Electrical activity of osphradial neurons of the greater pond snail Lymnaea stagnalis is modulated by the brominated flame retardant 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane

Electrical activity of osphradial neurons of the greater pond snail Lymnaea stagnalis is modulated by the brominated flame retardant 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Electrical activity of osphradial neurons of the greater pond snail Lymnaea stagnalis is modulated by the brominated flame retardant 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane Kaesy Lynne Enns, Gregg T. Tomy, W. Mark Fry This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4631370/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH) is a brominated flame retardant used as a chemical additive in commercial and industrial manufacturing to reduce product flammability. TBECH has previously been shown to be an endocrine disruptor of the gonadal and thyroid axes, however, its neurotoxic effects, including effects on electrical excitability of neurons, are understudied. Therefore, we investigated the potential of TBECH to modulate electrical activity of neurons from the chemosensory osphradial organ of Lymnaea stagnalis using a suction electrode and extracellular recording. Results Application of TBECH caused a variable response in osphradial nerve spike activity, whereby some recordings showed increased action potential firing and some showed decreased firing. This resulted no significant change in mean action potential frequency after TBECH treatment compared to control (n = 6 separate experiments). However, using semi-automated spike sorting analysis to identify individual spike types from each recording revealed that the frequency of some spike types increased and some decreased within each nerve recording, and that TBECH caused significant modulation of activity. These findings indicate that TBECH may represent an acutely neurotoxic environmental contaminant that has potential to interfere with neural signaling in animals. TBECH 1 2-dibromo-4-(1 2 dibromoethyl) cyclohexane brominated flame retardant neurotoxicity osphradium suction electrode extracellular recording action potential Figures Figure 1 Figure 2 Figure 3 Introduction Brominated flame retardants (BFRs) are used in industrial manufacturing to reduce flammability of manufactured goods. Legacy BFRs, including polybrominated diphenyl ethers and biphenyls (PBDE, PBB, TBBPA), and hexabromocyclododecanes (HBCD) have been restricted due to their persistence, bioaccumulation, and toxicity [1]. Approximately 30 novel BFRs have recently emerged and their use is increasing [2–5]. Of note is the BFR 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH), which has been shown to act an endocrine disruptor within the thyroid and gonadal axes [3, 6, for review see 7], an embryotoxic agent [8, 9], and a potential obesogen [10]. A recent study investigated acute neurophysiological effects of TBECH, and demonstrated that TBECH dose-dependently inhibited spontaneous action potentials in cultured rat Purkinje neurons [11]. These data, together with demonstrated bioaccumulation in terrestrial and aquatic animals and environments [12], warrant further investigation into the safety of TBECH’s use [ for review see 7]. Lymnaea stagnalis, the greater pond snail, is used as a model organism for an OECD reproductive toxicicity test [13], and is increasingly used as a model organism for neurobiology and neurotoxicology due to its accessible central nervous system, and emerging genomics [14]. Additional benefits include low cost of care and limited animal care regulations. The osphradium of Lymnaea is of particular interest, as it functions as chemosensory neural integrator of environmental molecules and stressors. Chemosensory neurons are found within the osphradium, and their axons carry sensory information to ganglia within the central nervous system through the short osphradial nerve which merges with right internal parietal nerve [15]. Action potential activity of osphradial neurons is modulated by chemical cues including secretions of predators, hypoxia and pH [16–19]; detection of these soluble molecules plays key roles in physiological functions including feeding, learning, and predator escape. In order to investigate the potential neurotoxicity of the environmental contaminant TBECH, we used a suction electrode to record extracellular action potentials (spikes) from a preparation of isolated osphradium and the emanating osphradial nerve following exposure to TBECH. Methods Lymnaea stagnalis snails were housed in aquatic habitats with a constant, gentle flow of dechlorinated water at 25°C. Their diet consisted of romaine lettuce, supplemented with trout pellets and cuttlebone for adequate calcium uptake. Each snail, 1.1g to 2.0g, was anesthetized with 0.5ml of freshly prepared 1.0M MgCl 2 injected into the foot. The shell was crushed and removed with forceps. The mantle was folded back to reveal the osphradial epithelial canal, just anterior to the pneumostome. Visual inspection confirmed the location of the osphradium on the ventral side of the mantle. An incision was made on the dorsal surface beneath the mantle, while submerging the preparation in artificial extracellular fluid (AECF) containing 4.1mM CaCl 2 , 1.5mM MgCl 2 ·6H 2 O, 5.0 HEPES, 1.7mM KCl, 51.3 NaCl, 5.0mM dextrose, pH 7.9. The osphradial nerve and tissue containing the osphradium were extirpated from the body. The tissue surrounding the osphradium was pinned to a 35mm Sylgard-lined dish equipped with an inlet and outlet permitting solution exchange. The isolated osphradial nerve was fitted to a bipolar suction electrode with a glass capillary bore measuring between 150-200mm in diameter such that the osphradial canal faced the solution inlet. The sample was continuously submerged and perfused with AECF at 1ml/min using a peristaltic pump. Electrical signals were amplified with a Model 1700 Differential AC Amplifier at 1000x gain (A-M Systems, Sequim, WA). Signals were filtered by a 1-500 Hz band pass filter, with a notch filter at 60Hz. Signals were digitized using Axon Instruments Digidata 1322A 50 kHz using Strathclyde Electrophysiology Software WinEDR (V4.0.2). We recorded electrophysiological activity in the presence of AECF alone to establish baseline activity (control). After at least 5 minutes of stable spike recording, 6ml of AECF containing either 7.2mg/ml yeast extract (containing a mix of amino acids and short peptides) (Kamardin et al., 1999; Fisher Scientific), or 100mM TBECH (Bosche Scientific, Rocky Hill NJ) was applied. We continued to record spike activity for at least two 3 minute intervals following application of yeast extract or TBECH. The length of the intervals was chosen such that the rhythmic activity of the osphradium would be accounted for while still observing overall changes in firing activity [16, 17]. After ten minutes of recovery in AECF alone, 6ml of 10mg/ml tricaine methanesulfonate (MS-222; Syndel Canada) was perfused over the osphradium and we recorded spike activity for a further two 3 minute intervals: MS-222 is a nerve blocking agent which antagonizes voltage gated Na+ channels and prevents action potentials in neurons and axons from vertebrates and invertebrates [20]. Raw data were imported to Spike2 6.12 software (CED, Milton England), and spikes were detected by setting a threshold approximately 10% greater than the peak-to-peak noise, at a minimum interval of 0.01s. Change in frequency of detected spikes was plotted as both raw frequencies, and normalized frequency (as the proportion relative to control). Our initial analysis did not reveal significant spike frequency changes after application of yeast extract or TBECH, however by eye we noticed changes in activity of specific spike waveforms. Therefore, we next identified and grouped individual spike types from each recording using the Spike6.12 semi-automated spike sorting routine. The frequency of each spike type with more than 30 spikes over the test period was analyzed separately before and following yeast extract or TBECH application. These changes were expressed as absolute value Log2 change in frequency to provide a linear representation of fold change, regardless of whether spikes were increasing or decreasing in frequency. For statistical testing, differences were considered significant at p<0.05. Results We tested the effect of MS-222 to ensure that the observed spike activity was indeed caused by action potentials in the axons within the isolated osphradial nerve. Application of MS-222 resulted in a rapid increase in mean firing rate in the first 3 minutes, followed by cessation of activity between 3 and 6 minutes (n=7; Figure. 1); the raw firing rate increased from 22.9±5.7 spikes per minute during control period to 57.3±11.0 spikes per minute during the first 3min and decreased to 1.5±0.7 during the 3-6 minutes interval. A repeated measures ANOVA was performed to compare the effect of MS-222 on raw firing raw rate. There was a statistically significant difference in raw firing rate (F = 23.0 (between groups df=1, within groups df=2), p < 0.001). Tukey’s post-hoc revealed firing rate during control period was significantly lower than during the first 3 minutes (p=0.0036), and greater than the rate during the 3-6 minute interval (p<0.001); control rate was not different than the rate during 3-6 minute interval. When firing rates were normalized to frequency observed during the control period (Figure 1A,C), the rates during the first 3 minute and 3-6 minute intervals are significantly different from control (p<0.001 each interval, one-way t-test, with null hypothesis mean =0). We carried out experiments applying 7.2mg/ml of yeast extract over the osphradial epithelium of L. stagnalis as a positive control to demonstrate that stimulation of osphradial neurons by a mixture of amino acids applied to the intact osphradium could be detected by suction electrode recording (Figure 2). We observed a variable and nonsignificant change in raw firing rate (Figure 2B) in both the first 3 minute interval and 3-6 minute interval following yeast extract application (repeated measures ANOVA, F=1.12.0, between groups df=1, within groups df=2; p=0.35). After normalization to control firing rates, the firing rate during the first 3 minute interval and the 3-6 minute interval after application of yeast extract was still not significantly different from the control, likely due to the variability of mean normalized responses (Figure 2C). Visual analysis of raw traces showed spikes of different waveforms followed different patterns of activity (where some spike types increased frequency, whereas some decreased frequency after yeast extract was applied). Therefore we analyzed the changes in activity of individual spike types (Fig. 2A, inset). Identification of the individual spike types from each experiment (n=8) yielded between 11-63 spike types with a total of 161 spike types. Expressing the change in frequency for each of these spike types as Log 2 -fold change revealed that application of yeast caused an increase in frequency of some types and a decrease in frequency of other types. Statistical comparison of the absolute value of the Log 2 fold change (Figure 2D) revealed that yeast extract caused a statistically significant change in frequency of electrical activity of osphradial neurons compared to baseline (regardless of whether the neurons were stimulated or inhibited; one sample t-test, DF=160, p<0.001 for both the first 3 minute interval and the 3-6 minute interval). Perfusion of TBECH to the osphradium also resulted in variable and nonsignificant changes in raw firing rates (Figure 3A). The mean raw firing rate observed under control conditions was 23.2±6.3 during the control period, 24.0±7.4 spikes per minute for the first 3 minutes following TBECH application and 24.1±6.6 spikes per minute for the 3-6 minutes following (Figure 3A; repeated measures