Perinatal undernutrition attenuates field excitatory postsynaptic potentials and influences dendritic spine density and morphology in hippocampus of male rat offspring

Neuroscience 244 (2013) 31–41

PERINATAL UNDERNUTRITION ATTENUATES FIELD EXCITATORY POSTSYNAPTIC POTENTIALS AND INFLUENCES DENDRITIC SPINE DENSITY AND MORPHOLOGY IN HIPPOCAMPUS OF MALE RAT OFFSPRING ZHANG Y. a,c WEI J. b AND YANG Z. c*

number of special dendritic spines. Thus, these changes in the density and the types of dendritic spines in CA1 pyramidal neurons may partly explain the impaired hippocampal synaptic plasticity as well as learning and memory disturbances commonly observed during undernourished rats. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, PR China b Department of Psychiatry, Ankang Hospital of Tianjin Public Security Bureau, Tianjin 300240, PR China c

School of Medicine, Nankai University, Tianjin 300071, PR China

Key words: hippocampus, learning and memory, excitatory postsynaptic potential, maternal undernutrition, Golgi impregnation, pyramidal neuron.

Abstract—Perinatal undernutrition affects the hippocampus, a brain region crucial for learning and memory. However, far less is known about the changes of dendritic spine density and morphology related to hippocampal synaptic plasticity. As dendritic spines are dynamic structures essential for synaptic plasticity and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory, the aim of the present study was to investigate whether the perinatal undernutrition affected hippocampal synaptic plasticity accompanied by the change of dendritic spines in anesthetized rats. An input–output curve was first determined using the measurements of field excitatory postsynaptic potential (fEPSP) slope in response to a series of stimulation intensities. Long-term potentiation (LTP) induced by high-frequency stimulation was recorded in the Schaffer collateral-CA1 pathway. Post-tetanic potentiation derived from the fEPSP slope was also measured immediately after LTP induction. Quantitative data of dendritic spines from hippocampal CA1 pyramidal cells were obtained using Golgi staining. The results showed that 50% perinatal food restriction (FR50) impaired the magnitude of LTP of the fEPSP slope in the Schaffer collateral-CA1 pathway. Additionally, FR50 reduced overall spine densities in both basal dendrites and apical dendrites of hippocampal CA1 pyramidal cells. Moreover, FR50 reduced type densities of thin and mushroom spines in apical dendrites, whereas a reduction in the type of mushroom spines was only observed in the basal dendrites of hippocampal CA1 pyramidal cells. These findings suggested that perinatal undernutrition decreased excitatory synaptic input and further affected the processing of information in a given network by selectively reducing the

INTRODUCTION The developmental brain is particularly susceptible to the intrauterine environment. The hippocampus is a brain region sensitive to undernutrition and is a necessary substrate for some forms of learning and memory. Previous studies showed that prenatal or perinatal nutrient restriction impaired hippocampal synaptic plasticity in the dentate gyrus area and adversely affected hippocampal-dependent learning and memory tasks in the postnatal period, and continued into adulthood (Austin et al., 1986; Bronzino et al., 1996, 1997). Related mechanisms included the changes in oxidative status (Partadiredja et al., 2005), cholinergic systems (Andrade and Paula-Barbosa, 1996), serotoninergic system (Chen et al., 1992), brain-derived neurotrophic factor (BDNF) (Wang and Xu, 2007), glutamic acid decarboxylase-67 (GAD-67) and nitric oxide synthase in the hippocampi of the young and adult rodent brains under prenatal or perinatal undernutrition (Dı´ az-Cintra et al., 2007; Zhang et al., 2010). However, far less is known about the morphological changes in dendritic spines related to the impaired hippocampal plasticity under perinatal undernutrition. At the electrophysiological level, synaptic plasticity is usually reflected by long-term potentiation (LTP), whereas LTP is frequently associated with changes of the morphology and the number of dendritic spines (Bourne and Harris, 2008). Dendritic spines are tiny membranous protrusions from neuronal dendrites that receive inputs from other neurons’ nerve terminals, which are the major sites of information processing and storage in the nervous system and are believed to provide an anatomical substrate for memory storage and synaptic transmission (Segal, 2005). There is

*Corresponding author. Address: School of Medicine, Nankai University, No. 94 Weijin Road, Tianjin 300071, PR China. Tel: +86-2223504364; fax: +86-22-23502554. E-mail address: [email protected] (Z. Yang). Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; E0, embryonic day 0; fEPSP, field excitatory postsynaptic potential; FR50, maternal 50% food restriction; HFS, highfrequency stimulation; I–O, input–output; IUGR, intrauterine growth restriction; LTP, long-term potentiation; PD, postnatal day; PD0, postnatal day 0; PD1, postnatal day 1; PTP, post-tetanic potentiation.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.03.061 31

