Influence of the gut microbiome on appetite-regulating neuropeptides in the hypothalamus: Insight from conventional, antibiotic-treated, and germ-free mouse models of anorexia nervosa.

Recent research highlights the profound impact of the gut microbiome on neuropsychiatric disorders, shedding light on its potential role in shaping human behavior. In this study, we investigate the role of the gut microbiome in appetite regulation using activity-based anorexia (ABA) mouse model of anorexia nervosa (AN) - a severe eating disorder with significant health consequences. ABA was induced in conventional, antibiotic-treated, and germ-free mice. Our results show the clear influence of the gut microbiome on the expression of four orexigenic (neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, and orexin) and four anorexigenic peptides (cocaine- and amphetamine-regulated transcript, corticotropin-releasing hormone, thyrotropin-releasing hormone, and pro-opiomelanocortin) in the hypothalamus. Additionally, we assessed alterations in gut barrier permeability. While variations were noted in germ-free mice based on feeding and activity, they were not directly attributable to the gut microbiome. This research emphasizes that the gut microbiome is a pivotal factor in AN's appetite regulation beyond just dietary habits or physical activity.

A transdiagnostic and translational framework for delineating the neuronal mechanisms of compulsive exercise in anorexia nervosa.

OBJECTIVE: The development of novel treatments for anorexia nervosa (AN) requires a detailed understanding of the biological underpinnings of specific, commonly occurring symptoms, including compulsive exercise. There is considerable bio-behavioral overlap between AN and obsessive-compulsive disorder (OCD), therefore it is plausible that similar mechanisms underlie compulsive behavior in both populations. While the association between these conditions is widely acknowledged, defining the shared mechanisms for compulsive behavior in AN and OCD requires a novel approach. METHODS: We present an argument that a better understanding of the neurobiological mechanisms that underpin compulsive exercise in AN can be achieved in two critical ways. First, by applying a framework of the neuronal control of OCD to exercise behavior in AN, and second, by taking better advantage of the activity-based anorexia (ABA) rodent model to directly test this framework in the context of feeding pathology. RESULTS: A cross-disciplinary approach that spans preclinical, neuroimaging, and clinical research as well as compulsive neurocircuitry and behavior can advance our understanding of when, why, and how compulsive exercise develops in the context of AN and provide targets for novel treatment strategies. DISCUSSION: In this article, we (i) link the expression of compulsive behavior in AN and OCD via a transition between goal-directed and habitual behavior, (ii) present disrupted cortico-striatal circuitry as a key substrate for the development of compulsive behavior in both conditions, and (iii) highlight the utility of the ABA rodent model to better understand the mechanisms of compulsive behavior relevant to AN. PUBLIC SIGNIFICANCE: Individuals with AN who exercise compulsively are at risk of worse health outcomes and have poorer responses to standard treatments. However, when, why, and how compulsive exercise develops in AN remains inadequately understood. Identifying whether the neural circuitry underlying compulsive behavior in OCD also controls hyperactivity in the activity-based anorexia model will aid in the development of novel eating disorder treatment strategies for this high-risk population.

Three daily intraperitoneal injections of sub-anesthetic ketamine ameliorate activity-based anorexia vulnerability of adult female mice.