ANOVA, F=0.48.0, between groups df=1, within groups df=2, p=0.62). Normalizing firing rate for each nerve tested revealed dramatic increases in firing rate of some nerves, and decreases in others, however there was no statistically significant difference from control firing rates (Figure 3B; one way t-tests, p=0.75). Spike sorting analysis of the 6 osphradial nerves yielded between 11 and 29 spike types for each nerve, for a total of 70 spike types identified. Statistical comparison of the absolute value of the Log 2 fold change revealed that TBECH caused a statistically significant change in frequency of electrical activity of osphradial neurons compared to baseline regardless of whether the neurons were stimulated or inhibited (Figure 3D; one sample t-test with null hypothesis mean =0, DF=69, p<0.001 for both the first 3 minutes and the 3-6 minutes following TBECH application). Discussion Using a relatively simple suction electrode and extracellular electrophysiological amplifier, we have demonstrated that the environmental contaminant TBECH alters action potential activity of the osphradial neurons of Lymnaea stagnalis. In contrast to our previous experiments where we used patch clamp electrophysiology to show that TBECH acutely modulates activity of single mammalian neurons growing in culture [11], here we use a simpler, but robust electrophysiological technique and an acute preparation of invertebrate neurons in a system to test neurotoxicity of this environmental contaminant. Importantly, while we observed clear changes in electrical activity of osphradial neurons, the frequency of some spike types increased and some decreased, making analysis considerably more involved and perhaps not suitable for a high throughput neurotoxicological testing model. Our data demonstrate for the first time the neurotoxicity of TBECH in aquatic animals. This finding is important in evaluating the overall toxicity of TBECH and its potential to harm sensitive ecosystems and human health. These data clearly indicate that further study of neurotoxic properties of TBECH is warranted. Indeed, recent reviews suggest that BFRs, including novel BFRs introduced since phase out of HDCDs and PBDEs, may be linked to human disorders including, neurobehavioral and developmental disorders, alteration in thyroid function and other illness [3, 21]. Because exposure to treatments or chemicals that alter spontaneous electrical activity of developing neurons are well-recognized to result in wide-ranging and long-lasting alteration of normal neuronal development [22], potential effects neurodevelopmental effects of exposure to TBECH should be investigated in detail. The change in spike activity following application of yeast extract included both simultaneous increases and decreases in frequency of different spike types, but this was not unexpected. Other studies have demonstrated at the single cell level that some stimuli excite osphradial neurons and others inhibit activity [16, 17], and therefore this pattern probably represents a level of neural processing for the biologically relevant yeast extract. Similar to the yeast extract, TBECH also caused simultaneous increases and decreases in frequency of different spike types, but the importance of this variable modulation is not clear. While the present data do not suggest a mechanism by which TBECH may alter electrical activity of neurons, other brominated have been shown to modulate voltage gated ion channels, modulate intracellular Ca++, generate reactive oxygen, and damage cell membranes [23–27]. It is possible that there is variability in the compliment of ion channels in individual opshradial neurons, and the neurons are modulated differently by TBECH. Alternatively, the types of channels modulated by TBECH may be narrow, and the variability in effect may be due to modulation of specific neurons within an osphradial network. Patch clamp electrophysiology will be required to dissect the ionic mechanism of TBECH action. 3. Limitations The study demonstrates that TBECH modulates activity of osphradial neurons, but the suction electrode technique does not allow for investigation of the ionic mechanism. The suction electrode technique is simpler and less expensive to implement than patch clamp electrophysiology, however the data analysis was unexpectedly more complex, requiring a more advanced technique of spike sorting because some spike types increased in frequency, and some decreased. It is unknown whether this is related to a broad spectrum of TBECH activity or neuronal network processing within the osphradium. Declarations Ethics approval and consent to participate No animal approval required Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Competing interests The authors declare that they have no competing interests Funding KLE was supported by a University of Manitoba Undergraduate Research Award; The project was supported by a University of Manitoba UGRP grant to WMF. Authours' contributions WMF and GTT conceptualized experiments. KLR carried out the experiments and analysed data. KLE wrote early versions of the manuscript, revised by GTT and WMF. Acknowledgements Not Applicable References UNEP. All POPs listed in the Stockholm Convention. The Stockholm Convention on Persistent Organic Pollutants. 2009. http://chm.pops.int/TheConvention/ThePOPs/AllPOPs/tabid/2509/Default.aspx. 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Dingemans MML, Heusinkveld HJ, de Groot A, Bergman Å, van den Berg M, Westerink RHS. Hexabromocyclododecane Inhibits Depolarization-Induced Increase in Intracellular Calcium Levels and Neurotransmitter Release in PC12 Cells. Toxicol Sci. 2009;107:490–7. Hendriks HS, Van kleef RGDM, Van den berg M, Westerink RHS. Multiple novel modes of action involved in the in vitro neurotoxic effects of tetrabromobisphenol-A. Toxicol Sci. 2012;128:235–46. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4631370","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":326835319,"identity":"5d147db9-8dce-4c54-a6c9-87af0d5d2155","order_by":0,"name":"Kaesy Lynne Enns","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Kaesy","middleName":"Lynne","lastName":"Enns","suffix":""},{"id":326835320,"identity":"4bd27ec9-4bf7-4c63-bf29-7c2d257be457","order_by":1,"name":"Gregg T. 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(A) Sample recording of osphradial nerve recorded with bipolar suction electrode following application of MS-222 (10 mg/ml) to osphradial epithelium. (B) Raw mean firing rate measured over control 3 minute interval prior to application of MS-222, and two 3 minute intervals following application (n = 7; Tukey’s post hoc test following repeated measures ANOVA). (C) Normalized mean firing rate over intervals measured in B, one way t-test with null hypothesis mean =1. (*, p\u0026lt;0.05; **, p\u0026lt;0.005; ***, p\u0026lt;0.001; ns, not significantly different).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4631370/v1/52b599d18d276ca87b224b80.png"},{"id":60839764,"identity":"ceff2572-cc53-4056-a697-b04a5dd8a22c","added_by":"auto","created_at":"2024-07-22 17:01:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":602698,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in osphradial nerve activity in response to yeast extract. (A) Sample recording of osphradial nerve recorded with bipolar suction electrode following application of yeast extract (7.2 mg/ml) to osphradial epithelium. Magnified samples from control, interval 1, 2 and 3 indicate multiple waveform types present. (B) Raw mean firing rate measured over a 3 minute interval prior to application of yeast and two 3 minute intervals following yeast application (n = 8; no significant differences using repeated measures ANOVA). (C) \u0026nbsp;Normalized mean firing rate over intervals measured in B (p=0.75 one way t-test with null hypothesis mean =1). (D) Identifying and sorting individual spikes revealed some spike types increased frequency, and some decreased.\u0026nbsp; Expressing change in frequency for each spike type as absolute value of Log2 (fold change) revealed significant change in frequency, regardless of whether frequency increased or decreased. (one way t-test null hypothesis mean =0).\u0026nbsp; For box plots: grey box represents 25\u003csup\u003eth\u003c/sup\u003e-75\u003csup\u003eth\u003c/sup\u003e percentile; whiskers represent 1\u003csup\u003est\u003c/sup\u003e -99\u003csup\u003eth\u003c/sup\u003e percentile; horizontal line represents median; solid square represents mean.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4631370/v1/1c29549c14640c4805a69a40.png"},{"id":60839766,"identity":"ede859ac-69aa-4abf-8c0b-c875fc113679","added_by":"auto","created_at":"2024-07-22 17:01:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":195059,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in osphradial nerve activity in response to TBECH. (A) Raw mean firing rate measured over a 3-minutes interval prior to application of TBECH, and two 3-minute intervals following TBECH application (n = 6; no significant differences using repeated measures ANOVA.). (B) \u0026nbsp;Normalized mean firing rate over intervals measured in B (p\u0026gt;0.05 one way t-test with null hypothesis mean =1). (C) Identifying and sorting individual spikes revealed some spike types increased frequency, and some decreased. Expressing change in frequency for each spike type as absolute value of Log2 (fold change) revealed significant change in frequency, regardless of whether frequency increased or decreased. (one way t-test with null hypothesis mean=0).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4631370/v1/b4429ee0c53c38e3af453b84.png"},{"id":93048037,"identity":"9f4f3a45-af77-4ae6-bb3f-0f067dafcf9d","added_by":"auto","created_at":"2025-10-08 13:39:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1307140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4631370/v1/ace940be-1ba0-411f-9dca-367eaa4b9c68.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrical activity of osphradial neurons of the greater pond snail Lymnaea stagnalis is modulated by the brominated flame retardant 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane","fulltext":[{"header":" Introduction","content":"\u003cp\u003eBrominated flame retardants (BFRs) are used in industrial manufacturing to reduce flammability of manufactured goods. Legacy BFRs, including polybrominated diphenyl ethers and biphenyls (PBDE, PBB, TBBPA), and hexabromocyclododecanes (HBCD) have been restricted due to their persistence, bioaccumulation, and toxicity\u0026nbsp;[1]. Approximately 30 novel BFRs have recently emerged and their use is increasing\u0026nbsp;[2\u0026ndash;5]. Of note is the BFR 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH), which has been shown to act an endocrine disruptor within the thyroid and gonadal axes\u0026nbsp;[3, 6, for review see 7], an embryotoxic agent\u0026nbsp;[8, 9], and a potential obesogen\u0026nbsp;[10]. A recent study investigated acute neurophysiological effects of TBECH, and demonstrated that TBECH dose-dependently inhibited spontaneous action potentials in cultured rat Purkinje neurons\u0026nbsp;[11]. These data, together with demonstrated bioaccumulation in terrestrial and aquatic animals and environments\u0026nbsp;[12], warrant further investigation into the safety of TBECH\u0026rsquo;s use [\u0026nbsp;for review see 7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLymnaea stagnalis,\u0026nbsp;\u003c/em\u003ethe greater pond snail, is used as a model organism for an OECD reproductive toxicicity test\u0026nbsp;[13], and is increasingly used as a model organism for neurobiology and neurotoxicology due to its accessible central nervous system, and emerging genomics\u0026nbsp;[14]. Additional benefits include low cost of care and limited animal care regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe osphradium of \u003cem\u003eLymnaea\u003c/em\u003e is of particular interest, as it functions as chemosensory neural integrator of environmental molecules and stressors. Chemosensory neurons are found within the osphradium, and their axons carry sensory information to ganglia within the central nervous system through the short osphradial nerve which merges with right internal parietal nerve [15]. Action potential activity of osphradial neurons is modulated by chemical cues including secretions of predators, hypoxia and pH [16\u0026ndash;19]; detection of these soluble molecules plays key roles in physiological functions including feeding, learning, and predator escape. \u0026nbsp;In order to investigate the potential neurotoxicity of the environmental contaminant TBECH, we used a suction electrode to record extracellular action potentials (spikes) from a preparation of isolated osphradium and the emanating osphradial nerve following exposure to TBECH.