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Y. Zhang et al. / Neuroscience 244 (2013) 31–41

evidence from animal models showing changes in dendritic spines, probably associated with synaptic disturbances and correlated with alterations in memory and learning abilities (Moser et al., 1994; Kolb et al., 2008). Especially, changes in the morphological pattern of dendritic spines have drawn a lot of interest, because the spine morphology is a very critical determinant of its function (Yuste et al., 2000; Kasai et al., 2003). Dendritic spines are characterized according to their sizes and shapes, (i.e., thin, stubby, mushroom, wide, branched, and double spines) (Bourne and Harris, 2008). In general, thin and mushroom spines have been considered to be the most efficient spines in transmitting synaptic impulses, attributed in part to their narrow neck (Koch et al., 1992). Specifically, mushroom-type spines have been hypothesized to represent physical substrates of long-term memories, and thin spines may represent the capacity for adaptive experiencedependent rewiring of neuronal circuits (Kasai et al., 2003). On the other hand, stubby and wide spines have generally been related to the regulation of excitability (Xu et al., 2009; Baye´s et al., 2011) by virtue of the fact that they have no neck to restrict the current flow from the postsynaptic density to the parental dendrite (Koch et al., 1992). Appropriate spine density and morphology are critical for the neuronal function that underlies learning and memory (Lynch et al., 2007; Baye´s et al., 2011). As such, a diverse spectrum of learning and memory disorders exhibits dendritic spine abnormalities, including neurodevelopmental disorders, such as autism, Down’s syndrome, non-syndromic mental retardation and neurodegenerative diseases (Fiala et al., 2002; Newey et al., 2005). Previous studies in experimental animals, mainly during early postnatal life, have shown that nutrition deprivation has profound effects on dendrites and dendritic spines in cortical neurons. Dobbin and Sands (1971) showed diminished dendritic arborizations of neurons of the fifth cortical layer in experimental animals with nutritional deprivation. Salas et al. (1973) found thinner dendrites and diminished spine numbers in neurons from the occipital cortex under neonatal food deprivation. These abnormalities also existed in cortical neurons in severe protein-calorie malnutrition infants whose ages ranged from 8 to 24 months (Benı´ tezBribiesca et al., 1999). Regretfully, up to now, we do not know whether maternal undernutrition reduces the number of dendritic spines of neurons in the hippocampus. If there are some decreases in the number of dendritic spines, which kind of spines is changed? Moreover, previous studies investigating the number of dendritic spines largely focused on the apical dendritic spines of cortex neurons induced by maternal undernutrition, the basal dendritic spines received less research attention. Recent studies have shown that the apical dendritic and basal dendritic spines may have different effects in synaptic plasticity (Spruston, 2008). One important question is whether maternal undernutrition produces a different profile of dendritic spines in the apical dendrites compared with that of basal dendrites.

In the present study, we first evaluated the effect of perinatal undernutrition on CA1 functional plasticity, as measured by the induction of post-tetanic potentiation (PTP) and LTP. Afterward, quantitative data of dendritic spines from hippocampal CA1 pyramidal cells were obtained from Golgi-stained sections. Only male animals were chosen for the study to avoid the hormonal fluctuations associated with female rats due to an estrous cycle.

EXPERIMENTAL PROCEDURES Animals and treatments Adult virgin female Wistar rats (body weight 250–280 g, 14-week-old) were obtained from the Chinese Academy of Medical Sciences (license No.: SCXK-2002-001). Animals were maintained under standard laboratory conditions under artificial 12-h light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.) and an ambient temperature of 21–23 °C. Food and water were available ad libitum. Two females were paired with one male (2:1) for a period of 4–5 days until mating was confirmed by observation of a copulatory plug or the presence of sperm in a vaginal rinse under a microscope. The day that mating was confirmed was recorded as gestation day 0 (GD0) or embryonic day 0 (E0). The day of birth was identified as postnatal day 0 (PD0). Wood shavings were provided to each dam, which was singly housed. The diet used in the experiment is based on the AIN-93G purified rodent diet. The composition of the standard rodent diet contains 20% protein, 5% fat, 65% carbohydrates, 5% fiber, 3.5% mineral mixture, 1% vitamin mixture, 0.3% DL-methionine and 0.2% choline bitartrate. All experiments were carried out according to the protocols approved by the Animal Care Committee of the Animal Center at the Chinese Academy of Sciences in Shanghai and in accordance with the principles outlined in the NIH guide for the Care and Use of Laboratory Animals. Maternal food restriction Two groups of pregnant rats were studied. In the control group (n = 9), dams were fed ad libitum during gestation, from embryonic day 1 (E1) to E21 for fetuses, and lactation, from postnatal day (PD) 1 to PD21 for pups. In the perinatal 50% food-restricted group, dams (n = 8) received 50% of the daily food intake of control mothers from E7 until the end of lactation and were returned to normal diet after lactation (on PD21) (Carney et al., 2004). Dams delivered spontaneously and the day of delivery was designated as PD0. Expected gestation length in our colony was 21.5– 23 days. The litter size was randomly culled to eight pups (four males and four females) to assure uniformity of litter size between FR50 and the control group. Each pup was marked with 1% methyl violet solution on the skin for identification on PD1 and was numbered on the tail with black ink on PD13. To assess the body weight before lactation, pups were observed between 9:00 and

Y. Zhang et al. / Neuroscience 244 (2013) 31–41

10:00 a.m. Afterward, they were immediately returned to their home cages.

Body weight tests Before weaning, an experimenter, blinded to the animal’s treatment condition, tested body weight from different litters for physical development on PD1, PD7, PD10, PD14 and PD21. Only one male and one female pup from each litter were used to obviate a putative litter effect. After lactation, the littermates were separated by gender and housed three to four males per cage until completion of the study. Body weights from dams were tested on E1, E7, E14 and E21.