OBJECTIVE: To identify ketamine's dosing schedule that ameliorates voluntary food restriction, hyperactivity and body weight loss of adult mice undergoing activity-based anorexia (ABA), an animal model of anorexia nervosa. METHOD: Female and male C57BL6 mice underwent three cycles of ABA, starting from mid-adolescence. ABA vulnerability was compared within and across two groups of animals: those injected intraperitoneally with 30 mg/kg ketamine for three consecutive days (30mgKetx3) during the second ABA in late adolescence (ABA2) or with vehicle only (Vx3). RESULTS: Vx3 females and males exhibited individual differences in wheel running and weight retention during first ABA in mid-adolescence (ABA1), ABA2, and third ABA in adulthood (ABA3). Their wheel running correlated with anxiety-like behavior. During ABA1 and ABA3, weight gain of Vx3 females (but not males) after food consumption correlated negatively with food-anticipatory activity (FAA) preceding the feeding hours, indicating that females with higher levels of running restrict feeding more and persistently. This paradoxical relationship confirms earlier findings of ABA females without ketamine treatment, capturing the maladaptive behaviors exhibited by individuals diagnosed with anorexia nervosa. By contrast, 30mgKetx3 had an effect on both sexes of reducing hyperactivity during the feeding hours acutely and reducing anxiety-like behavior's contribution to running. For females, only, 30mgKetx3 acutely improved the extent of compensatory food consumption relative to FAA and improved weight retention during ABA3, 12 days post ketamine in adulthood. DISCUSSION: Sub-anesthetic ketamine evokes behavior-specific ameliorative effects for adult mice re-experiencing ABA, supporting the notion that multiple doses of ketamine may be helpful in reducing relapse among adults with anorexia nervosa. PUBLIC SIGNIFICANCE STATEMENT: This study examined whether ketamine reduces anorexia-like behaviors in adult mice. Three daily sub-anesthetic ketamine injections suppress wheel running during and leading up to the hours of food availability and enable animals to compensate better for weight loss associated with excessive exercise by eating more. These findings suggest that ketamine may help adult females diagnosed with anorexia nervosa but also point to sex- and age-related differences in the action of ketamine.

Mutual facilitation between activity-based anorexia and schedule-induced polydipsia in rats.

The objective of this study was to evaluate the possible relationship between drinking (licks) in the schedule-induced polydipsia (SIP) phenomenon and running (turns in the wheel) in the activity-based anorexia (ABA) one. Within-subjects counterbalanced experiments were designed with male Wistar rats which underwent both behavioral procedures; half of them performed the ABA procedure first and the other half the SIP procedure first. In Experiment 1, the initial development of ABA facilitated the subsequent acquisition of SIP, whereas the first acquisition of SIP retarded the subsequent development of ABA. Given that SIP exposure implied food restriction, it could be that adaptation to the food regime contributed to lowering ABA manifestation. Thus, Experiment 2 was carried out in exactly the same way as Experiment 1, with the exception that animals which first went through SIP prior to undergoing the ABA procedure had no food restriction. In this case, both ABA and SIP as first experiences facilitated the further development of SIP and ABA, respectively. This suggests that running in ABA may be functionally similar to drinking in SIP; therefore, both behaviors can be thought of as induced by the schedule/regime of intermittent food availability.

Gut Microbiota and Brain Alterations after Refeeding in a Translational Anorexia Nervosa Rat Model.

The gut microbiota composition is causally involved in the regulation of body weight. Through the gut-brain axis, microbiota play a role in psychiatric disorders including anorexia nervosa (AN). Previously, we showed microbiome changes to be associated with brain volume and astrocyte reductions after chronic starvation in an AN animal model. Here, we analyzed whether these alterations are reversible after refeeding. The activity-based anorexia (ABA) model is a well-established animal model that mimics several symptoms of AN. Fecal samples and the brain were analyzed. Like previous results, significant alterations in the microbiome were observed after starvation. After refeeding, including the normalization of food intake and body weight, α- and ß-diversity, as well as the relative abundance of specific genera, were largely normalized in starved rats. Brain parameters appeared to normalize alongside microbial restitution with some aberrations in the white matter. We confirmed our previous findings of microbial dysbiosis during starvation and showed a high degree of reversibility. Thus, microbiome alterations in the ABA model appear to be mostly starvation-related. These findings support the usefulness of the ABA model in investigating starvation-induced effects on the microbiota-gut-brain axis to help comprehend the pathomechanisms of AN and potentially develop microbiome-targeted treatments for patients.

Expanded insights into the neural component of the activity-based anorexia animal model - morphological changes in the enteric nervous system and altered pain perception.