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eLymnaea stagnalis\u0026nbsp;\u003c/em\u003esnails were housed in aquatic habitats with a constant, gentle flow of dechlorinated water at 25\u0026deg;C. Their diet consisted of romaine lettuce, supplemented with trout pellets and cuttlebone for adequate calcium uptake. Each snail, 1.1g to 2.0g, was anesthetized with 0.5ml of freshly prepared 1.0M MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003einjected into the foot. The shell was crushed and removed with forceps. The mantle was folded back to reveal the osphradial epithelial canal, just anterior to the pneumostome. Visual inspection confirmed the location of the osphradium on the ventral side of the mantle. An incision was made on the dorsal surface beneath the mantle, while submerging the preparation in artificial extracellular fluid (AECF) containing 4.1mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1.5mM MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, 5.0 HEPES, 1.7mM KCl, 51.3 NaCl, 5.0mM dextrose, pH 7.9. The osphradial nerve and tissue containing the osphradium were extirpated from the body. The tissue surrounding the osphradium was pinned to a 35mm Sylgard-lined dish equipped with an inlet and outlet permitting solution exchange. The isolated osphradial nerve was fitted to a bipolar suction electrode with a glass capillary bore measuring between 150-200mm in diameter such that the osphradial canal faced the solution inlet. The sample was continuously submerged and perfused with AECF at 1ml/min using a peristaltic pump.\u003c/p\u003e\n\u003cp\u003eElectrical signals were amplified with a Model 1700 Differential AC Amplifier at 1000x gain (A-M Systems, Sequim, WA). Signals were filtered by a 1-500 Hz band pass filter, with a notch filter at 60Hz. Signals were digitized using Axon Instruments Digidata 1322A 50 kHz using Strathclyde Electrophysiology Software WinEDR (V4.0.2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe recorded electrophysiological activity in the presence of AECF alone to establish baseline activity (control). After at least 5 minutes of stable spike recording, 6ml of AECF containing either 7.2mg/ml yeast extract (containing a mix of amino acids and short peptides) (Kamardin et al., 1999; Fisher Scientific), or 100mM TBECH (Bosche Scientific, Rocky Hill NJ) was applied. We continued to record spike activity for at least two 3 minute intervals following application of yeast extract or TBECH. The length of the intervals was chosen such that the rhythmic activity of the osphradium would be accounted for while still observing overall changes in firing activity\u0026nbsp;[16, 17]. After ten minutes of recovery in AECF alone, 6ml of 10mg/ml tricaine methanesulfonate (MS-222; Syndel Canada) was perfused over the osphradium and we recorded spike activity for a further two 3 minute intervals: MS-222 is a nerve blocking agent which antagonizes voltage gated Na+ channels and prevents action potentials in neurons and axons from vertebrates and invertebrates\u0026nbsp;[20].\u003c/p\u003e\n\u003cp\u003eRaw data were imported to Spike2 6.12 software (CED, Milton England), and spikes were detected by setting a threshold approximately 10% greater than the peak-to-peak noise, at a minimum interval of 0.01s. Change in frequency of detected spikes was plotted as both raw frequencies, and normalized frequency (as the proportion relative to control). Our initial analysis did not reveal significant spike frequency changes after application of yeast extract or TBECH, however by eye we noticed changes in activity of specific spike waveforms. Therefore, we next identified and grouped individual spike types from each recording using the Spike6.12 semi-automated spike sorting routine. The frequency of each spike type with more than 30 spikes over the test period was analyzed separately before and following yeast extract or TBECH application. These changes were expressed as absolute value Log2 change in frequency to provide a linear representation of fold change, regardless of whether spikes were increasing or decreasing in frequency. For statistical testing, differences were considered significant at p\u0026lt;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe tested the effect of MS-222 to ensure that the observed spike activity was indeed caused by action potentials in the axons within the isolated osphradial nerve. Application of MS-222 resulted in a rapid increase in mean firing rate in the first 3 minutes, followed by cessation of activity between 3 and 6 minutes (n=7; Figure. 