Electrophysiology On PD70, male rat offsprings from control (n = 8) and FR50 group (n = 8) were anesthetized with sodium pentobarbital (50 mg/kg body weight, ip) and were then mounted in a stereotaxic apparatus. The rectal temperature was monitored and maintained at 37 ± 0.5 °C by a homeothermic blanket. Adult Wistar rats were implanted with a monopolar recording electrode in the stratum radiatum of the CA1 area (3.4 mm posterior to bregma and 2.5 mm lateral to the midline), and a bipolar stimulating electrode in the Schaffer collaterals of the dorsal hippocampus (4.2 mm posterior to the bregma and 3.5 mm lateral to the midline) via holes drilled through the skull (Cao et al., 2004). The optimal depth of the electrode in the stratum radiatum of the CA1 area of the dorsal hippocampus was determined by the maximal response to the Schaffer collateral pathway stimulus. Before LTP, the field excitatory postsynaptic potential (fEPSP) slope was recorded at a frequency of 0.033 Hz (30-s interval) by delivering a single current pulse (0.2 ms in duration) to the Schaffer collateral pathway. The stimulus intensity was adjusted to give a fEPSP value of 50% of maximum slope as analyzed by establishing an input–output (I–O) correlation. The I–O curve was constructed by measuring fEPSP slope of responses evoked by a series of stimulation intensities from 0.1 to 0.7 mA (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 mA). For LTP, each experiment consisted of a baseline measurement taken for 30 min, followed by a further measurement of evoked responses (120 min) after high-frequency stimulation (HFS) application. HFS consisted of ten trains of 20 stimuli at 200 Hz with a 2-s intertrain interval, with the same stimulation intensity used for the baseline recordings (Cao et al., 2004). There were no differences on the stimulus required to evoke 50% of maximum fEPSP between control and FR50 animals. The responses in every 2 min were averaged (four pulses). Baseline fEPSP slopes averaged across 30 min prior to LTP induction. PTP was derived from fEPSP slope measures taken immediately after LTP induction (2 min average) (Blalock et al., 2010). LTP was quantified by comparing the mean fEPSP slope over the 5–120 min HFS.

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Tissue preparation and Golgi impregnation A modified osmium-free version of the Golgi technique was used to study hippocampal pyramidal neurons (Gonza´lez-Burgos et al., 1992). After electrophysiological experiments, animals were anesthetized with 50 mg/kg i.p. sodium pentobarbital. Then, animals were perfused intracardially with 100 mL of a washing phosphate-buffered solution (pH 7.4; 0.01 M). Then, 200 mL of a fixing phosphate-buffered 4% formaldehyde solution was perfused. The animal’s brain was then removed and fixed for 48 h in 4% paraformaldehyde for 2 h and then transferred to a solution of 0.95 mM potassium dichromate in 80 mM formaldehyde diluted in distilled water for 48 h. Then, the tissue was immersed overnight in a solution of 0.95 mM potassium dichromate in 20 mM formaldehyde and then in a solution of 1.2 mM potassium dichromate in distilled water where the tissue remained for 5 days. After that, impregnated tissue was incubated in 0.44 mM silver nitrate in distilled water for 48 h. All the preceding steps were done in darkness and at room temperature. Finally the tissue was stored in 30% sucrose in 0.1 M phosphate buffer at 4 °C until coronal sectioning with a vibrating microtome at 100 lm. Sections were mounted on slides, pressed with blotting paper to avoid detachment during ethanol dehydration and xylene clearing, and coverslipped with Permount. Microscopic analysis For each rat, six CA1 pyramidal neurons were traced. Data from the six cells per rat were averaged, and the average of the eight animals per group was compared. Only those neurons showing a completely impregnated dendritic tree and that were relatively isolated from neighboring cells were selected for the analysis, and, as far as possible, to be clearly visible in one plane of focus. For the basilar region the number of dendritic spines per 10-lm segment of second-order basilar dendritic branch (a segment of branch that is found distal to a bifurcation in a branch that originated from the cell body) was calculated. Similarly, spine density for a segment of an apical dendritic branch was calculated from a portion distal to the first bifurcation of the primary apical dendrite (second-order bifurcation from the cell body). The spines were quantified in terms of the density of thin, stubby, mushroom, wide, branched and double spines (Gonza´lez-Burgos et al., 2009). The criteria for a thin spine was that the diameter of the neck was much less than the total length (the ratio of the diameter of the neck to the spine length was <0.5), and that the diameter of the head was not much greater than the neck diameter (the ratio of the head diameter to neck diameter was <1.3, head diameter <0.4 lm). The criterion for a stubby spine was that the diameter of the neck was greater than or equal to the spine length. A spine was classified as mushroom shaped if both the diameter and length of the neck were very much less than the head diameter (the ratio of the head diameter to neck diameter was >1.3, head diameter >0.6 lm), and if the length of the neck was less than that of thin