Anorexia nervosa (AN) is an eating disorder characterized by distinct etiopathogenetic concepts that are gradually being linked together to unravel the dominant pathophysiological pathways underlying the disease. Excessive food restrictions, often accompanied by over-exercise and undertaken to lose weight, lead to the development of numerous complications. The biological concept of neurohormonal dysfunction in AN seems incomplete without demonstrating or excluding the role of the enteric nervous system (ENS). Using an animal model of activity-based anorexia (ABA), we conducted the preliminary assessment of the ENS structure. Here we show, in preparations stained by immunohistochemistry with anti- ChAT, anti-NOS, anti-PGP 9.5, anti-c-fos, and anti-TH antibodies, a lower density of cholinergic and nitrergic nerve fibers as well as reduced neuronal activity in myenteric plexus. Such structural and functional damage to the ENS may be responsible for a number of gastrointestinal symptoms that worsen the course of the disease. In addition, we expanded the study to address the unresolved issue of mechanical and thermal pain sensitivity in AN. The Von Frey and hot plate tests revealed, that in ABA animals, the pain threshold for mechanical stimulus decreases while for thermal increases. In this way, we have significantly supplemented the background of AN with potentially observable nervous system changes which may influence the evolution of the therapeutic approach in the future.

Cortical reorganization of the glutamate synapse in the activity-based anorexia rat model: Impact on cognition.

Patients suffering from anorexia nervosa (AN) display altered neural activity, morphological, and functional connectivity in the fronto-striatal circuit. In addition, hypoglutamatergic transmission and aberrant excitability of the medial prefrontal cortex (mPFC) observed in AN patients might underpin cognitive deficits that fuel the vicious cycle of dieting behavior. To provide a molecular mechanism, we employed the activity-based anorexia (ABA) rat model, which combines the two hallmarks of AN (i.e., caloric restriction and intense physical exercise), to evaluate structural remodeling together with alterations in the glutamatergic signaling in the mPFC and their impact on temporal memory, as measured by the temporal order object recognition (TOOR) test. Our data indicate that the combination of caloric restriction and intense physical exercise altered the homeostasis of the glutamate synapse and reduced spine density in the mPFC. These events, paralleled by an impairment in recency discrimination in the TOOR test, are associated with the ABA endophenotype. Of note, after a 7-day recovery period, body weight was recovered and the mPFC structure normalized but ABA rats still exhibited reduced post-synaptic stability of AMPA and NMDA glutamate receptors associated with cognitive dysfunction. Taken together, these data suggest that the combination of reduced food intake and hyperactivity affects the homeostasis of the excitatory synapse in the mPFC contributing to maintain the aberrant behaviors observed in AN patients. Our findings, by identifying novel potential targets of AN, may contribute to more effectively direct the therapeutic interventions to ameliorate, at least, the cognitive effects of this psychopathology.

Rethinking the Approach to Preclinical Models of Anorexia Nervosa.

PURPOSE OF REVIEW: The goal of this review is to describe how emerging technological developments in pre-clinical animal research can be harnessed to accelerate research in anorexia nervosa (AN). RECENT FINDINGS: The activity-based anorexia (ABA) paradigm, the best characterized animal model of AN, combines restricted feeding, excessive exercise, and weight loss. A growing body of evidence supports the idea that pathophysiological weight loss in this model is due to cognitive inflexibility, a clinical feature of AN. Targeted manipulations that recapitulate brain changes reported in AN - hyperdopaminergia or hyperactivity of cortical inputs to the nucleus accumbens - exacerbate weight loss in the ABA paradigm, providing the first evidence of causality. The power of preclinical research lies in the ability to assess the consequences of targeted manipulations of neuronal circuits that have been implicated in clinical research. Additional paradigms are needed to capture other features of AN that are not seen in ABA.

Effects of adolescent experience of food restriction and exercise on spatial learning and open field exploration of female rats.