1); the raw firing rate increased from 22.9\u0026plusmn;5.7 spikes per minute during control period to 57.3\u0026plusmn;11.0 spikes per minute during the first 3min and decreased to 1.5\u0026plusmn;0.7 during the 3-6 minutes interval. A repeated measures ANOVA was performed to compare the effect of MS-222 on raw firing raw rate. There was a statistically significant difference in raw firing rate (F = 23.0 (between groups df=1, within groups df=2), p \u0026lt; 0.001). \u0026nbsp;Tukey\u0026rsquo;s post-hoc revealed firing rate during control period was significantly lower than during the first 3 minutes (p=0.0036), and greater than the rate during the 3-6 minute interval (p\u0026lt;0.001); control rate was not different than the rate during 3-6 minute interval. When firing rates were normalized to frequency observed during the control period (Figure 1A,C), the rates during the first 3 minute and 3-6 minute intervals are significantly different from control (p\u0026lt;0.001 each interval, one-way t-test, with null hypothesis mean =0). \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe carried out experiments applying 7.2mg/ml of yeast extract over the osphradial epithelium of \u003cem\u003eL. stagnalis\u003c/em\u003e as a positive control to demonstrate that stimulation of osphradial neurons by a mixture of amino acids applied to the intact osphradium could be detected by suction electrode recording (Figure 2). We observed a variable and nonsignificant change in raw firing rate (Figure 2B) in both the first 3 minute interval and 3-6 minute interval following yeast extract application (repeated measures ANOVA, F=1.12.0, between groups df=1, within groups df=2; p=0.35). After normalization to control firing rates, the firing rate during the first 3 minute interval and the 3-6 minute interval after application of yeast extract was still not significantly different from the control, likely due to the variability of mean normalized responses (Figure 2C).\u003c/p\u003e\n\u003cp\u003eVisual analysis of raw traces showed spikes of different waveforms followed different patterns of activity (where some spike types increased frequency, whereas some decreased frequency after yeast extract was applied). Therefore we analyzed the changes in activity of individual spike types (Fig. 2A, inset). Identification of the individual spike types from each experiment (n=8) yielded between 11-63 spike types with a total of 161 spike types. Expressing the change in frequency for each of these spike types as Log\u003csub\u003e2\u003c/sub\u003e-fold change revealed that application of yeast caused an increase in frequency of some types and a decrease in frequency of other types. Statistical comparison of the absolute value of the Log\u003csub\u003e2\u003c/sub\u003e fold change \u0026nbsp;(Figure 2D) revealed that yeast extract caused a statistically significant change in frequency of electrical activity of osphradial neurons compared to baseline (regardless of whether the neurons were stimulated or inhibited; one sample t-test, DF=160, p\u0026lt;0.001 for both the first 3 minute interval and the 3-6 minute interval).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePerfusion of TBECH to the osphradium also resulted in variable and nonsignificant changes in raw firing rates (Figure 3A). The mean raw firing rate observed under control conditions was 23.2\u0026plusmn;6.3 during the control period, 24.0\u0026plusmn;7.4 spikes per minute for the first 3 minutes following TBECH application and 24.1\u0026plusmn;6.6 spikes per minute for the 3-6 minutes following (Figure 3A; repeated measures ANOVA, F=0.48.0, between groups df=1, within groups df=2, p=0.62). Normalizing firing rate for each nerve tested revealed dramatic increases in firing rate of some nerves, and decreases in others, however there was no statistically significant difference from control firing rates (Figure 3B; one way t-tests, p=0.75). Spike sorting analysis of the 6 osphradial nerves yielded between 11 and 29 spike types for each nerve, for a total of 70 spike types identified. Statistical comparison of the absolute value of the Log\u003csub\u003e2\u003c/sub\u003e fold change revealed that TBECH caused a statistically significant change in frequency of electrical activity of osphradial neurons compared to baseline regardless of whether the neurons were stimulated or inhibited (Figure 3D; one sample t-test with null hypothesis mean =0, DF=69, p\u0026lt;0.001 for both the first 3 minutes and the 3-6 minutes following TBECH application).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUsing a relatively simple suction electrode and extracellular electrophysiological amplifier, we have demonstrated that the environmental contaminant TBECH alters action potential activity of the osphradial neurons of \u003cem\u003eLymnaea stagnalis.\u0026nbsp;\u003c/em\u003eIn contrast to our previous experiments where we used patch clamp electrophysiology to show that TBECH acutely modulates activity of single mammalian neurons growing in culture\u0026nbsp;[11], here we use a simpler, but robust electrophysiological technique and an acute preparation of invertebrate neurons in a system to test neurotoxicity of this environmental contaminant. Importantly, while we observed clear changes in electrical activity of osphradial neurons, the frequency of some spike types increased and some decreased, making analysis considerably more involved and perhaps not suitable for a high throughput neurotoxicological testing model. Our data demonstrate for the first time the neurotoxicity of TBECH in aquatic animals. This finding is important in evaluating the overall toxicity of TBECH and its potential to harm sensitive ecosystems and human health. These data clearly indicate that further study of neurotoxic properties of TBECH is warranted. Indeed, recent reviews suggest that BFRs, including novel BFRs introduced since phase out of HDCDs and PBDEs, may be linked to human disorders including, neurobehavioral and developmental disorders, alteration in thyroid function and other illness\u0026nbsp;[3, 21]. \u0026nbsp;Because exposure to treatments or chemicals that alter spontaneous electrical activity of developing neurons are well-recognized to result in wide-ranging and long-lasting alteration of normal neuronal development\u0026nbsp;[22], potential effects neurodevelopmental effects of exposure to TBECH should be investigated in detail.