Received: 3 August 2016
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Revised: 15 August 2017
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Accepted: 22 August 2017
DOI: 10.1002/hipo.22798
RESEARCH ARTICLE
Hippocampal mechanisms in impaired spatial learning and memory in male offspring of rats fed a low-protein isocaloric diet in pregnancy and/or lactation mez2* | N. J. Parga-Martínez3 | L. A. Reyes-Castro1* | E. Padilla-Go D. C. Castro-Rodríguez1 | G. L. Quirarte3 | S. Díaz-Cintra2 | P. W. Nathanielsz4 | E. Zambrano1 1
Departamento de Biología de la n, Instituto Nacional de Ciencias Reproduccio dicas y Nutricio n SZ, Me xico 14080, Me xico Me 2
Abstract Maternal nutritional challenges during fetal and neonatal development result in developmental programming of multiple offspring organ systems including brain maturation and function. A maternal
Departamento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional noma de Me xico, Juriquilla, Quere taro Auto xico 76230, Me
low-protein diet during pregnancy and lactation impairs associative learning and motivation. We
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Departamento de Neurobiología Conductual y Cognitiva, Instituto de Neurobiología, Universidad Nacional noma de Me xico, Juriquilla, Quere taro Auto xico 76230, Me
nancy, second lactation diet). We evaluated the behavior of young adult male offspring around
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Department of Animal Science, University of Wyoming, Laramie, Wyoming 820713684
ACTH were higher in RR vs. CC. In the MWM acquisition test CC offspring required two, RC three,
Correspondence Elena Zambrano, Departamento de Biología n, Instituto Nacional de de la Reproduccio dicas y Nutricio n Salvador Ciencias Me xico, Zubiran (INCMNSZ), Ciudad de Me C.P. 14080, Mexico, Mexico. Email:
[email protected]
entries than CC. RR and RC offspring spent less time in the target zone than CC. MF area, total,
Funding information Consejo Nacional de Ciencia y Tecnología, Grant Number: CONACYT-155166; n General Asuntos del Personal Direccio mico, Universidad Nacional Acade noma de Me xico UNAM-DGAPA, Auto Grant Number: PAPIIT-IN203616
evaluated effects of a maternal low-protein diet during gestation and/or lactation on male offspring spatial learning and hippocampal neural structure. Control mothers (C) ate 20% casein and restricted mothers (R) 10% casein, providing four groups: CC, RR, CR, and RC (first letter pregpostnatal day 110. Corticosterone and ACTH were measured. Males were tested for 2 days in the Morris water maze (MWM). Stratum lucidum mossy fiber (MF) area, total and spine type in basal dendrites of stratum oriens in the hippocampal CA3 field were measured. Corticosterone and and CR seven sessions to learn the maze. RR did not learn in eight trials. In a retention test 24 h later, RR, CR, and RC spent more time locating the platform and performed fewer target zone and thin spines were lower in RR, CR, and RC than CC. Mushroom spines were lower in RR and RC than CC. Stubby spines were higher in RR, CR, and RC than CC. We conclude that maternal lowprotein diet impairs spatial acquisition and memory retention in male offspring, and that alterations in hippocampal presynaptic (MF), postsynaptic (spines) elements and higher glucocorticoid levels are potential mechanisms to explain these learning and memory deficits. KEYWORDS
glucocorticoids, hippocampal mechanisms, learning, protein restriction, spatial memory
Abbreviations: ACTH, adrenocorticotropic hormone; AMPA, a amino-3 hydroxy-5 methyl 4-isoxazolepropionic acid receptor; BDNF, brain derived neurotrophic factor; C, control; CA, Cornu Ammonis; CC, control diet during pregnancy and lactation; CR, control diet during pregnancy and restricted during lactation; EDTA, ethylene diamine tetra acetic acid; DHT, dihydrotestosterone; GABA, gamma amino butyric acid; GLUR, glutamate receptor; GLUT, glutamate; HPA, hypothalamic dicas y Nutricio n Salvador Zubiran; MF, Mossy fiber; MWM, Morris water maze; NMDA, N methylpituitary adrenal; INCMNSZ, Instituto Nacional de Ciencias Me d-aspartate receptor; PND, postnatal day; PSDs, postsynaptic densities; R, restricted; RC, restricted diet during pregnancy and control during lactation; RR, restricted diet during pregnancy and lactation; SFv, tissue shrinkage factor; T, testosterone. *These authors equally contributed as first author.
