kohsaka, Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity, J. Neurochem. 88 (2004) 844–856, https://doi.org/10.1046/j.1471-4159.2003.02213.x. M.R. Lamprecht, D.M. Sabatini, A.E. Carpenter, CellProfiler: free, versatile software for automated biological image analysis, BioTechniques 42 (2007) 71–75, https:// doi.org/10.2144/000112257. F. Cerbai, D. Lana, D. Nosi, P. Petkova-Kirova, S. Zecchi, H.M. Brothers, G.L. Wenk, M.G. Giovannini, The neuron astrocyte-microglia triad in normal brain ageing and in a model of neuroinflammation in the rat hippocampus, PLoS One 7 (2012) e45250, , https://doi.org/10.1371/journal.pone.0045250. B. Rinaldi, F. Guida, A. Furiano, M. Donniacuo, L. Luongo, G. Gritti, K. Urbanek, G. Messina, S. Maione, F. Rossi, V. de Novellis, Effect of prolonged moderate exercise on the changes of nonneuronal cells in early myocardial infarction, Neural Plast. (2015) 265967, , https://doi.org/10.1155/2015/265967. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254, https://doi.org/10.1016/0003-2697(76)90527-3. B. Meczekalski, K. Katulski, A. Czyzyk, A. Podfigurna-Stopa, M. Maciejewska-Jeske, Functional hypothalamic amenorrhea and its influence on women’s health. J. Endocrinol Invest 37, 1049-1056. https://doi.org/10.1007/s40618-014-0169-3. U.K. Hanisch, Microglia as a source and target of cytokines, Glia 40 (2002) 140–155, https://doi.org/10.1002/glia.10161. A.J. Irving, J. Harvey, Leptin regulation of hippocampal synaptic function in health and disease, Philos. Trans. R. Soc. Lond., B, Biol. Sci. 369 (2014) 20130155, , https://doi.org/10.1098/rstb.2013.0155. T.M. Hsu, J.D. Hahn, V.R. Konanur, E.E. Noble, A.N. Suarez, J. Rhai, E.M. Nakamoto, S.E. Kanoski, Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways, Elife 4 (2015) e11190, , https://doi. org/10.7554/eLife.11190. P. Sweeney, Y. Yang, An excitatory ventral hippocampus to lateral septum circuit that suppress feeding, Nat. Commun. 15 (2015) 6, https://doi.org/10.1038/ ncomms10188 10188. Y.O. Henderson, G.P. Smith, M.B. Parent, Hippocampal neurons inhibit meal onset, Hippocampus 23 (2013) 100–107, https://doi.org/10.1002/hipo.22062. S. Kent, R.M. Bluthe, R. Dantzer, A.J. Hardwick, K.W. Kelley, N.J. Rothwell, J.L. Vannice, Different receptor mechanisms mediate the pyrogenic and behavioral effects of interleukin 1, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 9117–9120, https:// doi.org/10.1073/pnas.89.19.9117. J.H. Yao, S.M. Ye, W. Burgess, J.F. Zachary, K.W. Kelley, R.W. Johnson, Mice deficient in interleukin-1beta converting enzyme resist anorexia induced by central lipopolysaccharide, Am. J. Physiol. 277 (1999) R1435–R1443, https://doi.org/10. 1152/ajpregu.1999.277.5.R1435. M.S. Bienkowski, I. Bowman, M.Y. Song, L. Gou, T. Ard, K. Cotter, M. Zhu,

Behavioural Brain Research 363 (2019) 118–125

D. Ragu-Varman et al.

[43]

[44] [45] [46]

[47]

[48]

[49]

[50]

