Activity-based anorexia (ABA) is a widely used animal model for identifying the biological basis of excessive exercise and starvation, two hallmarks of anorexia nervosa (AN). Anxiety is correlated with exercise in AN. Yet the anxiety level of animals in ABA has not been reported. We asked: Does food restriction as part of ABA induction change the anxiety level of animals? If so, is the degree of anxiety correlated with degree of hyperactivity? We used the open field test before food restriction and the elevated plus maze test (EPM) during food restriction to quantify anxiety among singly housed adolescent female mice and determined whether food restriction alone or combined with exercise (i.e., ABA induction) abates or increases anxiety. We show that food restriction, with or without exercise, reduced anxiety significantly, as measured by the proportion of entries into the open arms of EPM (35.73 %, p= .04). Moreover, ABA-induced individuals varied in their open arm time measure of anxiety and this value was highly and negatively correlated to the individual’s food restriction-evoked wheel activity during the 24 hours following the anxiety test (R = − .75, p= .004, N = 12). This correlation was absent among the exercise-only controls. Additionally, mice with higher increase in anxiety ran more following food restriction. Our data suggest that food restriction-evoked wheel running hyperactivity can be used as a reliable and continuous measure of anxiety in ABA. The parallel relationship between anxiety level and activity in AN and ABA-induced female mice strengthens the animal model.

Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation and often linked with excessive exercise. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction (FR). Although certain rodents have been used successfully in this animal model, C57BL/6 mice are reported to be less susceptible to ABA. We re-examined the possibility that female C57BL/6 mice might exhibit ABA vulnerability during adolescence, the developmental stage/sex among the human population with particularly high AN vulnerability. After introducing the running wheel to the cage for three days, ABA was induced by restricting food access to 1 hour per day (ABA1, N=13) or 2 hours per day (ABA2, N=10). All 23 exhibited increased voluntary wheel running (p<0.005) and perturbed circadian rhythm within two days. Only one out of five survived ABA1 for three days, while ten out of ten survived ABA2 for three days and could subsequently restore their body weight and circadian rhythm. Exposure of recovered animals to a second ABA2 induction revealed a large range of vulnerability, even within littermates. To look for the cellular substrate of differences in vulnerability, we began by examining synaptic patterns in the hippocampus, a brain region that regulates anxiety as well as plasticity throughout life. Quantitative EM analysis revealed that CA1 pyramidal cells of animals vulnerable to the second ABA2 exhibit less GABAergic innervation on cell bodies and dendrites, relative to the animals resilient to the second ABA (p<0.001) or controls (p<0.05). These findings reveal that C57BL/6J adolescent females can be used to capture brain changes underlying ABA vulnerability, and that GABAergic innervation of hippocampal pyramidal neurons is one important cellular substrate to consider for understanding the progression of and resilience to AN.

Anorexia nervosa is a mental illness that emerges primarily during early adolescence, with mortality rate that is 200 times higher than that of suicide. The illness is characterized by intense fear of gaining weight, heightened anxiety, obstinate food restriction, often accompanied by excessive exercise, in spite of mounting hunger. The illness affects females nine times more often than males, suggesting an endocrine role in its etiology. Its relapse rate exceeds 25%, yet there are no accepted pharmacological treatments to prevent this. Here, I summarize studies from this laboratory that have used adolescent female rodents in activity-based anorexia (ABA), an animal model of anorexia nervosa, with the goal of identifying neurobiological underpinnings of this disease. We put forth a hypothesis that a GABAergic mechanism within the hippocampus is central to regulating an individual’s anxiety which, in turn, strongly influences the individual’s resilience/vulnerability to ABA. In particular, we propose that ionotropic GABAA receptors containing the subunits alpha4 and delta, are at play for exerting shunting inhibition upon hippocampal pyramidal neurons that become more excitable during ABA. Since these receptors confer insensitivity to benzodiazepines, this pharmacological profile of ABA fits with lack of report indicating efficacy of benzodiazepines in reducing the anxiety experienced by individuals with anorexia nervosa. The idea that the GABAergic system of the hippocampus regulates resilience/vulnerability to anorexia nervosa complements current opinions about the important roles of the prefrontal cortex, amygdala, striatum, gustatory pathways and feeding centers of the hypothalamus and of the neuromodulators, serotonin and dopamine, in the etiology of the disease.

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% to 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 4th 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 2 days 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.

