Using pERK immunostaining to quantify neuronal activity induced by stress in zebrafish larvae.

The larval zebrafish has emerged as a very useful model organism to study the neuronal circuits controlling neuroendocrine and behavioral responses to stress. This protocol describes how to expose zebrafish larvae to hyperosmotic stress and test whether candidate populations of neurons are activated or inhibited by the stressor using a relatively rapid immunofluorescence staining approach. This approach takes advantage of the phosphorylation of the extracellular signal-regulated kinase (ERK) upon neuronal activation. For complete details on the use and execution of this protocol, please refer to Corradi et al. (2022).

Hypothalamic Galanin-producing neurons regulate stress in zebrafish through a peptidergic, self-inhibitory loop.

Animals possess neuronal circuits inducing stress to avoid or cope with threats present in their surroundings, for instance, by promoting behaviors, such as avoidance and escape. However, mechanisms must exist to tightly control responses to stressors, since overactivation of stress circuits is deleterious for the wellbeing of an organism. The underlying neuronal dynamics responsible for controlling behavioral responses to stress have remained unclear. Here, we describe a neuronal circuit in the hypothalamus of zebrafish larvae that inhibits stress-related behaviors and prevents excessive activation of the neuroendocrine pathway hypothalamic-pituitary-interrenal axis. Central components of this circuit are neurons secreting the neuropeptide Galanin, as ablation of these neurons led to abnormally high levels of stress. Surprisingly, we found that Galanin has a self-inhibitory action on Galanin-producing neurons. Our results suggest that hypothalamic Galanin-producing neurons play an important role in fine-tuning stress responses by preventing potentially harmful overactivation of stress-regulating circuits.

Neuromodulation and Behavioral Flexibility in Larval Zebrafish: From Neurotransmitters to Circuits.

Animals adapt their behaviors to their ever-changing needs. Internal states, such as hunger, fear, stress, and arousal are important behavioral modulators controlling the way an organism perceives sensory stimuli and reacts to them. The translucent zebrafish larva is an ideal model organism for studying neuronal circuits regulating brain states, owning to the possibility of easy imaging and manipulating activity of genetically identified neurons while the animal performs stereotyped and well-characterized behaviors. The main neuromodulatory circuits present in mammals can also be found in the larval zebrafish brain, with the advantage that they contain small numbers of neurons. Importantly, imaging and behavioral techniques can be combined with methods for generating targeted genetic modifications to reveal the molecular underpinnings mediating the functions of such circuits. In this review we discuss how studying the larval zebrafish brain has contributed to advance our understanding of circuits and molecular mechanisms regulating neuromodulation and behavioral flexibility.

Early-Life Stress Regulates Cardiac Development through an IL-4-Glucocorticoid Signaling Balance.

Stressful experiences early in life can increase the risk of cardiovascular diseases. However, it remains largely unknown how stress influences susceptibility to the disease onset. Here, we show that exposure to brain-processed stress disrupts myocardial growth by reducing cardiomyocyte mitotic activity. Activation of the glucocorticoid receptor (GR), the primary stress response pathway, reduces cardiomyocyte numbers, disrupts trabecular formation, and leads to contractile dysfunction of the developing myocardium. However, a physiological level of GR signaling is required to prevent cardiomyocyte hyperproliferation. Mechanistically, we identify an antagonistic interaction between the GR and the cytokine interleukin-4 (IL-4) as a key player in cardiac development. IL-4 signals transcription of key regulators of cell-cycle progression in cardiomyocytes via signal transducer and activator of transcription 3 (Stat3). GR, on the contrary, inhibits this signaling system. Thus, our findings uncover an interplay between stress and immune signaling pathways critical to orchestrating physiological growth of the heart.

In Italy, healthy life expectancy drop dramatically: from 2004 to 2008 there was a 10 years drop among newborn girls.

INTRODUCTION: In this short essay, we would like to address a severe divergence observed in Italy between Life Expectancy (LE) and Healthy Life Expectancy (Healthy LE) and a unique trend of worsening in Healthy LE, compared to the other European countries. Both issues emerge in recent data by EUROSTAT Report. METHODS: The analysis used by the authors of the EUROSTAT report is based on Sullivan method which combines 2 type of variables: mortality and morbidity data. RESULTS: While several European countries started to deal with comparable data about LE since 1960, in Italy, analogous data were available for the first time in EUROSTAT Report only in 1985. In Italy, in the period 1985-2008, there was a good progressive increase in L.E., following the best European values. Nevertheless, while until 2004 Italy was among the European best countries in terms of both LE and Healthy LE at birth, four years later in 2008 there was a shocking loss of 10 years of Healthy LE at birth in newborn girls. In the process, they lost their 2-years previous advantage with respect to males (the latter lost only 6 years of Healthy LE, in the same time span). Looking at healthy LE at age 65 in respect to 2004, Italian women in 2008 could expect to live healthy only about 7 years (as much as men) versus the almost 15 years of the European best values (14 years for men). CONCLUSIONS: It is legitimate to wonder why no one official comment has been produced as a reaction after the first year of spectacular decline in Healthy Life Years in Italy: in counter-tendency with European values, from 2004 to 2008 there is a clear evidence of a 10 years drop in Healthy LE among newborn girls. The problem has not been taken into consideration even when the situation clearly appeared to worsen in the following years, dropping 4-6 more years for males and females in 2006 (for newborn babies); two more years of healthy life expectancy have been lost between 2006 and 2007 for each gender. One more year of Healthy Life Expectancy is lost in 2008. And data have not been made available any more, since then, from Italy.