N.L. Benavidez, S. Yamashita, J. Abu-Jaber, S. Azam, D. Lo, N.N. Foster, H. Hintiryan, H.W. Dong, Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks, Nat. Neurosci. 21 (2018) 1628–1643, https://doi.org/10.1038/s41593-018-0241-y. L.M. von Ziegler, N. Selevsek, R.Y. Tweedie-Cullen, E. Kremer, I.M. Mansuy, Subregion-specific proteomic signature in the hippocampus for recognition processes in adult mice, Cell Rep. 22 (2018) 3362–3374, https://doi.org/10.1016/j. celrep.2018.02.079. N. Vaisman, T. Hahn, Tumor necrosis factor-alpha and anorexia—cause or effect? Metabolism 40 (1991) 720–723, https://doi.org/10.1016/0026-0495(91)90090-J. P. Matthys, A. Billiau, Cytokines and cachexia, Nutrition 13 (1997) 763–770, https://doi.org/10.1016/S0899-9007(97)00185-8. K.G. Kahl, N. Kruse, P. Rieckmann, M.H. Schmidt, Cytokine mRNA expression patterns in the disease course of female adolescents with anorexia nervosa, Psychoneuroendocrinology 29 (2004) 13–20, https://doi.org/10.1016/S03064530(02)00131-2. W. Swardfager, S.E. Black, Dementia: a link between microbial infection and cognition? Nat. Rev. Neurol. 9 (2013) 301–302, https://doi.org/10.1038/nrneurol. 2013.93. S. Habbas, M. Santello, D. Becker, H. Stubbe, G. Zappia, N. Liaudet, F.R. Klaus, G. Kollias, A. Fontana, C.R. Pryce, T. Suter, A. Volterra, Neuroinflammatory TNFα impairs memory via astrocyte signaling, Cell 163 (2015) 1730–1741, https://doi. org/10.1016/j.cell.2015.11.023. M. Karczewska-Kupczewska, A. Adamska, A. Nikolajuk, E. Otziomek, M. Górska, I. Kowalska, M. Straczkowski, Circulating interleukin 6 and soluble forms of its receptors in relation to resting energy expenditure in women with anorexia nervosa, Clin. Endocrinol. 79 (2013) 812–816, https://doi.org/10.1111/cen.12118. T.E. Nelson, A. Olde Engberink, R. Hernández, A. Puro, S. Huitrón-Reséndiz, C. Hao,

[51]

[52]

[53]

[54]

[55]

[56]

125

P.N. De Graan, D.L. Gruol, Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6, Brain Behav. Immun. 26 (2012) 959–971, https://doi.org/10.1016/j.bbi.2012.05. 005. H. Wei, K.K. Chadman, D.P. McCloskey, A.M. Sheik, M. Malik, W.D. Brown, X. Li, Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors, Biochim. Biophys. Acta 1822 (2012) 831–842, https://doi.org/10.1016/ j.bbadis.2012.01.011. T.L. Baartman, T. Swanepoel, R.M. Barrientos, H.P. Laburn, D. Mitchell, L.M. Harden, Divergent effects of brain interleukin-1β in mediating fever, lethargy, anorexia and conditioned fear memory, Behav. Brain Res. 324 (2017) 155–163, https://doi.org/10.1016/j.bbr.2017.02.020. R.M. Barrientos, E.A. Higgins, J.C. Biedenkapp, D.B. Sprunger, K.J. WrightHardesty, L.R. Watkins, J.W. Rudy, S.F. Maier, Peripheral infection and aging interact to impair hippocampal memory consolidation, Neurobiol. Aging 27 (2006) 723–732, https://doi.org/10.1016/j.neurobiolaging.2005.03.010. R.M. Barrientos, M.G. Frank, A.M. Hein, E.A. Higgins, L.R. Watkins, J.W. Rudy, S.F. Maier, Time course of hippocampal IL-1 beta and memory consolidation impairments in aging rats following peripheral infection, Brain Behav. Immun. 23 (2009) 46–54, https://doi.org/10.1016/j.bbi.2008.07.002. M. Li, C. Li, H. Yu, X. Cai, X. Shen, X. Sun, J. Wang, Y. Zhang, C. Wang, Lentivirusmediated interleukin-1β (IL-1β) knock-down in the hippocampus alleviates lipopolysaccharide (LPS)-induced memory deficits and anxiety- and depression-like behaviors in mice, J. Neuroinflammation 14 (2017) 190, https://doi.org/10.1186/ s12974-017-0964-9. K. Hoshino, K. Hasegawa, H. Kamiya, Y. Morimoto, Synapse-specific effects of IL-1β on long-term potentiation in the mouse hippocampus, Biomed. Res. 38 (2017) 183–188, https://doi.org/10.2220/biomedres.38.183.