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Y. Zhang et al. / Neuroscience 244 (2013) 31–41

spines. A wide spine was very similar to a stubby one, but the length of a wide spine was greater than the diameter of the neck. Branched spines were recognized because two oblique necks ending as bulbous heads merged from a common single neck. Finally, a double spine was identified as a neck protruding from the parent dendrite and connected with a bulb, which was connected to another bulb by a second neck. For analysis of dendritic spine morphology, high magnification images were captured using a camera (DXC-390, Sony Corporation, Tokyo, Japan) attached to an Olympus upright microscope (BX60, Japanese). These images were magnified with the 100 oil immersion objective (NA = 1.25) using a magnification changer coupled to a light microscope. The final resolution was 0.125 lm. This present study did not assess spine density in a native manner, but rather in rats fixed by perfusion after HFS protocol. Although the study might overestimate the number of spines, it facilitated a direct comparison of different groups when they were analyzed in an identical manner. Data analysis Pup body weight was analyzed by univariate analysis of variance (ANOVA), with weight as the dependent effect, food restriction and sex as fixed effects. The main effect of group (Pfood) tested for effects of food restriction independent sex. The main effect of sex (Psex) tested for effects of sex differences independent group. The interaction term (Pfood
sex) tested whether the effects of food restriction differed in male and female pups. Repeated-measures ANOVA was used to analyze maternal body weight. For the I–O curves, data were compared between control and FR50 groups at each stimulation intensity using a two-way ANOVA with repeated measures. Baseline transmission, PTP, LTP and spine density were compared using independentsamples t-test (Ruszczycki et al., 2012). All the above data are presented as mean ± standard error of the mean (SEM). Analyses were performed using SPSS16.0 statistical software. In all cases, statistical significance was set at P < 0.05.

RESULTS Physiological data Body weight of dam during gestation was measured (Table 1). A repeated measures ANOVA revealed a significant main effect for food group (F = 35.31, df = 1/15, P < 0.001), time (F = 583.55, df = 3/45, P < 0.001), and their interaction (F = 106.20, df = 3/45, Table 1. Effects of maternal FR50 on body weight (g) of dam

P < 0.001). The body weights of mother rats on E14 (F = 125.95, df = 1/15, P < 0.001) and E21 (F = 61.81, df = 1/15, P < 0.001) were lower in the FR50 group than those in the control group, but no difference between E1 and E7 (Table 1). There were no significant differences in gestation length and litter size on PD0. Table 2 demonstrated the effects of FR50 on the body weight of both male and female pups during different developmental periods. Univariate ANOVA indicated that there were significant main effects on PD1, PD7, PD10, PD14 and PD21 for group (F = 19.62, df = 1/30, P < 0.001; F = 26.35, df = 1/30, P < 0.001; F = 30.188, df = 1/30, P < 0.001; F = 10.22, df = 1/30, P < 0.01; F = 20.14, df = 1/30, P < 0.001, respectively), and sex (F = 7.13, df = 1/30, P < 0.05; F = 8.20, df = 1/30, P < 0.01; F = 13.28, df = 1/30, P < 0.01; F = 5.35, df = 1/30, P < 0.05; F = 4.58, df = 1/30, P < 0.05, respectively). No significant interaction was found between food treatment and sex. The body weights of FR50 pups on PD1, PD7, PD10, PD14 and PD21 were significantly lower than those of the control group (Table 2). fEPSP data The relationships between fEPSP slopes and currents in the I–O curves were examined. A repeated measures ANOVA revealed no significant difference in basal transmission between control and FR50 groups (F = 0.384, df = 1/83, P = 0.537) (Fig. 1). Fig. 2A shows the waveform alterations before and after HFS induction in both groups, respectively. Fig. 2B illustrates the fEPSP slope in both control and FR50 groups. After 30-min baseline fEPSP recording, a HFS was applied to induce fEPSP of LTP. The fEPSP slope was measured between 20% and 80% of the peak amplitude and plotted versus time. The fEPSP slope was expressed as the absolute values for individual animals. Before HFS, the fEPSP slopes during the baseline were 6.741 ± 0.908 mV/ms and 6.946 ± 1.073 mV/ms for control and FR50 rats respectively. Apparently, differences between control and FR50 groups in the baseline values of the fEPSP slopes in the hippocampus were comparable (t = 0.144, P = 0.889). After HFS, the fEPSP slope was 8.367 ± 0.593 mV/ms in control rats versus 5.265 ± 0.818 mV/ms in FR50 rats. Notably, FR50 impaired hippocampal CA1 fEPSP slope of LTP compared with that of the control group (t = 3.069, P < 0.05). Similar results were seen on measures of PTP taken immediately following LTP induction, showing a significant difference (Control: 9.516 ± 0.788 mV/ms; FR50: 6.915 ± 0.655. t = 2.538, P < 0.05).

Parameters

Control

FR50

P-value

Dendritic spine density and type

E1 E7 E14 E21

259.34 ± 3.33 298.46 ± 2.59 335.13 ± 3.49 374.43 ± 3.99

261.84 ± 3.43 296.28 ± 3.45 278.94 ± 3.58 323.85 ± 5.15

n.s. n.s. <0.001 <0.001

In control rats, the density of dendritic spines of CA1 pyramidal cells was 14.88 ± 0.67 spines/10 lm in apical dendrites and 11.13 ± 1.01 spines/10 lm in basal dendrites. In FR50 rats, the density of dendritic spines of CA1 pyramidal cells was 12.63 ± 0.60 spines/

n.s. = Non-significant.

