The Severity of Acute Stress Is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons

Abstract

The hypothalamo-pituitary-adrenocortical (HPA) axis regulates stress physiology and behavior. To achieve an optimally tuned adaptive response, it is critical that the magnitude of the stress response matches the severity of the threat. Corticotropin-releasing hormone (CRH) released from the paraventricular nucleus of the hypothalamus is a major regulator of the HPA axis. However, how CRH-producing neurons in an intact animal respond to different stressor intensities is currently not known. Using two-photon calcium imaging on intact larval zebrafish, we recorded the activity of CRH cells, while the larvae were exposed to stressors of varying intensity. By combining behavioral and physiological measures, we first determined how sudden alterations in environmental conditions lead to different levels of stress axis activation. Then, we measured changes in the frequency and amplitude of Ca 2ϩ transients in individual CRH neurons in response to such stressors. The response magnitude of individual CRH cells covaried with stressor intensity. Furthermore, stressors caused the recruitment of previously inactive CRH neurons in an intensity-dependent manner, thus increasing the pool of responsive CRH cells. Strikingly, stressor-induced activity appeared highly synchronized among CRH neurons, and also across hemispheres. Thus, the stressor strength-dependent output of CRH neurons emerges by a dual mechanism that involves both the increased activity of individual cells and the recruitment of a larger pool of responsive cells. The synchronicity of CRH neurons within and across hemispheres ensures that the overall output of the HPA axis matches the severity of the threat.

Convergence on reduced stress behavior in the Mexican blind cavefish

“…Various stimuli such as predators, osmotic stress, and thermal alterations induce behavioral measures of stress, and lead to reduced exploration, prolonged immobility or freezing, and reduced social interaction (Facchin et al, 2015; Levin et al, 2007; Mateo, 2007; Ryu and De Marco, 2017; vom Berg-Maurer et al, 2016; Yeh et al, 2013). In fish, several behavioral assays exist for measuring stress in adults.…”

Performance on innate behaviour during early development as a function of stress level

“…The larvae’s neurosecretory preoptic-hypothalamic area (NPO) is homologous to the mammalian hypothalamic paraventricular nucleus, innervation of the pituitary by NPO cells is established by 3 days post fertilization, and ablation and activation of cortisol-producing interrenal cells reduces and increases cortisol upon stress, respectively 18 – 20 . Two-photon calcium imaging on intact larvae shows that the activity of NPO-cells producing corticotropin-releasing hormone, the primary stimulator of the HPA axis, is highly synchronized and co-varies with stressor intensity 21 . Interactions of GRs and serotonin signalling are present too 22 , and larvae show elaborate stress reactions 6 , 21 , 23 – 25 , including a reversible suppression of the feeding drive 13 , a common phenomenon among vertebrates also found in adult teleosts 26 , 27 .…”

“…Two-photon calcium imaging on intact larvae shows that the activity of NPO-cells producing corticotropin-releasing hormone, the primary stimulator of the HPA axis, is highly synchronized and co-varies with stressor intensity 21 . Interactions of GRs and serotonin signalling are present too 22 , and larvae show elaborate stress reactions 6 , 21 , 23 – 25 , including a reversible suppression of the feeding drive 13 , a common phenomenon among vertebrates also found in adult teleosts 26 , 27 . Furthermore, evidence also shows that serotonergic neurons in the dorsal raphe can modulate sensory responsiveness 28 as well as light/dark preference 29 , depending on their activity level.…”

“…Groups of thirty larvae in 30 mm Petri dishes were exposed to known stress protocols 6 , 11 – 13 , 21 , each based on one of four different stimulations: HCl (pH drop), NaCl (hyperosmotic medium), strong hydrodynamic flows or a squared pulse of light. They were then used for cortisol measurement or transferred to custom-made swimming chambers for behavioural testing.…”

An ecotoxicological view on neurotoxicity assessment

“…In this respect, model organisms such as the zebrafish are very helpful, because a large variety of genetic tools and genomic resources are available and many tests are already established for the analysis of human brain disorders [98], but they are not yet fully adopted for neurotoxicity testing. Additionally, emerging neuroscience techniques such as in vivo 2-photon calcium-imaging of neuronal activity [201–203] or optogenetics [204] might hold underexplored opportunities for the mechanistic dissection of complex neurotoxicological processes. Moreover, large-scale toxicity screenings using the zebrafish model has been implemented in the framework of ToxCast and Tox21 [205–208], but more efforts are needed to increase the specificity of tests for sensory neurotoxicity in larval zebrafish and implement them in a high-throughput manner in order to keep pace with toxicity testing of the vast number of newly registered chemicals.…”