Changes in dendritic spine turnover are a major mechanism of experience-dependent plasticity in the adult neocortex. Dendritic spine plasticity may also contribute to functional recovery after stroke, but in that setting its expression may be complicated by alterations in local tissue perfusion, especially around the infarct. Using adult Thy-1 GFP-M mice, we simultaneously recorded long-term spine dynamics in apical dendrites from layer 5 pyramidal cells and blood flow from surrounding capillaries with in vivo two-photon microscopy in peri-infarct cortex before and after unilateral middle cerebral artery occlusion. Blood flow in periinfarct cortex decreased significantly immediately after stroke and improved gradually over time, in a distance-dependent manner from the epicenter of the infarct. However, local tissue perfusion was never fully restored even after a 3 month recovery period. On average, surviving layer 5 pyramidal neurons experienced a ϳ20% decrease in spine density acutely after stroke but eventually recovered. The dynamics of this improvement were different depending on the degree of tissue perfusion acutely after arterial occlusion. Cells in ischemic areas closer to the infarct returned to normal spine density levels slowly by retaining spines, while cells in more remote regions with preserved blood flow recovered faster by adding more spines, eventually surpassing baseline spine density by 15%. Our data suggest that maintaining tissue perfusion in the area surrounding the infarct could hasten or augment synaptic plasticity and functional recovery after stroke.

Activity-based anorexia (ABA) is an animal model for anorexia nervosa that has revealed genetic links to anxiety traits and neurochemical characteristics within the hypothalamus. However, few studies have used this animal model to investigate the biological basis for vulnerability of pubertal and adolescent females to ABA, even though the great majority of the anorexia nervosa cases are females exhibiting the first symptoms during puberty. GABAergic inhibition of the hippocampus strongly regulates anxiety as well as plasticity throughout life. We recently showed that the hippocampal CA1 of female mice undergo a dramatic change at puberty onset – from expressing virtually none of the non-synaptic α4βδ GABAA receptors (GABARs) pre-pubertally to expressing these GABARs at approximately 7% of the CA1 dendritic spine membranes at puberty onset. Furthermore, we showed that this change underlies the enhanced modulation of anxiety, neuronal excitability and NMDA receptor-dependent synaptic plasticity in the hippocampus by the stress neurosteroid, THP (3a-OH-5α[β]-pregnan-20-one or [allo]pregnanolone). Here, we used quantitative electron microscopy to determine whether ABA induction in female rats during adolescence also elevates the expression of α4 and δ subunits of α4βδ GABARs, as was observed at puberty onset for mice. Our analysis revealed that rats also exhibit a rise of α4 and δ subunits of α4βδ GABARs at puberty onset, in that these subunits are detectable at approximately 6% of the dendritic spine membranes of CA1 pyramidal cells at puberty onset (postnatal day 32–36; P32–36) but this drops to about 2% by P40 – P44. The levels of α4 and δ subunits at the CA1 spines remained low following exposure of P40 females to either of the two environmental factors needed to generate ABA - food restriction and access to a running wheel for four days (P40 to P44). This pattern contrasted greatly from those of ABA animals, for which the two environmental factors were combined. Within the hippocampus of ABA animals, 12% of the spine profiles were labeled for α4, reflecting a six-fold increase, relative to hippocampi of age-matched (P44) control females [p<0.005]. Concurrently, 7% of the spine profiles were labeled for δ, reflecting a 130% increase from the control values of 3% (p=0.01). No measurable change was detected for spine size. The observed magnitude of increase in the α4 and δ subunits at spines is sufficient to increase both tonic inhibition of hippocampus and anxiety during stress, thereby likely to exacerbate hyperactivity and weight loss.

Cajal–Retzius (CR) neurons play a critical role in cortical neuronal migration, but their exact fate after the completion of neocortical lamination remains a mystery. Histological evidence has been unable to unequivocally determine whether these cells die or undergo a phenotypic transformation to become resident interneurons of Layer 1 in the adult neocortex. To determine their ultimate fate, we performed chronic in vivo two-photon imaging of identified CR neurons during postnatal development in mice that express the green fluorescent protein (GFP) under the control of the early B-cell factor 2 (Ebf2) promoter. We find that, after birth, virtually all CR neurons in mouse neocortex express Ebf2. Although postnatal CR neurons undergo dramatic morphological transformations, they do not migrate to deeper layers. Instead, their gradual disappearance from the cortex is due to apoptotic death during the second postnatal week. A small fraction of CR neurons present at birth survive into adulthood. We conclude that, in addition to orchestrating cortical layering, a subset of CR neurons must play other roles beyond the third postnatal week.

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 irrational fears of gaining weight. There is no accepted pharmacological treatment for AN, and AN has the highest mortality rate among psychiatric illnesses. Anorexia nervosa most commonly affects females during adolescence, suggesting an effect of sex and hormones on vulnerability to the disease. Activity-based anorexia (ABA) is a rodent model of AN that shares symptoms with AN, including over-exercise, elevation of stress hormones, and genetic links to anxiety traits. We previously reported that ABA in adolescent female rats results in increased apical dendritic branching in CA1 pyramidal cells of the ventral hippocampus at postnatal day 44 (P44). To examine the long-term effects of adolescent ABA (P44) in female rats, we compared the apical branching in the ventral hippocampal CA1 after recovery from ABA (P51) and after a relapse of ABA (P55) with age-matched controls. To examine the age-dependence of the hippocampal plasticity, we examined the effect of ABA during adulthood (P67). We found that while ABA at P44 resulted in increased branching of ventral hippocampal pyramidal cells, relapse of ABA at P55 resulted in decreased branching. Activity-based anorexia induced during adulthood did not have an effect on dendritic branching, suggesting an age-dependence of the vulnerability to structural plasticity. Cells from control animals were found to exhibit a dramatic increase in branching, more than doubling from P44 to P51, followed by pruning from P51 to P55. The proportion of mature spines on dendrites from the P44-ABA animals is similar to that on dendrites from P55-CON animals. These results suggest that the experience of ABA may cause precocious anatomical development of the ventral hippocampus. Importantly, we found that adolescence is a period of continued development of the hippocampus, and increased vulnerability to mental disorders during adolescence may be due to insults during this developmentally critical period.

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Differences in the prevalence and presentation of psychiatric illnesses in men and women suggest that neurobiological sex differences confer vulnerability or resilience in these disorders. Rodent behavioral models are critical for understanding the mechanisms of these differences. Reward processing and punishment avoidance are fundamental dimensions of the symptoms of psychiatric disorders. Here we explored sex differences along these dimensions using multiple and distinct behavioral paradigms. We found no sex difference in reward-guided associative learning but a faster punishment-avoidance learning in females. After learning, females were more sensitive than males to probabilistic punishment but less sensitive when punishment could be avoided with certainty. No sex differences were found in reward-guided cognitive flexibility. Thus, sex differences in goal-directed behaviors emerged selectively when there was an aversive context. These differences were critically sensitive to whether the punishment was certain or unpredictable. Our findings with these new paradigms provide conceptual and practical tools for investigating brain mechanisms that account for sex differences in susceptibility to anxiety and impulsivity. They may also provide insight for understanding the evolution of sex-specific optimal behavioral strategies in dynamic environments.