The hippocampus carries out multiple functions: spatial cognition dorsally (DH) and regulation of emotionality-driven behavior ventrally (VH). Previously, we showed that dendrites of DH and VH pyramidal neurons of female rats are still developing robustly during adolescence and are altered by the experience of food restriction and voluntary exercise on a wheel. We tested whether such anatomical changes during adolescence impact anxiety-like behavior and spatial cognition. Four groups of female rats were evaluated for these behaviors: those with wheel access in its cage from postnatal day (P) 36-44 (EX); those with food access restricted to 1 hr per day, from P40 to 44 (FR); those with EX from P36 to 44, combined with FR from P40 to 44, which we will refer to as EX + FR; and controls, CON (no EX, no FR). Open field test for anxiety-like behavior and active place avoidance test for spatial cognition were conducted at P47-49, the age when food restricted animals have restored body weight, or at P54-56, to identify more enduring effects. Anxiety-like behavior was elevated for the EX and FR groups at P47-49 but not for the EX + FR group. By P54-56, the EX + FR and EX groups exhibited less anxiety-like behavior, indicating a beneficial delayed main effect of exercise. There was a beneficial main effect of food restriction upon cognition, as the FR group showed cognition superior to CONs' at P44-46 and P54-56, while the EX + FR animals also showed enhanced spatial learning at P54-56. EX + FR animals with best adaptation to the feeding schedule showed the best spatial learning performance but with a delay. The EX group exhibited only a transient improvement. These findings indicate that FR, EX, and EX + FR in mid-adolescence are all beneficial in reducing anxiety-like behavior and improving spatial cognition but with subtle differences in the timing of their manifestation, possibly reflecting the protracted maturation of the hippocampus.

Differential effects of heat in the phases of the light-dark cycle in the activity-based anorexia model.

BACKGROUND: This research builds on the studies on ambient temperature as a key influence in the recovery of rodents exposed to the activity-based anorexia (ABA) model. The ABA model is an experimental paradigm in which rodents under a restricted feeding schedule and with free access to an activity wheel show signs that parallel those of anorexia nervosa in humans. OBJECTIVE: The present study focuses on the effects of applying heat during the different phases of the dark-light cycle in the activity levels, body weight, food intake, body temperature, and recovery rates of 30 male rats submitted to ABA. METHOD: After reaching a 20% weight loss criterion, animals were randomly assigned to three experimental conditions: (a) continuous warming, (b) warming exclusively during the light phase, or (c) warming exclusively during the dark phase. RESULTS: Differential effects were found depending on the modalities of warming: in comparison with either light or dark warming, continuous warming significatively decreased activity, facilitated weight gain, and maintained body temperature. Transient effects of warming were found both in the groups warmed either during light or dark periods exclusively. DISCUSSION: The results suggest that both light and dark warming did not promote recovery in animals exposed to ABA. Evidence about the beneficial effects of continuous warming are in line with previous research and reinforces adding external heat as a useful tool in the treatment of anorexia nervosa.

Sexually dimorphic behavioral effects of maternal separation in anorexic rats.

Exposure to negative events during the neonatal period is one of the leading factors contributing to the development of psychiatric disorders, including anorexia nervosa. In this study, we investigated the effects of maternal separation (MS) on the development of anorexia in rodents using the mild-stress form of the activity-based anorexia (ABA) model (2 hr of free access to a running wheel and a 1-hr feeding test) in both male and female rats. We assessed anxiety-like and locomotor behavior and hyperactivity with the open field and elevated plus maze tests. Our results showed that ABA rats of both sexes displayed hyperactive behavior associated with reduced anxiety-like behavior when compared to controls. However, a sexually dimorphic effect of MS emerged in anorexic rats: while the females exposed to MS + ABA were hyperactive with diminished anxiety-related behaviors compared to females of the ABA group, MS in males attenuated or did not alter the effects of the ABA protocol. In conclusion, our data reveal that the synergistic effects of MS and ABA on physical activity and anxiety-like behavior act in opposite directions in the two sexes.

Impaired brain endocannabinoid tone in the activity-based model of anorexia nervosa.

