α4βδ-GABAARs in the hippocampal CA1 as a biomarker for resilience to activity-based anorexia

Abstract

Anorexia nervosa (AN) is a psychiatric illness characterized by restricted eating and an intense fear of gaining weight. Most individuals with AN are females, diagnosed first during adolescence, 40-80% of whom exhibit excessive exercise, and an equally high number with a history of anxiety disorder. We sought to determine the cellular basis for individual differences in AN vulnerability by using an animal model, activity-based anorexia (ABA), that is induced by combining food restriction (FR) with access to a running wheel that allows voluntary exercise. Previously, we showed that by the fourth day of FR, the ABA group of adolescent female rats exhibit >500% greater levels of non-synaptic α4βδ-GABAARs at the plasma membrane of hippocampal CA1 pyramidal cell spines, relative to the levels found in age-matched controls that are not FR and without wheel access. Here, we show that the ABA group exhibits individual differences in body weight loss, with some losing nearly 30%, while others lose only 15%. The individual differences in weight loss are ascribable to individual differences in wheel activity that both precedes and concurs with days of FR. Moreover, the increase in activity during FR correlates strongly and negatively with α4βδ-GABAAR levels (R=-0.9, p<0.01). This negative correlation is evident within 2days of FR, before body weight loss approaches life-threatening levels for any individual. These findings suggest that increased shunting inhibition by α4βδ-GABAARs in spines of CA1 pyramidal neurons may participate in the protection against the ABA-inducing environmental factors of severe weight loss by suppressing excitability of the CA1 pyramidal neurons which, in turn, is related indirectly to suppression of excessive exercise. The data also indicate that, although exercise has many health benefits, it can be maladaptive to individuals with low levels of α4βδ-GABAARs in the CA1, particularly when combined with FR.

Keywords: adolescence; alpha4-GABA-receptor; anxiety; hippocampus; neuromodulation; stress.

Figure 1. GABA_AR α4 subunits in the hippocampal CA1 at P42 occur along the extracellular surface of the plasma membrane and intracellularly

Panel A. Spine s1 protruding from a dendritic shaft (curved arrow) exhibits a thick postsynaptic density (PSD), indicating that it is postsynaptic to a terminal (t) forming an excitatory synapse. This spine exhibits two silver-intensified gold particles (SIG) along the cytoplasmic surface of the PSD and one near a mitochondrion within the parent dendritic shaft (sh). Another dendritic shaft in the near vicinity contains five SIG particles but no SIG particle within the spine (s2) emanating from the shaft (curved arrow). White arrows in this and other panels highlight SIG particles that occur intracellularly. Panel B. The spine that is postsynaptic to the terminal exhibits an SIG particle along the extracellular surface of the plasma membrane (black arrow). Panel C. SIG immunolabeling was analyzed along the extreme surface of the vibratome section, as is indicated by the broken membranes and disappearance of the neuropil at the transition from the section surface to the pure EPON zone (asterisks here and in other panels). Spine s1 exhibits a single cluster of SIG particles that lies directly over the plasma membrane, while spine s2 exhibits no SIG that is within 10 nm from the extracellular surface. The asterisk in this and other panels depict the transition from tissue to vibratome section surface. Panel D. Two spines, s1 and s2, are postsynaptic to a terminal, of which s2 exhibits an SIG particle along the intracellular surface of the plasma membrane. A third spine, s3, exhibits two SIG particles along its plasma membrane. Panel E. Of the three spines, s3 is the only one that exhibits an SIG particle that resides directly over the plasma membrane. The calibration bar in panel E indicates 500 nm and applies to all panels.

Figure 2. α4-immunoreactivity in the hippocampus of ABA animals at P44 correlates negatively with wheel activity

Panel A. Membranous SIG correlates with the extent of rats’ activity during the entire experimental period, spanning from the days before and through food restriction. The correlation is negative - those animals that exhibit the greatest elevation in α4 immunoreactivity run the least. Panel B. Membranous SIG also correlates with the last day of food restriction. Panel C. Total SIG level (membranous or intracellular) correlates negatively with the hyperactivity evoked by food restriction during the latter two days of the food restriction. Panel D. The intracellular portion of the SIG correlates with the food restriction-evoked hyperactivity during the last two out of the four days of food restriction. Panel E. The membranous portion of SIG counts correlates with wheel activity before food restriction was imposed. Panel F. Total SIG level correlates negatively and most strongly with hyperactivity during the last day of food restriction. Panel G. Weight loss correlates strongly and positively with the increase in wheel running activity that is evoked by food restriction. Panel H. Weight loss also correlates strongly but negatively with the intracellular level of α4 expression. Those individuals that fail to up-regulate α4 levels intracellularly lose the most amount of body weight. In all panels, the grey arrow indicates the average level of α4 measured within or on the plasma membrane of CA1 dendritic spines of the P44 CON group. The y-axes of the three graphs in panels C, D and F are drawn to the same scale so as to facilitate comparison of values.

Figure 3. Body weight and exercise of ABA animals, relative to age-matched controls

Panel A. The body weight was measured daily and compared for the 8 ABA, 7 EX, 8 FR, and 8 CON animals. Panel B. The daily activity of 8 ABA and 7 EX animals was measured as the distance run on the wheel. Animals were acclimated to the wheel starting the age of postnatal day 37. The red line along the x-axis in panels A and B indicate the dates of food restriction for the ABA and FR groups. For both graphs, the values represent mean ± SEM. ** indicates p<0.001, comparing ABA to CON in panel A and ABA to EX in panel B.

