SUMMARYSerotonergic systems play important roles in modulating stress-induced arousal and vigilance behaviours. The pond snail, Lymnaea, shows multiple defensive vigilance behaviours in response to the stress associated with predator detection. Predator detection elicited by crayfish effluent (CE), increases the time to re-emerge from the shell and enhances the shadow withdrawal response. More importantly, in Lymnaea, CE enhances the ability to form long-term memory (LTM). We investigated the role of the serotonergic system in these anti-predator responses in Lymnaea. Using a serotonin-receptor antagonist, mianserin, we found that two defensive vigilance behaviours (e.g. increasing the time to re-emerge from their shell and shadow response) elicited by CE were not observed when the serotonergic system was disrupted. Also, methysergide, another serotonin antagonist, blocked the enhanced LTM formation after training in CE. Importantly, mianserin did not alter LTM formation in pond water (PW). These data suggest that a serotonergic system is activated only when Lymnaea detect a predator. When snails were trained in CE using a training procedure that in PW produces a 24-h LTM, a more persistent form of LTM (5days) occurred. This more persistent form of LTM was abolished after mianserin treatment. Increasing 5-HT levels in the snail by the injection of 5-HT was also associated with enhanced LTM formation. Lastly, we tested whether the osphradium is implicated in CE detection and subsequent enhanced formation of LTM. Cutting the osphradial nerve to the CNS resulted in the loss of the ability to form enhanced LTM in CE. Together, these findings support the hypothesis that the serotonergic system plays a key role in modulating the predator-induced stress responses in Lymnaea.

Neurotrophic factors (NTFs) support neuronal survival, differentiation, and even synaptic plasticity both during development and throughout the life of an organism. However, their precise roles in central synapse formation remain unknown. Previously, we demonstrated that excitatory synapse formation in Lymnaea stagnalis requires a source of extrinsic NTFs and receptor tyrosine kinase (RTK) activation. Here we show that NTFs such as Lymnaea epidermal growth factor (L-EGF) act through RTKs to trigger a specific subset of intracellular signalling events in the postsynaptic neuron, which lead to the activation of the tumor suppressor menin, encoded by Lymnaea MEN1 (L-MEN1) and the expression of excitatory nicotinic acetylcholine receptors (nAChRs). We provide direct evidence that the activation of the MAPK/ERK cascade is required for the expression of nAChRs, and subsequent synapse formation between pairs of neurons in vitro. Furthermore, we show that L-menin activation is sufficient for the expression of postsynaptic excitatory nAChRs and subsequent synapse formation in media devoid of NTFs. By extending our findings in situ, we reveal the necessity of EGFRs in mediating synapse formation between a single transplanted neuron and its intact presynaptic partner. Moreover, deficits in excitatory synapse formation following EGFR knock-down can be rescued by injecting synthetic L-MEN1 mRNA in the intact central nervous system. Taken together, this study provides the first direct evidence that NTFs functioning via RTKs activate the MEN1 gene, which appears sufficient to regulate synapse formation between central neurons. Our study also offers a novel developmental role for menin beyond tumour suppression in adult humans.

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Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.

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We conclude that the neuroprotective effect of Epo prevents oxidative damage in the brain and cardiorespiratory disorders induced by CIH. Considering that Epo is used in clinics to treat chronic kidney disease and stroke, our data show convincing evidence suggesting that Epo may be a promising alternative drug to treat sleep-disorder breathing.

Since the evolution of aerobic metabolism, cellular requirements for molecular oxygen have been the major driver for the development of sophisticated mechanisms underlying both invertebrate and vertebrate respiratory behaviour. Among the most important characteristics of respiration is its adaptability, which allows animals to maintain oxygen homeostasis over a wide range of environmental and metabolic conditions. In all animals, the respiratory behaviour is controlled by neural networks often termed respiratory central pattern generators (rCPG). While rCPG neurons are intrinsically capable of generating rhythmical outputs, the respiratory needs are generally "sensed" by either central or peripheral chemoreceptive neurons. The mechanisms by which chemoreceptors respond to changes in oxygen and modulate central respiratory control centers have been the focus of decades of research. However, our understanding of these mechanisms has been limited due to an inability to precisely locate oxygen chemoreceptor populations, combined with the overwhelming complexity of vertebrate neural circuits. Although mammalian models remain the gold standard for research in general, invertebrates do nevertheless offer greatly simplified neural networks that share fundamental similarities with vertebrates. The following review will provide evidence for the existence of oxygen chemoreceptors in many invertebrate groups and reveal the mechanisms by which these neurons may "perceive" environmental oxygen and drive central rCPG activity. For this, we will specifically highlight an invertebrate model, the pond snail Lymnaea stagnalis whose episodic respiratory behaviour resembles that of diving mammals. The rCPG neurons have been identified and fully characterized in this model both in vivo and in vitro. The Lymnaea respiratory network has also been reconstructed in vitro and the contributions of individual rCPG neurons towards rhythm generation characterized through direct intracellular recordings. We now provide evidence for the presence of genuine peripheral oxygen chemoreceptors in Lymnaea, and demonstrate that these neurons respond to hypoxia in a manner analogous to that of mammalian carotid bodies. These chemoreceptor cells not only drive the activity of the rCPG neurons but their synaptic connections also exhibit hypoxia-induced plasticity. The lessons learned from this model will likely reveal fundamental principles underlying both peripheral and central respiratory control mechanisms, which may be conserved in both invertebrate and vertebrate species.

Respiratory behaviour relies critically upon sensory feedback from peripheral oxygen chemoreceptors. During environmental or systemic hypoxia, chemoreceptor input modulates respiratory central pattern generator activity to produce reflex-based increases in respiration and also shapes respiratory plasticity over longer timescales. The best-studied oxygen chemoreceptors are undoubtedly the mammalian carotid bodies; however, questions remain regarding this complex organ's role in shaping respiration in response to varying oxygen levels. Furthermore, many taxa possess distinct oxygen chemoreceptors located within the lungs, airways and cardiovasculature, but the functional advantage of multiple chemoreceptor sites is unclear. In this study, it is demonstrated that a distributed network of peripheral oxygen chemoreceptors exists in Lymnaea stagnalis and significantly modulates aerial respiration. Specifically, Lymnaea breath frequency and duration represent parameters that are shaped by interactions between hypoxic severity and its time-course. Using a combination of behaviour and electrophysiology approaches, the chemosensory pathways underlying hypoxia-induced changes in breath frequency/duration were explored. The current findings demonstrate that breath frequency is uniquely modulated by the known osphradial ganglion oxygen chemoreceptors during moderate hypoxia, while a newly discovered area of pneumostome oxygen chemoreception serves a similar function specifically during more severe hypoxia. Together, these findings suggest that multiple oxygen chemosensory sites, each with their own sensory and modulatory properties, act synergistically to form a functionally distributed network that dynamically shapes respiration in response to changing systemic or environmental oxygen levels. These distributed networks may represent an evolutionarily conserved strategy vis-à-vis respiratory adaptability and have significant implications for the understanding of fundamental respiratory control systems.