OBJECTIVE: Despite the growing knowledge on the functional relationship between an altered endocannabinoid (eCB) system and development of anorexia nervosa (AN), to date no studies have investigated the central eCB tone in the activity-based anorexia (ABA) model that reproduces key aspects of human AN. METHOD: We measured levels of two major eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), those of two eCB-related lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the cannabinoid type-1 receptor (CB1R) density in the brain of female ABA rats, focusing on areas involved in homeostatic and rewarding-related regulation of feeding behavior (i.e., prefrontal cortex, nucleus accumbens, caudato putamen, amygdala, hippocampus and hypothalamus). Analysis was carried out also at the end of recovery from the ABA condition. RESULTS: At the end of the ABA induction phase, 2-AG was significantly decreased in ABA rats in different brain areas but not in the caudato putamen. No changes were detected in AEA levels in any region, whereas the levels of OEA and PEA were decreased exclusively in the hippocampus and hypothalamus. Furthermore, CB1R density was decreased in the dentate gyrus of hippocampus and in the lateral hypothalamus. After recovery, both 2-AG levels and CB1R density were partially normalized in some areas. In contrast, AEA levels became markedly reduced in all the analyzed areas. DISCUSSION: These data demonstrate an altered brain eCB tone in ABA rats, further supporting the involvement of an impaired eCB system in AN pathophysiology that may contribute to the maintenance of some symptomatic aspects of the disease.

Colonic Mucosal Proteome Signature Reveals Reduced Energy Metabolism and Protein Synthesis but Activated Autophagy during Anorexia-Induced Malnutrition in Mice.

Anorexia nervosa is an eating disorder often associated with intestinal disorders. To explore the underlying mechanisms of these disorders, the colonic proteome was evaluated during activity-based anorexia. Female C57Bl/6 mice were randomized into three groups: Control, Limited Food Access (LFA) and Activity-Based Anorexia (ABA). LFA and ABA mice had a progressive limited access to food but only ABA mice had access to an activity wheel. On colonic mucosal protein extracts, a 2D PAGE-based comparative proteomic analysis was then performed and differentially expressed proteins were identified by LC-ESI-MS/MS. Twenty-seven nonredundant proteins that were differentially expressed between Control, LFA, and ABA groups were identified. ABA mice exhibited alteration of several mitochondrial proteins involved in energy metabolism such as dihydrolipoyl dehydrogenase and 3-mercaptopyruvate sulfurtransferase. In addition, a downregulation of mammalian target of rapamycin (mTOR) pathway was observed leading, on the one hand, to the inhibition of protein synthesis, evaluated by puromycin incorporation and mediated by the increased phosphorylation of eukaryotic elongation factor 2, and on the other hand, to the activation of autophagy, assessed by the increase of the marker of autophagy, form LC3-phosphatidylethanolamine conjugate/Cytosolic form of Microtubule-associated protein 1A/1B light chain 3 (LC3II/LC3I) ratio. Colonic mucosal proteome is altered during ABA suggesting a downregulation of energy metabolism. A decrease of protein synthesis and an activation of autophagy were also observed mediated by mTOR pathway.

Exposure to activity-based anorexia impairs contextual learning in weight-restored rats without affecting spatial learning, taste, anxiety, or dietary-fat preference.

UNLABELLED: Relapse rates are high amongst cases of anorexia nervosa (AN) suggesting that some alterations induced by AN may remain after weight restoration. OBJECTIVE: To study the consequences of AN without confounds of environmental variability, a rodent model of activity-based anorexia (ABA) can be employed. We hypothesized that exposure to ABA during adolescence may have long-term consequences in taste function, cognition, and anxiety-like behavior after weight restoration. METHODS: To test this hypothesis, we exposed adolescent female rats to ABA (1.5 h food access, combined with voluntary running wheel access) and compared their behavior to that of control rats after weight restoration was achieved. The rats were tested for learning/memory, anxiety, food preference, and taste in a set of behavioral tests performed during the light period. RESULTS: Our data show that ABA exposure leads to reduced performance during the novel object recognition task, a test for contextual learning, without altering performance in the novel place recognition task or the Barnes maze, both tasks that test spatial learning. Furthermore, we do not observe alterations in unconditioned lick responses to sucrose nor quinine (described by humans as "sweet" and "bitter," respectively). Nor Do we find alterations in anxiety-like behavior during an elevated plus maze or an open field test. Finally, preference for a diet high in fat is not altered. DISCUSSION: Overall, our data suggest that ABA exposure during adolescence impairs contextual learning in adulthood without altering spatial leaning, taste, anxiety, or fat preference.