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Y. Zhang et al. / Neuroscience 244 (2013) 31–41 Table 2. Effects of food restriction during pregnancy and lactation on mean pup body weights (g) Control

PD1 PD7 PD10 PD14 PD21

FR50

Males

Females

Males

Females

5.98 ± 0.21 12.86 ± 0.58 16.59 ± 0.68 24.12 ± 0.88 36.17 ± 1.89

5.54 ± 0.25 11.36 ± 0.46 14.16 ± 0.48 21.98 ± 1.05 31.43 ± 1.58

5.20 ± 0.10 10.28 ± 0.27 13.13 ± 0.34 21.15 ± 0.99 27.99 ± 0.81

4.59 ± 0.18 9.05 ± 0.50 11.50 ± 0.62 18.96 ± 0.75 26.44 ± 1.14

Pfood

Psex

Psex
food

<0.001 <0.001 <0.001 <0.01 <0.001

<0.05 <0.01 <0.01 <0.05 <0.05

n.s. n.s. n.s. n.s. n.s.

n.s. = Non-significant.

A

1.0

Normalized fEPSP slope

Control FR50

0.8

0.6

0.4

0.2 0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Stimulation intensity (mA)

10 lm in apical dendrites and 10.25 ± 0.88 spines/10 lm in basal dendrites. Obviously, the spine density for both apical and basal dendritic trees of CA1 pyramidal cells was reduced in FR50 rats (Apical: P < 0.01; basal: P < 0.05, respectively) (Figs. 3 and 4). In different spine types in apical dendrites, FR50 rats exhibited fewer density of thin (7.13 ± 0.40 spines/10 lm in control CA1 neurons, 5.88 ± 0.39 spines/10 lm in FR50 CA1 neurons, P < 0.01) and mushroom spines (2.75 ± 0.25 spines/10 lm in control CA1 neurons, 1.73 ± 0.45 spines/10 lm in FR50 CA1 neurons, P < 0.05) (Figs. 5 and 6), whereas no changes in the types of stubby (2.01 ± 0.38 spines/10 lm in control CA1 neurons, 1.25 ± 0.16 spines/10 lm in FR50 CA1 neurons, P = 0.090), branched (0.27 ± 0.19 spines/ 10 lm in control CA1 neurons, 0.13 ± 0.11 spines/ 10 lm in FR50 CA1 neurons, P = 0.120), wide (1.25 ± 0.25 spines/10 lm in control CA1 neurons, 1.00 ± 0.27 spines/10 lm in FR50 CA1 neurons, P = 0.506) and double spines (0.38 ± 0.18 spines/ 10 lm in control CA1 neurons, 0.50 ± 0.27 spines/ 10 lm in FR50 CA1 neurons, P = 0.705) were observed. In contrast, in the basal dendrites of CA1 pyramidal neurons, FR50 resulted in a significant reduction in the type of mushroom spines (3.25 ± 0.37 spines/10 lm in control CA1 neurons,

B Absolute fEPSP slope (mv/ms)

Fig. 1. Input–output curves in both groups of FR50 rats and controls. There was no significant difference in baseline transmission between control and FR50 groups (2-way repeated measure ANOVA, P = 0.537). Error bars represent standard errors.

11

Control FR50

10 9 8 7 6 5 0

20

40

60

80

100 120 140 160

Time (min) Fig. 2. The fEPSP in the CA1 area of adult control and FR50 offspring before and after HFS. The fEPSP slopes of the Schaffer collateral-CA1 field potentials were plotted against time. After baseline responses were stable for 30 min, HFS was given by electrical stimulation. (A) Representative fEPSP waveforms before (left) and after HFS (right) inductions in control and FR50 rats. The waveforms in each case are from a single experiment. Scale bar = 1 mV, and 5 ms. (B) fEPSP slopes (mV/ms) evoked by a single 0.2-ms stimulus to the Schaffer collaterals. Arrow at 30 min indicates the application of HFS. The fEPSPs were collected at 0.033 Hz. The fEPSP slopes were averaged in 2-min bins for individual animals. Error bars represent standard errors of the group means. Baseline fEPSP slopes averaged across 30 min prior to LTP induction. PTP was derived from fEPSP slope measures taken immediately after LTP induction (2 min average). LTP was quantified by comparing the mean fEPSP slope over the 5–120 min HFS. FR50 rats showed a decreased fEPSP slope of long-term potentiation (LTP) in hippocampal CA1 area. Similar results were seen on measures of PTP taken immediately following LTP induction.

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Y. Zhang et al. / Neuroscience 244 (2013) 31–41

Fig. 3. Representative images of Golgi-impregnated dendritic spines from apical and basal dendrites of CA1 pyramidal neurons, showing differences in spine density. (a) Spines from apical dendrites of CA1 pyramidal neurons in control rats. (b) Spines from apical dendrites of CA1 pyramidal neurons in FR50 rats. (c) Spines from basal dendrites of CA1 pyramidal neurons in control rats. b: Spines from basal dendrites of CA1 pyramidal neurons in FR50 rats. Scale bar = 5 lm. Maternal FR50 decreased the density of dendritic spines from apical and basal dendrites of CA1 pyramidal neurons.