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe change in spike activity following application of yeast extract included both \u0026nbsp;simultaneous increases and decreases in frequency of different spike types, but this was not unexpected. Other studies have demonstrated at the single cell level that some stimuli excite osphradial neurons and others inhibit activity\u0026nbsp;[16, 17], and therefore this pattern probably represents a level of neural processing for the biologically relevant yeast extract.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to the yeast extract, TBECH also caused simultaneous increases and decreases in frequency of different spike types, but the importance of this variable modulation is not clear. While the present data do not suggest a mechanism by which TBECH may alter electrical activity of neurons, other brominated have been shown to modulate voltage gated ion channels, modulate intracellular Ca++, generate reactive oxygen, and damage cell membranes\u0026nbsp;[23\u0026ndash;27]. It is possible that there is variability in the compliment of ion channels in individual opshradial neurons, and the neurons are modulated differently by TBECH. Alternatively, the types of channels modulated by TBECH may be narrow, and the variability in effect may be due to modulation of specific neurons within an osphradial network. Patch clamp electrophysiology will be required to dissect the ionic mechanism of TBECH action. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. \u003cem\u003eLimitations\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eThe study demonstrates that TBECH modulates activity of osphradial neurons, but the suction electrode technique does not allow for investigation of the ionic mechanism.\u003c/li\u003e\n \u003cli\u003eThe suction electrode technique is simpler and less expensive to implement than patch clamp electrophysiology, however the data analysis was unexpectedly more complex, requiring a more advanced technique of spike sorting because some spike types increased in frequency, and some decreased. It is unknown whether this is related to a broad spectrum of TBECH activity or neuronal network processing within the osphradium. \u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eEthics approval and consent to participate\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eNo animal approval required\u003cbr\u003e\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eConsent for publication\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAvailability of data and materials\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests\u003cbr\u003e\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eKLE was supported by a University of Manitoba Undergraduate Research Award; The project was supported by a University of Manitoba UGRP grant to WMF.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAuthours\u0026apos; contributions\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eWMF and GTT conceptualized experiments. KLR carried out the experiments and \u0026nbsp;analysed data. KLE wrote early versions of the manuscript, revised by GTT and WMF.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAcknowledgements\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eNot Applicable\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eUNEP. All POPs listed in the Stockholm Convention. The Stockholm Convention on Persistent Organic Pollutants. 2009. http://chm.pops.int/TheConvention/ThePOPs/AllPOPs/tabid/2509/Default.aspx. Accessed 8 May 2018.\u003c/li\u003e\n\u003cli\u003e,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane | C8H12Br4 | CID 18728 - PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/18728. Accessed 24 May 2024.\u003c/li\u003e\n\u003cli\u003eDong L, Wang S, Qu J, You H, Liu D. New understanding of novel brominated flame retardants (NBFRs): Neuro(endocrine) toxicity. Ecotoxicol Environ Saf. 2021;208.\u003c/li\u003e\n\u003cli\u003eTang B, Xiong SM, Zheng J, Wang MH, Cai FS, Luo WK, et al. Analysis of polybrominated diphenyl ethers, hexabromocyclododecanes, and legacy and emerging phosphorus flame retardants in human hair. Chemosphere. 2021;262.\u003c/li\u003e\n\u003cli\u003eMuir DCG, Howard PH. Are there other persistent organic pollutants? A challenge for environmental chemists. Environ Sci Technol. 2006;40:7157\u0026ndash;66.\u003c/li\u003e\n\u003cli\u003ePark BJ, Palace V, Wautier K, Gemmill B, Tomy G. Thyroid axis disruption in juvenile brown trout (Salmo trutta) exposed to the flame retardant beta-tetrabromoethylcyclohexane (beta-TBECH) via the diet. Environ Sci Technol. 2011;45:7923\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eMarteinson SC, Bodnaryk A, Fry M, Riddell N, Letcher RJ, Marvin C, et al. A review of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane in the environment and assessment of its persistence, bioaccumulation and toxicity. Environ Res. 2021;195.\u003c/li\u003e\n\u003cli\u003eMarteinson SC, Letcher RJ, Graham L, Kimmins S, Tomy G, Palace VP, et al. The Flame Retardant \u0026beta;-1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane: Fate, Fertility, and Reproductive Success in American Kestrels ( \u003cem\u003eFalco sparverius\u003c/em\u003e ). Environ Sci Technol. 2012;46:8440\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003ePradhan A, Kharlyngdoh JB, Asnake S, Olsson P-E. The brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor, alters gene transcription and causes developmental disturbances. Aquat Toxicol. 2013;142\u0026ndash;143:63\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eMarteinson SC, Fernie KJ. Is the current-use flame retardant, DBE-DBCH, a potential obesogen? Effects on body mass, fat content and associated behaviors in American kestrels. Ecotoxicol Environ Saf. 2019;169:770\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eStojak BL, van Ginkel RA, Ivanco TL, Tomy GT, Fry WM. Acute \u0026beta;-tetrabromoethylcyclohexane (\u0026beta;-TBECH) treatment inhibits the electrical activity of rat Purkinje neurons. Chemosphere. 