Anorexia-Induced Hypoleptinemia Drives Adaptations in the JAK2/STAT3 Pathway in the Ventral and Dorsal Hippocampus of Female Rats.

Leptin is an appetite-regulating adipokine that is reduced in patients with anorexia nervosa (AN), a psychiatric disorder characterized by self-imposed starvation, and has been linked to hyperactivity, a hallmark of AN. However, it remains unknown how leptin receptor (LepR) and its JAK2-STAT3 downstream pathway in extrahypothalamic brain areas, such as the dorsal (dHip) and ventral (vHip) hippocampus, crucial for spatial memory and emotion regulation, may contribute to the maintenance of AN behaviors. Taking advantage of the activity-based anorexia (ABA) model (i.e., the combination of food restriction and physical activity), we observed reduced leptin plasma levels in adolescent female ABA rats at the acute phase of the disorder [post-natal day (PND) 42], while the levels increased over control levels following a 7-day recovery period (PND49). The analysis of the intracellular leptin pathway revealed that ABA rats showed an overall decrease of the LepR/JAK2/STAT3 signaling in dHip at both time points, while in vHip we observed a transition from hypo- (PND42) to hyperactivation (PND49) of the pathway. These changes might add knowledge on starvation-induced fluctuations in leptin levels and in hippocampal leptin signaling as initial drivers of the transition from adaptative mechanisms to starvation toward the maintenance of aberrant behaviors typical of AN patients, such as perpetuating restraint over eating.

Taking better advantage of the activity-based anorexia model.

The lack of specific treatments for anorexia nervosa (AN) is partly driven by an inadequate understanding of the neurobiological drivers of the condition. The activity-based anorexia (ABA) model recapitulates key characteristics of AN in rats and mice, and can be used to understand factors that predispose, maintain, and rescue anorectic behaviour. With the rapidly evolving suite of technologies to manipulate and record neural activity during the development of ABA, we are better placed than ever before to take advantage of this unique biobehavioural model in order to develop and refine novel treatments for AN. This will require a collective effort to bridge research disciplines in order to capitalise on knowledge gains from genetics, neurobiology, metabolism, and cognition.

Adolescent activity-based anorexia has a substantial and prolonged impact on social behavior in young adult female rats.

Activity-based anorexia (ABA) is a rodent model of anorexia nervosa (AN) that induces several key components of AN, including voluntary reduction in food intake, reduced body weight, hyperactivity, and alterations to the hypothalamic-pituitary-adrenal (HPA) axis. Previous research has demonstrated persistently increased anxiety-like behavior in the elevated plus maze (EPM), a test measuring avoidance of novel and open areas in adult female rats that experienced ABA during adolescence and are weight-restored in adulthood. Whether the same behavioral effects of two bouts of adolescent ABA emerge in response to different anxiety-provoking stimuli, however, has not been explored. We used the social partition (SP), novelty suppressed feeding (NSF), marble burying, and EPM tests to explore whether two bouts of adolescent ABA have persistent effects on anxiety-like behavior in weight restored young adult female rats. One-way ANOVA analyses revealed that female rats that experienced two bouts of ABA during adolescence had increased anxiety-like behavior in the EPM and SP tests in young adulthood following weight restoration compared with controls. These data demonstrate that the enduring behavioral effects of two bouts of adolescent ABA are specific to particular anxiety-provoking stimuli and suggest that adolescent ABA has enduring effects on social relationships.

Rethinking Therapeutic Strategies for Anorexia Nervosa: Insights From Psychedelic Medicine and Animal Models.