Figure 4. α4-immunoreactivity in the hippocampus of ABA animals at P42 is negatively correlated to wheel-activity

The food restriction-evoked increase in wheel activity was quantified as the distance run during the 2 days after food restriction began minus the distance run during the 2 days preceding food restriction. All animals exercised more after food restriction. Panel A. The extent of increase in activity after food restriction correlates strongly with the body weight lost following food restriction. Each data point in Panel A and all other panels represent the values of one animal’s food restriction-evoked hyperactivity during the 2 days of food restriction, correlated with the percent of body weight lost during the same 2 days. Panel B. α4-immunoreactivity within spines was quantified by counting all silver-intensified immunogold particles (SIG) that occur along the extracellular surface of the plasma membrane and intracellularly (i.e., Total count) for every group of 10 spines encountered. Repeated measures of this SIG count per 10 spines correlates negatively with the hyperactivity that is evoked by food restriction. Each point represents data from one animal. Here and in other panels, α4 immunoreactivity of tissue from 7 CON animals are shown for comparison with the values of ABA animals. Panel C. Total SIG count correlates less strongly with the percent of weight lost during the 2 days of food restriction. Panel D. The proportion of spines exhibiting α4-immunoreactivity for every group of 10 spines encountered also correlates strongly with the hyperactivity evoked by food restriction. Panel E: The counts of SIG particles that occur specifically at the extracellular surface of spines’ plasma membranes correlates with the total level of activity during the 2 days that follows food restriction. Panel F. SIG at the spine membrane correlates only weakly with the extent of hyperactivity after food restriction. Panel G. SIG levels that reside in the cytoplasm correlate very strongly with the food restriction-evoked hyperactivity.

Figure 5. Summary of results

This schematic summarizes the α4-immunoreactivity levels observed at the plasma membrane and intracellularly within dendritic spines of the CA1 pyramidal cells across two stages of ABA induction, P42 and P44. The spine head shows a PSD, where excitatory axons target. The shafts to which the spines are connected are depicted by the presence of a mitochondrion. The pattern of immunoreactivity correlated negatively with the animal’s activity on the wheel. The spines filled with red, green and blue colors depict the α4-immunoreactivity observed within brains of animals that exhibited the greatest (red lines in graphs), intermediate (green lines in graphs) and lowest (blue lines in graphs) activity on the wheel, respectively, following ABA induction. Each grey dot represents the level roughly equal to 0.5 SIG particle per 10 spines. The activities (in km per day) are actual values recorded from 6 representative animals. ABA induced de novo synthesis of α4 subunits but the increased expression among the P42 group was not yet statistically significantly different from those of CON animals (depicted with a colorless cytoplasm) that never received food restriction or the wheel access. α4-immunoreactivity within spines of the P42 dendrites is in flux, as is indicated by the black arrows. By P44, following four days of food restriction, there was a significant increase in the level of α4, relative to age-matched controls. Based on the consistent correlation between activity and α4-immunoreactivity, it is likely that those individuals that exhibited the least activity (the Blue group) already expressed higher levels of α4 before ABA induction and were able to respond to food restriction with the greatest increase in α4 expression. The response in α4-immunoreactivity was more rapid for the intracellular domain than for the membrane domain. This spatio-temporal pattern is depicted by individual differences within the cytoplasmic domain at P42, followed by an increase in the membranous domain by P44.

Figure 6. The hypothesized sequence of events that link up-regulation of α4βδ-GABA_ARs to suppression of food restriction-evoked hyperactivity

It has long been recognized that food restriction evokes a paradoxical increase of wheel running upon rodents (Routtenberg and Kuzneof, 1967; Barbarich-Marsteller et al., 2013; Gutierrez, 2013) and multiple plausible explanations have been offered as to why food-restricted rodents run (reviewed in Gutierrez, 2013). Some of the explanations are that running is a motivated foraging behavior, manifestation of an excessively activated reward system, or is a thermoregulatory behavior. The present study supports a complementary view – namely, that wheel activity is a manifestation of heightened anxiety. This idea is supported by the following findings: (1) wheel running correlates with α4βδ-GABA_ARs expression at CA1 spines (data presented in this paper) and GABAergic input upon CA1 pyramidal cells (Chowdhury et al., 2013); (2) Increased expression of α4βδ-GABA_ARs at CA1 spines reduces excitability of CA1 pyramidal cells (Shen et al., 2007; Shen et al., 2010); and (3) reduction of hippocampal excitability is anxiolytic (Huttunen and Myers, 1986; Kataoka et al., 1991; Talaenko, 1993). This view is further supported by our preliminary observation, indicating that hyperactivity correlates positively with anxiety traits, as measured by the behavior of mice on the elevated plus maze after a day of food restriction (Gauri Wable, Jung-yun Min and Chiye Aoki, unpublished observations). This schematic also summarizes the possible mechanisms leading to the emergence of α4βδ-GABA_ARs at spines of the CA1 pyramidal cells. One likely possibility is that food restriction causes BDNF release (Lee et al., 2002) which, in turn, augments the synthesis of α4 and δ subunits (Roberts et al., 2006). Stress associated with food restriction may also lead to the elevation of allopregnanolone, another agent that potently increases the expression of α4 and δ subunits of GABA_ARs (Smith et al., 1998, Smith et al., 2007). This working hypothesis can be tested by determining whether ABA vulnerability can be reduced through experimental manipulations that up-regulate α4 and δ subunit expressions.