14

**

12

*

10 8

Spine density (10 µm dendrite)

a

Control FR50

Control FR50

8

**

6 4

*

2 0 th

mu

st

6

wi

do

br

Spine types

4

b

2 0 Apical

Basal

Dendrite Fig. 4. FR50 reduced overall spine densities in both the basal dendrites and apical dendrites of hippocampal CA1 pyramidal cells. The spine density (number of spines) was counted in a dendrite segment 10 lm in length of pyramidal neurons from the hippocampal CA1 field. ⁄P < 0.05, ⁄⁄P < 0.01, FR50 group vs control group. Error bars represent standard errors.

1.51 ± 0.53 spines/10 lm in FR50 CA1 neurons, P < 0.01) (Figs. 5 and 6), although there were no differences in the types of thin (6.50 ± 0.42 spines/ 10 lm in control CA1 neurons, 5.75 ± 0.53 spines/ 10 lm in FR50 CA1 neurons, P = 0.285), stubby (1.38 ± 0.32 spines/10 lm in control CA1 neurons, 1.25 ± 0.31 spines/10 lm in FR50 CA1 neurons, P = 0.802), branched (0.38 ± 0.18 spines/10 lm in control CA1 neurons, 0.25 ± 0.16 spines/10 lm in FR50 CA1 neurons, P = 0.619), wide (0.75 ± 0.16 spines/10 lm in control CA1 neurons, 1.13 ± 0.23 spines/10 lm in FR50 CA1 neurons, P = 0.201), and double spines (0.25 ± 0.16 spines/ 10 lm in control CA1 neurons, 0.16 ± 0.13 spines/ 10 lm in FR50 CA1 neurons, P = 0.554) compared with those of the control group.

Spine density (10 µm dendrite)

Spine density/10 µm dendrite

16

Control FR50

6 4

* *

2 0 th

mu

st

wi

do

br

Spine types Fig. 5. FR50 selectively affected spine-type densities in both basal dendrites and apical dendrites of hippocampal CA1 pyramidal cells. The spine density (the number of spines) was counted in a dendrite segment 10 lm in length of pyramidal neurons from the hippocampal CA1 field. (a) The spine density from apical dendrites of CA1 pyramidal neurons. (b) The spine density from basal dendrites of CA1 pyramidal neurons. The letters th, mu, st, wi, do and br exemplify thin, mushroom, stubby, wide, double and branched spines, respectively. ⁄ P < 0.05, ⁄⁄P < 0.01, FR50 group vs control group. Error bars represent standard errors.

DISCUSSION The perinatal period, the period around the time of birth, is critical for brain development in most mammalian species. Maternal undernutrition during the perinatal period is known to cause a deficit in fetal development, of which intrauterine growth restriction (IUGR) is one manifestation. IUGR is associated with neurodevelopmental delay after birth (Taylor and Howie, 1989; McIntire et al., 1999), childhood stunting,

Y. Zhang et al. / Neuroscience 244 (2013) 31–41

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Fig. 6. Representative images (a, b, c and d) of spine morphological types, showing visible differences in spine classification. Scale bar = 3 lm. The letters th, mu, st, wi, do and br exemplify thin, mushroom, stubby, wide, double and branched spines, respectively. Note that in the present pictures, Scale bar = 3 lm. However, scale bar of Fig. 3 = 5 lm.

intellectual decrease, behavioral dysfunctions and school achievement (Chang et al., 2002). One of the target sites of nutritional insults in the CNS is the hippocampus that is involved in learning and memory. In the excitatory trisynaptic pathway of the hippocampus, two synaptic areas, the CA1 and dentate gyrus, exhibit NMDA receptor-dependent LTP (Bliss and Collingridge, 1993). Although these two synaptic areas are all involved in learning and memory behavior, the CA1 area is crucial for spatial learning. This issue is not only proven by both lesion studies and transgenic mice models (Silva et al., 1992; Zola-Morgan et al., 1992; Giese et al., 1998; Remondes and Schuman, 2004; Westerink et al., 2012), but also in normal rats. For example, water-maze learning resulted in significant increases in agmatine in the CA1 (about 60–80%) and DG (about 20%), which appeared to be consistent with the roles of these two areas in spatial learning and memory processing (Liu et al., 2008). On the other hand, they may have distinct roles in spatial learning and memory tested in the water-maze task. Using recombinant Sindbis viral vectors encoding the unedited form of GluR2 (GluR2Q) in which an arginine (R) in the Q/R site of the edited GluR2 were replaced with glutamine (Q) (Sommer et al., 1991; Seeburg, 1993; Hollmann and Heinemann, 1994; Ozawa et al., 1998). Okada demonstrated that postsynaptic enhancement of Ca2+ influx through newly expressed Ca2+-permeable a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the hippocampal CA1 area facilitated learning behavior, whereas that involving neurons in the dentate gyrus impaired it (Okada et al., 2003). When the animals are trained to find a platform that is kept constant throughout all training sessions, spatial reference memory requires neural activity in the CA1 area (Jo et al., 2007). The dentate gyrus area is characterized by orthogonalization of sensory inputs to create a metric spatial representation (Rolls and Kesner, 2006). Previous studies on hippocampal synaptic plasticity induced by maternal undernutrition largely focused on hippocampal dentate gyrus. Perinatal food restriction or

