NR2A- and NR2B-NMDA receptors and drebrin within postsynaptic spines of the hippocampus correlate with hunger-evoked exercise

Abstract

Hunger evokes foraging. This innate response can be quantified as voluntary wheel running following food restriction (FR). Paradoxically, imposing severe FR evokes voluntary FR, as some animals choose to run rather than eat, even during limited periods of food availability. This phenomenon, called activity-based anorexia (ABA), has been used to identify brain changes associated with FR and excessive exercise (EX), two core symptoms of anorexia nervosa (AN), and to explore neurobiological bases of AN vulnerability. Previously, we showed a strong positive correlation between suppression of FR-evoked hyperactivity, i.e., ABA resilience, and levels of extra-synaptic GABA receptors in stratum radiatum (SR) of hippocampal CA1. Here, we tested for the converse: whether animals with enhanced expression of NMDA receptors (NMDARs) exhibit greater levels of FR-evoked hyperactivity, i.e., ABA vulnerability. Four groups of animals were assessed for NMDAR levels at CA1 spines: (1) ABA, in which 4 days of FR was combined with wheel access to allow voluntary EX; (2) FR only; (3) EX only; and (4) control (CON) that experienced neither EX nor FR. Electron microscopy revealed that synaptic NR2A-NMDARs and NR2B-NMDARs levels are significantly elevated, relative to CONs'. Individuals' ABA severity, based on weight loss, correlated with synaptic NR2B-NMDAR levels. ABA resilience, quantified as suppression of hyperactivity, correlated strongly with reserve pools of NR2A-NMDARs in spine cytoplasm. NR2A- and NR2B-NMDAR measurements correlated with spinous prevalence of an F-actin binding protein, drebrin, suggesting that drebrin enables insertion of NR2B-NMDAR to and retention of NR2A-NMDARs away from synaptic membranes, together influencing ABA vulnerability.

Keywords: Anorexia nervosa; Eating disorder; Exercise; Food restriction; Foraging; Receptor trafficking.

Fig. 1

Body weight and wheel activity of CON, ABA, FR, and EX groups. a Timeline of the environmental conditions of the four experimental groups. Animals were food restricted (FR), given ad libitum access to a running wheel to allow for voluntary exercise (EX), induced to undergo activity-based anorexia (ABA) by combining wheel access and food restriction (ABA), or served as controls (CON) that neither exercised nor were food restricted. All animals were shipped to NYU's animal facility at postnatal day (P) 28. Wheel access for the ABA and the EX group began on experimental day 1 when animals were P35 or 36. Food restriction for the ABA and the FR groups began on experimental day 1 (FR1), which corresponded to the fifth experimental day when animals were P39 or 40. All animals were euthanized by transcardial perfusion under anesthesia on P43/44. b Body weights of all groups are shown from ED1 through ED8, as mean ± SEM. Hash indicates a main effect of food restriction with p < 0.05. There was no significant interaction between the effect of food restriction and wheel access. The dashed line along the x-axis indicates the food-restricted period, beginning on ED4. c Average voluntary running wheel activity is displayed for the EX and ABA groups for the last 6 of the 8 days of wheel access. Asterisk indicates significant difference between EX and ABA at p < 0.05. d, e Average weight of the FR and ABA groups across the first four experimental days (ED1 through ED4) is compared to the average weight across the last four food-restricted days (ED5 through ED8). Each gray line represents data from a single animal, while the heavier line represents the group average as mean ± SD. f, g The average running activity per 24 h during the first 4 days of wheel access was compared to the average activity during the last 4 days of wheel access in the EX and the ABA groups. Each gray line represents a single subject, while the heavier lines represent the group average as mean ± SD. In the ABA group, the latter 4 days of wheel access were accompanied by food restriction. 1 wheel count equaled 0.64 m

Fig. 2

Categorization of subcellular sites in the vicinity of axo-spinous asymmetric synapses where NR2A and NR2B immunoreactivities were observed. a through e are examples of electron micrographs taken from tissue immunolabeled for the NR2A subunits, while f, g are examples of micrographs taken from tissue immunolabeled for the NR2B subunits. a–d, f are micrographs taken from EX tissue, while e, g were taken from ABA tissue. The numbers depict PEG particles associated with axo-spinous asymmetric synapses. The presynaptic sides are indicated by t for axon terminal. Calibration bar is equal to 200 nm and applies to all panels. The cartoon in the center describes the categorization of subcellular location of PEG particles. The subcellular position for each particle in these examples were categorized as follows: 1 at PSD; 2 at extra-synaptic spine plasma membrane, 3 and 4 near PSD, 5 as cytoplasmic on the presynaptic side, 6 and 7 at extra-synaptic spine plasma membrane, 8 at cleft, 9 at/near presynaptic plasma membrane, 10 (two particles) as cytoplasmic within the postsynaptic spine, 11 near PSD, 12 at cleft, 13 at/near presynaptic axon terminal, 14 at/near presynaptic plasma membrane, 15 near PSD, and 16 at extra-synaptic spine plasma membrane