Many, but not all, adolescent female mice that are exposed to a running wheel while food restricted (FR) become excessive wheel runners, choosing to run even during the hours of food availability, to the point of death. This phenomenon is called activity-based anorexia (ABA). We used electron microscopic immunocytochemistry to ask whether individual differences in ABA resilience may correlate with the lengths of axo-somatic contacts made by GABAergic axon terminals onto layer 5 pyramidal neurons (L5P) in the prefrontal cortex. Contact lengths were, on average, 40% greater for the ABA-induced mice, relative to controls. Correspondingly, the proportion of L5P perikaryal plasma membrane contacted by GABAergic terminals was 45% greater for the ABA mice. Contact lengths in the anterior cingulate cortex correlated negatively and strongly with the overall wheel activity after FR (R = -0.87, P < 0.01), whereas those in the prelimbic cortex correlated negatively with wheel running specifically during the hours of food availability of the FR days (R = -0.84, P < 0.05). These negative correlations support the idea that increases in the glutamic acid decarboxylase (GAD) terminal contact lengths onto L5P contribute toward ABA resilience through suppression of wheel running, a behavior that is intrinsically rewarding and helpful for foraging but maladaptive within a cage.

Adolescent females are particularly vulnerable to mental illnesses with comorbidity of anxiety, such as anorexia nervosa (AN). We used an animal model of AN, called activity-based anorexia (ABA), to investigate the neurobiological basis of vulnerability to repeated, food restriction (FR) stress-evoked anxiety. Twenty-one of 23 adolescent female mice responded to the 1st FR with increased wheel running activity (WRA), even during the limited period of food access, thereby capturing AN's symptoms of voluntary FR and over-exercise. Baseline WRA was an excellent predictor of FR-elicited WRA (severity of ABA, SOA), with high baseline-runners responding to FR with minimal SOA (i.e., negative correlation). Nine gained resistance to ABA following the 1st FR. Even though allopregnanolone (3α-OH-5α-pregnan-20-one, THP), the metabolite of progesterone (P4), is a well-recognized anxiolytic agent, subcutaneous P4 to these ABA-resistant animals during the 2nd FR was exacerbative, evoking greater WRA than the counterpart resistant group that received oil vehicle, only. Moreover, P4 had no WRA-reducing effect on animals that remained ABA-vulnerable. To explain the sensitizing effect of P4 upon the resistant mice, we examined the relationship between P4 treatment and levels of the α4 subunit of GABAARs at spines of pyramidal cells of the hippocampal CA1, a parameter previously shown to correlate with resistance to ABA. α4 levels at spine membrane correlated strongly and negatively with SOA during the 1st ABA (prior to P4 injection), confirming previous findings. α4 expression levels were greater among P4-treated animals that had gained resistance than of vehicle-treated resistant animals or of the vulnerable animals with or without P4. We propose that α4-GABAARs play a protective role by counterbalancing the ABA-induced increase in excitability of CA1 pyramidal neurons, and although exogenous P4's metabolite, THP, enhances α4 expression, especially among those that can gain resistance, it also interferes with α4-GABAARs’ protective role by desensitizing α4-GABAARs.

Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4-containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma −2.8 mm) or caudal (Bregma −3.8 mm) portion of the basal amygdala. Sholl analysis of golgi-stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal-like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals’ anxiety.

The vermis or “spinocerebellum” receives input from the spinal cord and motor cortex for controlling balance and locomotion, while the longitudinal hemisphere region or “cerebro-cerebellum” is interconnected with non-motor cortical regions, including the prefrontal cortex that underlies decision-making. Noradrenaline release in the cerebellum is known to be important for motor plasticity but less is known about plasticity of the cerebellar noradrenergic (NA) system, itself. We characterized plasticity of dopamine β-hydroxylase-immunoreactive NA fibers in the cerebellum of adolescent female rats that are evoked by voluntary wheel running, food restriction (FR) or by both, in combination. When 8 days of wheel access was combined with FR during the last 4 days, some responded with excessive exercise, choosing to run even during the hours of food access: this exacerbated weight loss beyond that due to FR alone. In the vermis, exercise, with or without FR, shortened the inter-varicosity intervals and increased varicosity density along NA fibers, while excessive exercise, due to FR, also shortened NA fibers. In contrast, the hemisphere required the FR-evoked excessive exercise to evoke shortened inter-varicosity intervals along NA fibers and this change was exhibited more strongly by rats that suppressed the FR-evoked excessive exercise, a behavior that minimized weight loss. Presuming that shortened inter-varicosity intervals translate to enhanced NA release and synthesis of norepinephrine, this enhancement in the cerebellar hemisphere may contribute towards protection of individuals from the life-threatening activity-based anorexia via relays with higher-order cortical areas that mediate the animal’s decision to suppress the innate FR-evoked hyperactivity.