prenatal protein restriction impaired LTP in hippocampal dentate gyrus of adult rodents (Jordan and Clark, 1983; Austin et al., 1986; Bronzino et al., 1997). Prenatal food restriction impaired LTP in hippocampal dentate gyrus of 15-day-old and 30-day-old rats (Bronzino et al., 1996). In fact, CA1 neurons are also sensitive to undernutrition (Lister et al., 2005; Dı´ az-Cintra et al., 2007). Taking account of the CA1 area is more crucial for spatial learning, we observed hippocampal CA1 fEPSP slope of LTP induced by FR50. FR50 impaired CA1 fEPSP slope of LTP of adult rats similar to previous observation in the dentate gyrus area. In the present study, we also observed the slopes of PTP between control and FR50 rats. PTP is another type of synaptic potentiation that can be induced in the CA1 area of the hippocampus by tetanisation. PTP is different from LTP in that its induction is independent of the NMDA receptor activation. It is observed immediately after tetanisation, declines over a relatively short period of time (Stevens et al., 1994) and reflects the clearance of Ca2+ that accumulates in the presynaptic terminals during tetanisation (Zucker, 1999). The slope of PTP decreased in FR50 rats, suggesting that perinatal undernutrition might impair the presynaptically mediated synaptic potentiation. Future study should explore the mechanisms involved. It is now well established that, in hippocampal CA1 area, the LTP induction at Schaffer-collateral pathway is mainly due to the postsynaptic mechanism depending on the dendritic spines. Dendritic spines receive the majority of excitatory connections in the central nervous system, and, thus, they are key structures in the regulation of neural activity. In our studies, Wistar rats submitted to perinatal undernutrition display decreased fEPSP slope paralleled by decreased hippocampal spine density. The currently available data further prove that there is a strong correlation between synaptic plasticity and dendritic spines. A number of factors might account for the observed decreases in spine density in hippocampal neurons. Numerous studies have demonstrated that spine density is regulated by glutamatergic transmission and

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glutamate receptor types located on dendritic spine heads (Fischer et al., 2000; Segal and Andersen, 2000; Norrholm and Ouimet, 2001). In addition, a series of in vitro studies have shown that N-methyl-D-aspartic acid (NMDA) receptors mediate the destabilization of filamentous actin (f-actin) associated with dendritic spine loss (Halpin et al., 1998; Norrholm and Ouimet, 2001). Recent evidence has shown that prenatal or perinatal undernutrition led to the profound changes in the level of free radicals (Partadiredja et al., 2005), cholinergic systems (Andrade and Paula-Barbosa, 1996) and nitric oxide synthase in hippocampus of the young and adult rodent brains. These changes are caused by different mechanisms but may share a final common pathway to dendritic spines due to changes in glutamatergic transmission or the glutamate receptor’s associated ion channels (Aizenman et al., 1990; Marino et al., 1998). BDNF is another well-characterized determinant of dendritic spine number and morphology (Stranahan, 2011). Regulation of BDNF has been reported to be very sensitive to undernutrition in the hippocampus (Wang and Xu, 2007). Thus, it is tempting to speculate that the observed reduction in spine density in CA1 pyramidal neurons may involve alterations of NMDA receptor-mediated responses following maternal undernutrition. Therefore, it is also possible that maternal undernutrition might alter BDNF and its downstream signaling targets in the dendrites of CA1 pyramidal neurons. Further studies are in progress to address the molecular mechanisms involving alterations in spine density. Analysis of spine density showed that perinatal undernutrition did not lead to the same effect on the apical dendrites (P < 0.01) compared with basal dendrites (P < 0.05). The apical dendritic spine seems more sensitive to undernutrition than the basal dendritic spine. In the literature, differential vulnerability of spines between basal and apical dendrites due to exogenous or endogenous factors has been reported although the mechanisms involved are not clear. For instance, Santos et al. (2004) reported that neonatal rats exposed repetitively to low doses of paroxon (an organophosphate-type cholinesterase inhibitor) lost dendritic spines selectively in basal dendrites with no changes in apical dendrites of CA1 pyramidal neurons. Moreover, normal aging also results in decreases of the spine density on basal but not apical dendrites in C57BL/6 mice (von Bohlem und Halbach et al., 2006). CNQX (cyano-nitroquinoxaline-dione) did not inhibit the estradiol-induced increase in the spine density in apical dendrites of CA1 pyramidal neurons; however, CNQX completely suppressed the increase in the spine density in basal dendrites. MK-801 completely suppressed the increase in the spine density in the apical dendrites; however, MK-801 only partially suppressed the increase in the spine density in basal dendrites (Murakami et al., 2006). Regretfully, previous studies investigating undernutrition, rarely focused on the basal dendritic spines neurons. Indeed, anatomical and functional differences between the apical and basal dendrites have long been illustrated (Arai et al., 1994; Yuste et al.,

