We wish to thank Mark Evans and Rosie Sammons for help with 3D tracing in Fiji, Annisa Chand for instructions on nose plug manufacture, and Maxim Volgushev for Matlab code. Venki Murthy and all members of the Grubb, Murthy and Galliano laboratories provided helpful discussions, while Juan Burrone and Sue Jones made invaluable comments on the manuscript.

Domoic acid (DA) is one of the best known marine toxins, causative of important neurotoxic alterations. DA effects are documented both in wildlife and experimental assays, showing that this toxin causes severe injuries principally in the hippocampal area. In the present study we have addressed the long-term toxicological effects (30 days) of DA intraperitoneal administration in rats. Different histological techniques were employed in order to study DA toxicity in heart, an organ which has not been thoroughly studied after DA intoxication to date. The presence of DA was detected by immunohistochemical assays, and cellular alterations were observed both by optical and transmission electron microscopy. Although histological staining methods did not provide any observable tissue damage, transmission electron microscopy showed several injuries: a moderate lysis of myofibrils and loss of mitochondrial conformation. This is the first time the association between heart damage and the presence of the toxin has been observed.

In the glomerular layer of the olfactory bulb, local dopaminergic interneurons play a key role in regulating the flow of sensory information from nose to cortex. These dual dopamine‐ and GABA‐releasing cells are capable of marked experience‐dependent changes in the expression of neurotransmitter‐synthesising enzymes, including tyrosine hydroxylase (TH). However, such plasticity has most commonly been studied in cell populations identified by their expression of the enzyme being studied and after long periods of sensory deprivation. Here, instead, we used brief 1‐ or 3‐day manipulations of olfactory experience in juvenile mice, coupled with a conditional genetic approach that labelled neurons contingent upon their expression of the dopamine transporter (DAT‐tdTomato). This enabled us to evaluate the potential for rapid changes in neurotransmitter‐synthesising enzyme expression in an independently identified neuronal population. Our labelling strategy showed good specificity for olfactory bulb dopaminergic neurons, while revealing a minority sub‐population of non‐dopaminergic DAT‐tdTomato cells that expressed the calcium‐binding protein calretinin. Crucially, the proportions of these neuronal subtypes were not affected by brief alterations in sensory experience. Short‐term olfactory manipulations also produced no significant changes in immunofluorescence or whole‐bulb mRNA for the GABA‐synthesising enzyme GAD67/Gad1. However, in bulbar DAT‐tdTomato neurons, brief sensory deprivation was accompanied by a transient, small drop in immunofluorescence for the dopamine‐synthesising enzyme dopa decarboxylase (DDC) and a sustained decrease for TH. Deprivation also produced a sustained decrease in whole‐bulb Th mRNA. Careful characterisation of an independently identified, genetically labelled neuronal population therefore enabled us to uncover rapid experience‐dependent changes in dopamine‐synthesising enzyme expression.

9 10 In the glomerular layer of the olfactory bulb, local dopaminergic interneurons play a key role in 11regulating the flow of sensory information from nose to cortex. These dual dopamine-and GABA-12 releasing cells are capable of marked experience-dependent changes in the expression of 13 neurotransmitter-synthesising enzymes, including tyrosine hydroxylase (TH). However, such 14 plasticity has most commonly been studied in cell populations identified by their expression of the 15 enzyme being studied, and after long periods of sensory deprivation. Here, instead, we used brief 1-16 or 3-day manipulations of olfactory experience in juvenile mice, coupled with a conditional genetic 17 approach that labelled neurons contingent upon their expression of the dopamine transporter . This enabled us to evaluate the potential for faster changes in neurotransmitter-19 synthesising enzyme expression in an independently identified population of neurons. Our labelling 20 strategy showed good specificity for olfactory bulb dopaminergic neurons, whilst also revealing a 21 minority sub-population of non-dopaminergic DAT-tdTomato cells that expressed the calcium-22 binding protein calretinin. Crucially, the proportions of these neuronal subtypes were not affected 23 by brief alterations in sensory experience. Short-term olfactory manipulations also produced no 24 significant changes in immunofluorescence for the GABA-synthesising enzyme GAD67. However, in 25 bulbar DAT-tdTomato neurons brief sensory deprivation was accompanied by a transient drop in 26 immunofluorescence for the dopamine-synthesising enzyme dopa decarboxylase (DDC), and a 27 sustained decrease in TH expression. Careful characterisation of an independently identified, 28 genetically labelled neuronal population therefore enabled us to uncover experience-dependent 29 changes in neurotransmitter-synthesising enzyme expression that are more rapid than previously 30 appreciated. 31 in olfactory bulb TH-positive neurons, without any accompanying change in their density within the 65 glomerular layer (Galliano et al., 2020). 66 A significant caveat is that, to date, these changes in neurotransmitter-synthesising enzyme 67 expression have all been detected either by employing tissue-level analyses that encompass the full 68 range of bulbar cell types, or by using the measured variable itself (e.g. TH immunofluorescence) to 69 identify individual cells to be studied. Ideally, gaining cell-type-specific information regarding 70 plasticity in enzyme expression would instead use an independent marker of cell identity. This 71 would enable experience-dependent changes to be identified amongst a consistently identified 72 group of neurons, without the potential distortion that can result when a plastic variable determines 73 which cells to measure. Here, we therefore took advantage of a conditional mouse transgenic line, 74 DAT IREScre (Bäckman et al., 2006), which when crossed with an appropriate reporter line generates 75 selective fluorescent label in cells that express...

Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.