Anorexia nervosa (AN) has the highest mortality rate of any psychiatric disease, yet available pharmacological treatments are largely ineffective due, in part, to an inadequate understanding of the neurobiological drivers that underpin the condition. The recent resurgence of research into the clinical applications of psychedelic medicine for a range of mental disorders has highlighted the potential for classical psychedelics, including psilocybin, to alleviate symptoms of AN that relate to serotonergic signaling and cognitive inflexibility. Clinical trials using psychedelics in treatment-resistant depression have shown promising outcomes, although these studies are unable to circumvent some methodological biases. The first clinical trial to use psilocybin in patients with AN commenced in 2019, necessitating a better understanding of the neurobiological mechanisms through which psychedelics act. Animal models are beneficial in this respect, allowing for detailed scrutiny of brain function and behavior and the potential to study pharmacology without the confounds of expectancy and bias that are impossible to control for in patient populations. We argue that studies investigating the neurobiological effects of psychedelics in animal models, including the activity-based anorexia (ABA) rodent model, are particularly important to inform clinical applications, including the subpopulations of patients that may benefit most from psychedelic medicine. Appeared originally in Front Neurosci 2020; 14:43.

Ketamine ameliorates activity-based anorexia of adolescent female mice through changes in GluN2B-containing NMDA receptors at postsynaptic cytoplasmic locations of pyramidal neurons and interneurons of medial prefrontal cortex.

Anorexia nervosa (AN) is a mental illness with high rates of mortality and relapse, and no approved pharmacotherapy. Using the activity-based anorexia (ABA) model of AN, we previously showed that a single sub-anesthetic intraperitoneal injection of ketamine (30 mg/kg-KET, but not 3 mg/kg-KET), has an immediate and long-lasting effect of reducing anorexia-like behavior among adolescent female mice. We also showed previously that excitatory outflow from medial prefrontal cortex (mPFC) engages hunger-evoked hyperactivity, leading to the ABA condition of severe weight loss. Ketamine is known to target GluN2B-containing NMDARs (NR2B). Might synaptic plasticity involving NR2B in mPFC contribute to ketamine's ameliorative effects? We addressed this question through electron microscopic immunocytochemical quantification of GluN2B at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (IN) in mPFC layer 1 of animals that underwent recovery from a second ABA induction (ABA2), 22 days after ketamine injection during the first ABA induction. The 30 mg/kg-KET evoked synaptic plasticity that differed for PN and IN, with changes revolving the cytoplasmic reserve pool of NR2B more than the postsynaptic membrane pool. Those individuals that suppressed hunger-evoked wheel running the most and increased food consumption during recovery from ABA2 the most showed the greatest increase of NR2B at PN and IN excitatory synapses. We hypothesize that 30 mg/kg-KET promotes long-lasting changes in the reserve cytoplasmic pool of NR2B that enables activity-dependent rapid strengthening of mPFC circuits underlying the more adaptive behavior of suppressed running and enhanced food consumption, in turn supporting better weight restoration.

Development of activity-based anorexia requires PKC-δ neurons in two central extended amygdala nuclei.

Anorexia nervosa (AN) is a serious psychiatric disease, but the neural mechanisms underlying its development are unclear. A subpopulation of amygdala neurons, marked by expression of protein kinase C-delta (PKC-δ), has previously been shown to regulate diverse anorexigenic signals. Here, we demonstrate that these neurons regulate development of activity-based anorexia (ABA), a common animal model for AN. PKC-δ neurons are located in two nuclei of the central extended amygdala (EAc): the central nucleus (CeA) and oval region of the bed nucleus of the stria terminalis (ovBNST). Simultaneous ablation of CeAPKC-δ and ovBNSTPKC-δ neurons prevents ABA, but ablating PKC-δ neurons in the CeA or ovBNST alone is not sufficient. Correspondingly, PKC-δ neurons in both nuclei show increased activity with ABA development. Our study shows how neurons in the amygdala regulate ABA by impacting both feeding and wheel activity behaviors and support a complex heterogeneous etiology of AN.