Hippocampus. 2017;1–13.
wileyonlinelibrary.com/journal/hipo
C 2017 Wiley Periodicals, Inc. V
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REYES-CASTRO
1 | INTRODUCTION
ET AL.
hypothesized that exposure of the developing rat to a moderate maternal protein restricted diet during pregnancy and lactation impairs mem-
During development, there are critical periods of vulnerability to sub-
ory and spatial learning accompanied by changes in hippocampal fine
optimal conditions. Developmental programming is defined as “The
structure (MF area and spine density) in the CA3 area and glucocorti-
response to a specific challenge to the mammalian organism during a criti-
coids production.
cal developmental time window that alters the trajectory of development with resulting effects on health that persist throughout life” (Zambrano &
2 | EXPERIMENTAL PROCEDURES
Nathanielsz, 2013). Fetal and neonatal exposure to various maternal challenges, such as hypoxia (Giussani & Davidge, 2013), suboptimal maternal nutrition (Langley-Evans, 2015), and maternal obesity and over nutrition (Nathanielsz et al., 2013; Rodriguez-Gonzalez et al., 2015; Santos et al., 2015; Taylor, Samuelsson, & Poston, 2014; Vega et al., 2015; Zambrano & Nathanielsz, 2013) alter offspring phenotype as a result of epigenetic gene/environment interactions (Hanson & Gluckman, 2014; Peter et al., 2016). Reduced maternal nutrition is the most extensively studied programming challenge of phenotypic plasticity (Cripps et al., 2009; Fernandez-Twinn et al., 2005; Pinheiro, Salvucci, Aguila, & Mandarim-de-Lacerda, 2008; Qasem, Li, Tang, Pontiggia, & D’mello, 2016; Rodriguez-Gonzalez et al., 2014; Vega et al., 2016). Previous studies have demonstrated that maternal moderate lowprotein (10%) in pregnancy and lactation increases serum corticosterone levels (Reyes-Castro et al., 2012a, 2012b) and impairs learning acquisition and offspring motivation (Reyes-Castro et al., 2011, 2012a). The hippocampal formation is closely involved in the control of both short and long term memory as well as in memories associated with special learning and planning (Buzsaki, 2015; Lynch, 2004). Ageing and over or undernutrition in the rat are known to produce anatomic and functional hippocampal alterations (Castro-Chavira et al., 2016) which impair maintenance of long-term memory (Martinez et al., 2009). Severe maternal protein restriction (6%) in the rat negatively programs the time course of offspring development of the dentate gyrus (Debassio, Kemper, Galler, & Tonkiss, 1994; Debassio, Kemper, Tonkiss, & Galler, 1996), the morphology of hippocampal cells (Diaz-Cintra et al., 1991), the number and distribution of neurotransmitter receptors (Almeida, Tonkiss, & Galler, 1996), mossy fiber (MF) axonal area (Andrade, Cadete-Leite, Madeira, & Paula-Barbosa, 1991; GranadosRojas et al., 2002) and synaptic spine complexity (Diaz-Cintra et al., 1991). Changes in density and types of dendritic spines in CA1 pyramidal neurons (Zhang, Wei, & Yang, 2013) may partly explain the altered modulation of cell excitability, as well as of the emotional, motivational, and memory disturbances commonly observed in pre- (da Silva Hernandes, Francolin-Silva, Valadares, Fukuda, & Almeida, 2005) and postnatally malnourished (6% protein diet) rats (da Silva Hernandes et al., 2005; Francolin-Silva, da Silva Hernandes, Fukuda, Valadares, & Almeida, 2006). Similar morphological effects of poor nutrition have been found in CA3 pyramidal cells (Cintra, Diaz-Cintra, Galvan, Kemper, & Morgane, 1990; Diaz-Cintra et al., 1991; Garcia-Ruiz, Diaz-Cintra, Cintra, & Corkidi, 1993; Granados-Rojas et al., 2002). However, much less is known about the direct effects of this important programming
2.1 | Care and maintenance of animals All procedures were approved by the Animal Experimentation Ethics dicas y Nutricio n, Committee of the “Instituto Nacional de Ciencias Me xico. Animals maintenance was in Salvador Zubiran” (INCMNZ), Me accordance with the NIH guide for care and use of laboratory animals. Details of maternal diet, breeding, and management of groups of offspring have been published in detail (Zambrano et al., 2005b). Briefly, mothers were virgin female albino Wistar rats aged 15–17 weeks and weighing 220–260 g obtained from INCMNSZ. Mothers were maintained on normal laboratory chow diet (Zeigler Rodent RQ 22–5, USA) under controlled lighting (lights on from 7:00 am to 7:00 pm at 22– 23 8C) and mated overnight with proven male breeders. The day on which spermatozoa were present in a vaginal smear was designated as conception (day 0). Only females impregnated within 5 days of introducing the male were retained in the study. Our record for the animal room indicates that the fertility rate is 75–80% for both, control and protein restricted female rats. Pregnant rats were transferred to individual cages and allocated at random to one of two groups fed either a 20% casein (control diet C) or an isocaloric (4 kcal g21) 10% casein (restricted diet R) (Zambrano et al., 2005a). Food and water were available ad libitum. Pregnant rats were weighed daily during pregnancy and lactation. Food was provided as flat biscuits and food intake was measured daily. Delivery occurred at all hours of post-conceptual day 22, but more commonly in the morning, only 15% of C group and 40% of R group was after noon time. The day of delivery was considered postnatal day (PND) 0. Pup weight was recorded at birth. To assign sex, ano-genital distance was measured with calipers as previously described (Zambrano et al., 2006). Rats with litters of more than 12 or less than 10 pups were not included in the study. Litters were adjusted to 10 pups per mother, while maintaining as close to a 1:1 sex ratio as possible and excluding the smallest and the biggest pup of the litter. After weaning three animals from the same sex and group were housed in the same cage. For the lactation period, half of each group was assigned to C or R diet. Four groups were established: CC, RR, CR, and RC (first letter maternal diet received during pregnancy and second letter maternal diet during lactation) composed of 10 animals, none of which were litter mates, in each group. After weaning (PND 21), all pups were fed standard rodent chow diet. We evaluated male offspring only.