2019;231:301\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eTomy GT, Pleskach K, Arsenault G, Potter D, McCrindle R, Marvin CH, et al. Identilication of the novel cycloaliphatic brominated flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane in Canadian Arctic beluga (Delphinapterus leucas). Environ Sci Technol. 2008;42:543\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eDucrot V, Askem C, Azam D, Brettschneider D, Brown R, Charles S, et al. Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda). Regul Toxicol Pharmacol. 2014;70:605\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eRosato M, Hoelscher B, Lin Z, Agwu C, Xu F. Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages. BMC Genomics. 2021;22.\u003c/li\u003e\n\u003cli\u003eZaitseva O V. Principles of the structural organization of the chemosensory systems of freshwater gastropod mollusks. Neurosci Behav Physiol. 1999;29:581\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003eWedemeyer H, Schild D. Chemosensitivity of the osphradium of the pond snail Lymnaea stagnalis. J Exp Biol. 1995;198 Pt 8:1743\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eKamardin NN, Shalanki Y, Sh.-Rozha K, Nozdrachev AD. Studies of chemoreceptor perception in mollusks. Neurosci Behav Physiol. 2001;31:227\u0026ndash;35.\u003c/li\u003e\n\u003cli\u003eOrr M V., El-Bekai M, Lui M, Watson K, Lukowiak K. Predator detection in Lymnaea stagnalis. J Exp Biol. 2007;210 Pt 23:4150\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eKarnik V, Braun M, Dalesman S, Lukowiak K. Sensory input from the osphradium modulates the response to memory-enhancing stressors in Lymnaea stagnalis. J Exp Biol. 2012;215 Pt 3:536\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eStanley CE, Adams R, Nadolski J, Amrit E, Barrett M, Bohnett C, et al. The effects of tricaine mesylate on arthropods: crayfish, crab and Drosophila. Invert Neurosci. 2020;20.\u003c/li\u003e\n\u003cli\u003eKim YR, Harden FA, Toms LML, Norman RE. Health consequences of exposure to brominated flame retardants: a systematic review. Chemosphere. 2014;106:1\u0026ndash;19.\u003c/li\u003e\n\u003cli\u003eMoody WJ, Bosma MM. Ion channel development, spontaneous activity, and activity-dependent development in nerve and muscle cells. Physiol Rev. 2005;85:883\u0026ndash;941.\u003c/li\u003e\n\u003cli\u003eFeiteiro J, Rocha SM, Mariana M, Maia CJ, Cairrao E. Vascular Response of Tetrabromobisphenol a in Rat Aorta: Calcium Channels Inhibition and Potassium Channels Activation. Toxics. 2022;10.\u003c/li\u003e\n\u003cli\u003eHendriks HS, Meijer M, Muilwijk M, Van Den Berg M, Westerink RHS. A comparison of the in vitro cyto- and neurotoxicity of brominated and halogen-free flame retardants: Prioritization in search for safe(r) alternatives. Arch Toxicol. 2014;88:857\u0026ndash;69.\u003c/li\u003e\n\u003cli\u003eDingemans MML, van den Berg M, Bergman \u0026Aring;, Westerink RHS. Calcium-related processes involved in the inhibition of depolarization-evoked calcium increase by hydroxylated PBDEs in PC12 cells. Toxicol Sci. 2009;114:302\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eDingemans MML, Heusinkveld HJ, de Groot A, Bergman \u0026Aring;, van den Berg M, Westerink RHS. Hexabromocyclododecane Inhibits Depolarization-Induced Increase in Intracellular Calcium Levels and Neurotransmitter Release in PC12 Cells. Toxicol Sci. 2009;107:490\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eHendriks HS, Van kleef RGDM, Van den berg M, Westerink RHS. Multiple novel modes of action involved in the in vitro neurotoxic effects of tetrabromobisphenol-A. Toxicol Sci. 2012;128:235\u0026ndash;46.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, brominated flame retardant, neurotoxicity, osphradium, suction electrode extracellular recording, action potential","lastPublishedDoi":"10.21203/rs.3.rs-4631370/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4631370/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003e1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH) is a brominated flame retardant used as a chemical additive in commercial and industrial manufacturing to reduce product flammability. TBECH has previously been shown to be an endocrine disruptor of the gonadal and thyroid axes, however, its neurotoxic effects, including effects on electrical excitability of neurons, are understudied. Therefore, we investigated the potential of TBECH to modulate electrical activity of neurons from the chemosensory osphradial organ of \u003cem\u003eLymnaea stagnalis\u003c/em\u003e using a suction electrode and extracellular recording.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eApplication of TBECH caused a variable response in osphradial nerve spike activity, whereby some recordings showed increased action potential firing and some showed decreased firing. This resulted no significant change in mean action potential frequency after TBECH treatment compared to control (n\u0026thinsp;=\u0026thinsp;6 separate experiments). However, using semi-automated spike sorting analysis to identify individual spike types from each recording revealed that the frequency of some spike types increased and some decreased within each nerve recording, and that TBECH caused significant modulation of activity. These findings indicate that TBECH may represent an acutely neurotoxic environmental contaminant that has potential to interfere with neural signaling in animals.\u003c/p\u003e","manuscriptTitle":"Electrical activity of osphradial neurons of the greater pond snail Lymnaea stagnalis is modulated by the brominated flame retardant 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 17:01:54","doi":"10.21203/rs.3.rs-4631370/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c95804cc-07b4-4789-8714-bc395e7fdb5c","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-08T13:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 17:01:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4631370","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4631370","identity":"rs-4631370","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}