Fig. 3

ABA elevates both NR2A and NR2B immunoreactivity specifically at the postsynaptic membrane. The histograms depict group comparisons of the number of PEG particles encountered per-10 synapses, at and near axo-spinous junctions, reflecting NR2A (a) and NR2B (b) immunoreactivitynormalized to the average of the CON values. Asterisk depicts significance of difference at p < 0.05 by two-way ANOVA, followed by Fisher's LSD post hoc analysis. The original, pre-normalized CON values for NR2A and NR2B immunoreactivity are shown in Table 2

Fig. 4

Correlations between ABA vulnerability and NR2A immunoreactivity. Pearson correlation analyses were run to determine the relationship between ABA vulnerability and the level of NR2A-PEG particle labeling. a, b ABA vulnerability was measured as the extent of body weight change, following 4 days of food restriction. c, d ABA vulnerability was measured as the average daily running during the 4 days of food restriction. e ABA vulnerability was measured as the weight change during the last day of food restriction, on ED 8. a, c Significant correlations between ABA vulnerability and NR2A immunoreactivity within the spine cytoplasm. b, d, e Lack of correlation between postsynaptic NR2A and ABA vulnerability. All values were normalized to a single value derived from an average of all CON tissue that were immunolabeled by the PEG procedure in parallel with the experimental group (ABA, FR, or EX). The shape of the unfilled symbols indicates the experimental group with which the CON tissue runs. R values of the Pearson correlation are indicated for each graph. Asterisk indicates p < 0.05, and double asterisk indicates p < 0.01. Solid lines represent trend lines for correlations with p < 0.05, while dashed lines represent trend lines for correlations with p > 0.05 but ≤0.1

Fig. 5

Correlations between ABA vulnerability and NR2B immunoreactivity. Pearson correlation analyses were run to determine the relationship between ABA vulnerability and the level of NR2B-PEG particle labeling, just as was conducted for NR2A immunolabeling. a, b ABA vulnerability was measured as the extent of body weight change during the four days of food restriction. c, d ABA vulnerability was measured as the average daily running during the four days of food restriction. e ABA vulnerability was measured as the weight change during the last day of food restriction, on ED 8. The only measure of vulnerability that shows correlation to NR2B immunoreactivity is the extent of weight change on ED 8 (e). All values were normalized to a single value derived from an average of all CON tissue that were immunolabeled by the PEG procedure in parallel with the experimental group (ABA, FR or EX). The shape of the unfilled symbols indicates the experimental group with which the CON tissue runs. f Outcome of the Pearson correlation analysis comparing normalized cytoplasmic NR2A to normalized postsynaptic NR2B. R values of the Pearson correlation are indicated for each graph. Asterisk indicates p < 0.05; solid lines represent trend lines for correlations with p < 0.05; and hash and dashed lines indicate p > 0.05 but ≤0.1

Fig. 6

Correlations between drebrin at PSDs and NMDAR subunits at locations associated with ABA vulnerability. a (from an ABA animal) and b (from a CON animal) show examples of drebrin immunoreactivity at the PSD. The mitochondrial profile is indicated by m1 and m2 in the panels. The levels of drebrin immnuoreactivity were categorized to be the following for the 12 PSDs shown in the two panels. a 1,2,3, and 6 are intensely labeled, 4 is moderately labeled, and 5 and 7 are unlabeled. b 1 is intensely labeled, 4 is moderately labeled, and 2, 3 and 5 are unlabeled. Calibration bar equals 500 nm and applies to both panels. c shows intensity (gray value) difference between PSD and mitochondria normalized to the gray value of the mitochondria for the three categories of drebrin-immunolabeled PSDs. Mean gray values were sampled from 11 micrographs of ABA tissue (from 3 ABA animals) and 10 micrographs of CON tissue (from 3 CON animals). Mean gray values of PSDs categorized as intensely labeled were significantly greater than values of PSDs categorized as unlabeled or moderately labeled. Asterisk indicates significance (p < 0.05). d Quantification of the proportion of intensely drebrin-immunolabeled PSDs for ABA and CON groups. 768 and 384 spines were sampled from eight ABA and four CON tissues, respectively. e Normalized cytoplasmic NR2A labeling levels of the 8 ABA animals (the same as the values shown in Fig. 4a and c) are negatively correlated to the proportion of spines immunolabeled intensely over the PSD. This correlation is significant (p < 0.05). f shows that normalized postsynaptic NR2B labeling (the same as those shown in Fig. 5b and e) of the eight ABA animals is related to the proportion of spines immunolabeled intensely over the PSD. This correlation approached significance (p = 0.08), and is indicated by a dashed line

Fig. 7

Summary of the levels of NR2A-containing NMDARs, NR2B-containing NMDARs, and drebrin at postsynaptic versus cytoplasmic locations reported for spines belonging to hippocampi of ABA-vulnerable and ABA-resilient individuals. The strongly correlating pools of synaptic proteins are shown as putatively interacting through direct contacts. The correlations suggest that the inactive pool of cytoplasmic NR2A level is actively retained from becoming inserted into the PSD through interaction with drebrin, and that drebrin interacts relatively more strongly with NR2B-containing NMDARs at the PSD than with NR2A-containing NMDARs