High-Salt Exposure During Perinatal Development Enhances Stress Sensitivity

Abstract

Excess consumption of dietary sodium during pregnancy has been shown to impair offspring cardiovascular function and enhance salt preference in adulthood, but little is known regarding the long-term impact of this nutritional surplus on offspring brain morphology and behavior. Using a combination of cellular and behavioral approaches, we examined the impact of maternal salt intake during the perinatal period on structural plasticity in the prefrontal cortex (PFC) and nucleus accumbens (NAc) in weanling and adult offspring as well as reward- and stress-driven behaviors in adult offspring. We found that weanling rats born to 4% NaCl-fed dams exhibited an increase and decrease in thin spine density in the infralimbic PFC (IL-PFC) and prelimbic PFC (PL-PFC), respectively, as well as an increase in mushroom spine density in the NAc shell, compared to 1% NaCl-fed controls. Structural changes in the IL-PFC and NAc shell persisted into adulthood, the latter of which is a phenotype that has been observed in rats exposed to early life stress. There was no effect of maternal salt intake on reward-driven behaviors, including sucrose preference, conditioned place preference (CPP) for cocaine, and forced swim stress (FSS)-induced reinstatement of cocaine-induced CPP. However, rats born to high-salt fed dams spent less time swimming in the FSS and displayed heightened plasma CORT levels in response to the FSS compared to controls, suggesting that early salt exposure increases stress sensitivity. Overall, our results suggest that perinatal salt exposure evokes lasting impacts on offspring physiology and behavior.

Keywords: dendritic spines; dietary salt; nucleus accumbens; perinatal development; prefrontal cortex; stress.

Figure 1.. Model of Dietary Manipulation.

A. Experimental timeline. Sexually mature male and female Wistar rats were housed together and female estrous cycles were monitored until pregnancy was confirmed. Females were then separated and fed either a 1% or 4% NaCl diet throughout gestation and lactation (denoted in gray). At weaning, offspring were either immediately euthanized for structural analysis or put into group housing until experimentation at PND60. Experimentation included cocaine conditioned place preference (CPP), sucrose preference testing (SPT), corticosterone (CORT) analysis or open field test. B. Maternal food consumption of 1% (white circles, n=8) and 4% (black squares, n=8) fed rats during dietary manipulation. C. Weight of offspring during postnatal development born to 1% (white circles, n=23/6, where first and second numbers indicate the number of animals and dams, respectively) and 4% (black squares, n=42/9) fed dams.

Figure 2.. Spine Analysis of the Prelimbic Prefrontal Cortex (PL-PFC).

A. Schematic of region analyzed and example dendrite with depiction of individual spine types. B. Representative images of terminal basal dendrites from DiI stained pyramidal cells of 1% (left) and 4% (right) NaCl fed offspring at postnatal day 21 (PND21, top) and PND60 (bottom). C. Representative images of terminal apical dendrites from DiI stained pyramidal cells of 1% (left) and 4% (right) NaCl fed offspring at PND21 (top) and PND60 (bottom). D. Quantification of spine types analyzed from terminal basal dendrites in 1% (white, n=24/3/3 for each time point, where first, second, and third numbers indicate the number of total dendrites analyzed, number of animals, and number of dams respectively) and 4% (black, n=24/3/3 for each time point) NaCl fed offspring at PND 21 and PND60. E. Quantification of spine types analyzed from terminal apical dendrites in 1% and 4% NaCl fed offspring at PND 21 and PND60. Values represent the mean ± SEM (*p<0.05).

Figure 3.. Spine Analysis of the Infralimbic Prefrontal Cortex (IL-PFC).

A. Schematic of region analyzed. B. Representative images of terminal basal dendrites from DiI stained pyramidal cells of 1% (left) and 4% (right) NaCl fed offspring at postnatal day 21 (PND21, top) and PND60 (bottom). C. Representative images of terminal apical dendrites from DiI stained pyramidal cells of 1% (left) and 4% (right) NaCl fed offspring at PND21 (top) and PND60 (bottom). D. Quantification of spine types analyzed from terminal basal dendrites in 1% (white, n=24/3/3 for each time point, where first, second, and third numbers indicate the number of total dendrites analyzed, number of animals, and number of dams respectively) and 4% (black, n=24/3/3 for each time point) NaCl fed offspring at PND 21 and PND60. E. Quantification of spine types analyzed from terminal apical dendrites in 1% and 4% NaCl fed offspring at PND 21 and PND60. Values represent the mean ± SEM (*p<0.05).

Figure 4.. Spine Analysis of the Nucleus Accumbens (NAc).

A. Schematic of regions analyzed. B. Representative images of terminal dendrites from DiI stained medium spiny neurons in the nucleus accumbens shell (NAc-Sh) of 1% (left) and 4% (right) NaCl fed offspring at postnatal day 21 (PND21, top) and PND60 (bottom). C. Representative images of terminal dendrites from DiI stained medium spiny neurons in the nucleus accumbens core (NAc-C) of 1% (left) and 4% (right) NaCl fed offspring at PND21 (top) and PND60 (bottom). D. Quantification of spine types analyzed from terminal basal dendrites in 1% (white, n=24/3/3 for each time point, where first, second, and third numbers indicate the number of total dendrites analyzed, number of animals, and number of dams respectively) and 4% (black, n=24/3/3 for each time point) NaCl fed offspring at PND 21 and PND60. E. Quantification of spine types analyzed from terminal dendrites in the NAc-C in 1% and 4% NaCl fed offspring at PND 21 and PND60. Values represent the mean ± SEM (*p<0.05).

Figure 5.. Conditioned Place Preference (CPP) for Cocaine.

A. Plasma corticosterone levels basally and following 3-min and 5-min FSS in offspring born to 1% (white, n=3/3, 4/4, and 3/3, for basal, 3-min FFS, 5-min FFS, respectively, where first and second numbers indicate the number of animals and dams, respectively) and 4 % (black, n=3/3, 6/6, and 5/5 for basal, 3-min FFS, 5-min FFS) NaCl fed dams. B. Basal locomotion measured as photobeam breaks in a 30-min open field test of offspring born to 1% (white, n=6/6) and 4% NaCl (black, n=6/6) fed dams. C. Experimental timeline for cocaine CPP. Animals were habituated to the 3-chambered CPP apparatus for 15 minutes on day 1 (H). The following day initial preference (IP) was determined by how much time the animal spent in each chamber. Test 1 (T1) occurred 24 hr after the last training session and was followed by an extinction period. Test 2 (T2) occurred 24 hr after a 3-min forced swim stress (FSS). D. Time spent on cocaine-paired side during IP, T1, Last extinction, and T2 in 1% (white, n=9/4) and 4% (black, n=11/5) NaCl fed adult offspring. E. Locomotor behavior during the 3-min FSS for 1% (white) and 4% (black) fed offspring expressed as time mobile and immobile. Values represent the mean ± SEM. *p<0.05 indicates group difference. #p<0.05 indicates that both groups displayed CPP for cocaine, as measured by a significant increase in time spent in the cocaine-paired chamber from IP to T1.

Figure 6.. Sucrose Preference Test.

A. Experimental Timeline. Animals were acclimated to having 2 bottles in their cage for 24 hr. During the next two days, they were given ad libitum access to two bottles, one containing 2% sucrose dissolved in H2O, one containing tap water. Consumption of both solutions was measured. B. Consumption of 2% sucrose solution (left) and water (right) for 1% (white, n=4/4, where first and second numbers indicate the number of animals and dams, respectively) and 4% (black, n=4/4) NaCl fed offspring.