2.2 | Morris water maze (MWM) task
challenge on spatial learning and changes of dendritic spine density
Around PND 110 (range from 105 to 115), 10 male offspring per group
and morphology related to CA3 hippocampal synaptic plasticity. We
from different litters were tested on two consecutive days for spatial
REYES-CASTRO
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ET AL.
learning and memory in the MWM (Quirarte et al., 2009). Spatial MWM training was carried out in a cylindrical, black-plastic water tank,
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2.4 | Tissue collection for Timm’s silver sulfide technique
154 cm in diameter and 60 cm high, located in a dimly illuminated room (236 3 225 3 242 cm) and elevated 60 cm above the floor, with posters and figures on the inside walls of the room as landmarks. The tank was filled with water (25 6 0.5 8C) to a depth of 26 cm. A 12 cm square, transparent acrylic platform was placed inside the tank at a fixed location in the center of the southwest quadrant and submerged under 1 cm of water. As the platform could not be seen or detected by smell, the rats required spatial navigation achieved by using the distal cues as landmarks to learn the platform location. A tracking system (SMART, San Diego Instruments, CA) located in an adjacent room was used to record the latency to reach the platform. MWM training consisted of an 8-trial session in which each animal from each group (CC, RR, CR, and RC) was introduced into the pool, always facing the nearest wall of the room but at different starting points for each trial. The animal was allowed to swim for a maximum of 60 s or until it climbed onto the platform, where it was allowed to remain for 20 s. If on the first trial, the subject did not find the platform within this time limit, it was placed on the platform by hand and allowed to rest for 20 s. After the 20 s rest, the animal was towel dried and placed in a waiting cage
A total of 20 animals from different litters (five per group: CC, RR, CR, RC) were studied to estimate the MF area of the suprapyramidal bundle with the Timm histological technique for demonstrating metal ions, especially zinc (West, Coleman, & Flood, 1988). Rats were perfused through the left ventricle for 2 min with a buffered sodium sulfide solution (5.85 g Na2S, 5.95 g NaH2PO4 H2O in 500 mL distilled water) followed by 1% paraformaldehyde and 1.25% glutaraldehyde in 0.10 M phosphate buffer, pH 7.4 (as described by West et al., 1988) for 10 min. Brains were removed, weighed, and post-fixed in the same fixative containing 30% sucrose in which they remained until they sank to the bottom of the container. The brain was dissected, and the hippocampal formation frozen and cut into coronal sections a 40-lm thickness. Sections were serially mounted and processed in darkness in a 12:6:2 mixture of Arabic gum (20%), hydroquinone (5.6%), citrate buffer and 1 mL of a 17% silver nitrate solution (Padilla-Gomez et al., 2012). Each slide was assigned a random number to ensure the observer was blind to treatments.
began. This sequence was repeated until the eight trials were com-
2.5 | Area of the mossy fiber system in the hippocampal suprapyramidal bundle
pleted. The latency to locate the platform (Acquisition test) on each
The MF area in the suprapyramidal bundle, corresponding to the stra-
trial was used as an index of performance. Escape latency was meas-
tum lucidum, was estimated in sections selected from the dorsal hippo-
ured on each trial for every animal. To measure memory, 24 h after the
campus. Every fifth section was obtained from a random starting
last training session (retention test), a single-trial 60 s session was con-
position within the first five serially mounted sections. For each sec-
ducted under training conditions similar to those of the learning task.
tion, the boundaries of the suprapyramidal bundle were defined
The only variation was that the platform was removed, and the meas-
according to Gaarskjaer (1985) and Amaral and Dent (1981), and its
ured parameter was the time spent in the quadrant where the platform
area was determined in right-side hippocampal sections with a 43
was previously localized.
(Plan-Apochromat® 0.17 NA) objective lens using the computer-
under a heating lamp for 30 s in an adjacent room, and then a new trial
assisted Image J (NIH) analysis system. The mean section thickness
2.3 | Corticosterone and ACTH measurements To determine ACTH and corticosterone levels 30 min after the memory session, animals were anesthetized with a combination of a dissociative anesthetic (ketamine) 40 mg/kg and the tranquilizer and relaxing non-narcotic analgesic (xylazine) at dose of 3.5 mg/kg. A blood sample was obtained by heart left ventricle puncture (within approximately 1 min) and transferred to EDTA tubes on ice. Blood samples were centrifuged at 4 8C for 15 min at 3500 rpm, and plasma collected and stored at 220 8C until analyzed within 1 month. Corticosterone was determined by radioimmunoassay using a commercial kit designed for rat blood, DPC Coat-a-count (TKRC1) from Diagnostic Products (Los Angeles, CA, USA). Intra- and interassay variability was