1994). In the stratum radiatum (apical side) of the hippocampal CA1 area, Schaffer collaterals, originating from CA3 pyramidal neurons, project to dendrites of CA1 neurons. In the stratum oriens (basal side) of the hippocampal CA1 area, recurrent collaterals (CA1 to CA1 connections) project to dendrites of CA1 pyramidal neurons (Murakami et al., 2006). Therefore, apical dendritical spines of hippocampus CA1 pyramidal cells may be a dendritic trigger zone for regenerative Ca2+ spikes or a current amplifier for synaptic events (Yuste et al., 1994), whereas basal dendritic spines of hippocampal CA1 pyramidal cells may lead to stronger enhancement of cellular activity (excitability) imposed by recurrent collaterals (local feedforward excitation) (Papp et al., 2004). Future studies will be required to evaluate the mechanistic basis of differential vulnerability in the reduction of spine density between CA1 basal and apical dendrites induced by perinatal undernutrition. In contrast to spine morphology, in the apical dendrites of CA1 pyramidal neurons, maternal undernutrition resulted in a significant decrease in the types of both thin and mushroom spines. However, in the basal dendrites of CA1 pyramidal neurons, only a reduction in the type of mushroom spines was exhibited induced by maternal undernutrition. Recently, there has been a growing awareness of functional differences between thin and mushroom spines. Thin spines maintain the structural flexibility to enlarge and stabilize after LTP and can accommodate new, enhanced or recently weakened inputs, making them candidate ‘‘learning spines’’ (Bourne and Harris, 2007; Peebles et al., 2010). By decreasing the density of learning spines in the apical dendrites, perinatal undernutrition may therefore decrease a neuron’s ability to form new synapses. Age-related reductions in thin spines have been observed in rhesus monkeys, with cognitive performance inversely proportional to thin spine volume (Dumitriu et al., 2010). Mushroom spine affects the diffusion and compartmentalization of membraneassociated proteins (Hugel et al., 2009) as well as the expression of higher levels of AMPA receptors (Matsuzaki et al., 2001; Ganeshina et al., 2004; Nimchinsky et al., 2004; Ashby et al., 2006). In particular, mushroom dendritic spines have larger postsynaptic densities (Harris et al., 1992) and the length of the spine neck seems to be a key regulator of spinodendritic Ca2+ signaling (Majewska et al., 2000; Yuste et al., 2000; Hayashi and Majewska, 2005; Noguchi et al., 2005; Schmidt and Eilers, 2009) and of the transmission of membrane potentials (Araya et al., 2006). In addition, mushroom spines seem to form more stable synapses, which have higher motility and form more transient contacts (Konur and Yuste, 2004). Therefore, mushroom spines may be involved in the management of previously acquired information and sustain memory storage (Kasai et al., 2003; Matsuzaki et al., 2004; Bourne and Harris, 2007). By decreasing the density of memory spines in the apical and basal dendrites, perinatal undernutrition may decrease a neuron’s ability to form stable connections and sustain acquired information in the dendrites. Thus, it can be

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speculated that maternal undernutrition may influence the processing of synaptic impulses, including changes in excitatory synaptic connectivity, synaptic structure, ionic conductance, neurotransmitter responsiveness and calcium-handling in the CA1 region and further leads to the altered synaptic efficacy. Although our findings are suggestive for understanding the role of disturbed synaptic plasticity induced by undernutrition, they should be interpreted with some caution. It should be noted that spine analyses in the present study were performed in rats fixed by perfusion after high-HFS, but not in a native manner. Therefore, the study might overestimate the number of spines. Dendritic spines have attracted increasing interest of neuroscientists, as their particular shape and density seem to impact heavily on the network connectivity, and many neurological diseases present alterations of dendritic spines. These results undoubtedly enrich current knowledge of synaptic plasticity and provide insight into the mechanisms of learning and memory process. However, there are some limitations. Among the many publications involving spine-type classification, many show a variety of definitions, and thus an even variability in the results. Moreover, resolution limits of light microscopy affect results. The small sizes of these protrusions, with heads between 0.2 and 1.2 lm in diameter, are close to the resolution limits of light microscopy. Thus, the evaluation of the spines may be affected. A new imaging technology really needs to be developed in the future.

CONCLUSION Maternal undernutrition had a negative effect on the apical and basal spine density of hippocampal pyramidal neurons. This indicates that spines and, hence, probably excitatory synaptic contacts are lost. Spines of the thin and mushroom type, characterized by a long spine neck, were particularly affected. The decrease in the thin and mushroom spines may in turn contribute to a corresponding decrease in the total number of putative glutamatergic terminals forming asymmetric synapses as well as related impaired synaptic plasticity. Recently, some studies demonstrated that the nature and/or extent of spine remodeling can be manipulated by various interventional strategies such as enriched housing, transplants, and NT-3. The interventions, especially a combination of the three, increased the complexity of synaptic connectivity by adding more spines and promoted the maturation of synaptic structures. This highlights an interesting possibility that structural remodeling of dendritic spines in the hippocampus can be targeted as a future strategy to improve neuroplasticity induced by undernutrition. Acknowledgements—The study was supported by Grants from the National Natural Science Foundation of China (81271224) and Ph.D. programs Foundation of Logistics University of Chinese People’s Armed Police Force (WYB201107).

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(Accepted 30 March 2013) (Available online 6 April 2013)