Cultural differences in affective and cognitive intrinsic motivation could pose challenges for global public health campaigns, which use cognitive or affective goals to evoke desired attitudes and proactive health-promoting actions. This study aimed to identify cross-cultural differences in affective and cognitive intrinsic motivation and discuss the potential value of this information for public health promotion.A cross-sectional survey using cross-culturally validated need for affect (NFA) and need for cognition (NFC) scales was carried out among 1166 Chinese participants, and the results were compared with published data from 980 American participants. Additionally, we assessed a highly prevalent symbolic geriatric health condition, hearing loss, in 500 Chinese community-dwelling seniors. The Chinese NFA scale was developed following the translation-back translation procedure, and the psychometric evaluation was performed by applying confirmatory factor analysis (CFA), exploratory structural equation modeling (ESEM), correlation analysis and multigroup invariance test. MANOVA and Hedge’s g statistic were employed to compare the NFA and NFC levels between individuals from different countries and between Chinese seniors with and without hearing loss. The relation of early hearing intervention intention to NFA and NFC was also explored in the Chinese sample.A basic two-factor model of NFA adequately fit the sample data from Chinese and American cultures. The questionnaire demonstrated reasonable invariance of the factor structure and factor loadings across the groups. Those in the primary Chinese sample had lower NFA and NFC than their American peers. This difference held in the senior sample. Moreover, Chinese seniors with hearing loss had even lower NFA and NFC than those without hearing loss. Their early hearing intervention intention was low but was associated with intrinsic motivation.The Need for Affect (NFA) construct may be generalized beyond its Western origins. There was a general lack of affective and cognitive intrinsic motivation in Chinese individuals, particularly in seniors with hearing loss, compared with their American peers. These differences point to a potential challenge in framing effective messages for some cultures in the geriatric public health domain. Ideally, recognizing and understanding this challenge will inspire the consideration of novel persuasive strategies for these audiences.The online version contains supplementary material available at 10.1186/s12889-021-10689-w. Many disciplines rely on persuasive communication with target audiences around affective goals  or cognitive goals . An implicit assumption is that such affective goals are universal. Rather, affective and cognitive orientation (indexed by need for affect and need for cognition) differs across individuals and is associated with the effectiveness of messages in persuasive communication , 2 and cAffective and cognitive orientations are indexed by self-report questionnaires. The Need for Affect (NFA) questionnaire assesses the tendency to approach or avoid emotion-inducing situations and activities. It focuses on the motivation to engage in the affective process rather than assessing emotional ability or emotional style . People Geriatric health campaign messages are often geared towards affective goals , as evidence has shown that affective outcome expectancies have a significantly more positive impact on older people’s intentions to take proactive actions than rationale outcome expectancies . HoweverMake listening safe: avoid noise-induced hearing loss”, “Earcare can avoid hearing loss”, “Proper use of hearing aids”, and “Act now, here is how”, while the more recent themes have emphasized the emotional side, e.g., “Hearing for life: don’t let hearing loss limit you”, “Hear the future”, “Make a sound investment”, and “Healthy hearing, happy life”. Although advocacy of early hearing intervention to reduce the long-term adverse effects of hearing loss has been carried out for over 2 decades using the same WHO messages, the hearing intervention rate in geriatrics remains low at < 2% in China [Overall, these data suggest that the baseline levels of affective and cognitive intrinsic motivation may be low in certain cultures, so conventional emotion/cognition-based messaging is likely to be less effective. A prototypical example of diverse outcomes across cultures, given similar campaign content and service accessibility, is in the hearing healthcare domain. There are cross-cultural public health outreach efforts spanning China and the U.S. The prevalence of hearing loss in elderly individuals is 45 ~ 63% in both countries . The WHOin China , whereasin China . PotentiThe current study examines whether NFA and NFC differ systematically across relatively large Chinese and American samples. The objectives are to 1) develop a Chinese NFA scale based on Appel et al.’s English language version  and estaage = 45.4 ± 17.6 yrs., range = 20 ~ 90 yrs., 65.6% female), 695  were recruited through an online survey tool (TengXun Survey) and completed a web-based survey in return for an instant and direct deposit of RMB¥2 RMB (USD$0.3) to their WeChat payment account. Five hundred older participants  were recruited offline at a local community health center during their annual mandatory physical examination. The community-based participants completed hearing assessments in addition to the NFA and NFC surveys. They were reimbursed ¥20 RMB for their participation and travel. Our American sample  was from Arceneaux and Vander Wielen [We recruited 1195 Chinese adults to participate in the study from April to July 2019. This sample size was comparable to those in the original NFA scale development studies , 22. Of r Wielen . The 100The English 10-item short version consists of approach and avoidance subscales with 5 items in each . The scaThe Chinese version of the NFA see  was rigor = 0.52, p < 0.01), which was in line with Cacioppo et al. [Cacioppo et al.’s  18-item o et al. . The sumo et al. .The purpose of assessing hearing impairment in the Chinese sample was to make claims regarding the extent to which, in Chinese culture, low levels of NFA are specifically present in the subpopulation that would be targeted by hearing campaigns. Two hearing-related assessments were administered to the community-based seniors: 1) a simplified pure tone examination  using anChinese participants responded to the Chinese NFA questionnaire developed in the present study  and the Chinese NFC scale. The 500 community-based Chinese seniors completed the hearing assessments as well. Forty-seven of the 500 participants completed the battery twice with an interval of 3 ~ 4 weeks; on each occasion, they completed the NFA, NFC, and the single question about early hearing intervention intention. American participants in Arceneaux and Vander Wielen’s  study reOnly 5.9% of cases had missing data; overall, therefore, 0.25% of the values were missing in our samples. Missing values were managed via a full information maximum likelihood (FIML) approach. The psychometrics of the Chinese NFA questionnaire were examined in terms of test-retest reliability, internal consistency and Cronbach’s alpha. Convergent validity was indexed by the correlation between the NFA and NFC scales . The conIn the present study, the following cut-off values were adopted: 1) comparative fit index (CFI), comparative fit index (TLI), and Turker and Lewis’s Index of fit (NFI) of 0.90 , 35; 2) The configural, construct-level metric and scalar invariance of the basic NFA model between the Chinese and American samples were tested following a stepwise procedure , 42. SpeTo test our hypothesis about cultural differences in NFA, the scores of our samples and those of 4 independent European samples reported by Appel et al.  were comTo explore whether seniors with a highly prevalent public health problem  have reasonably high intrinsic motivation, as assumed by public health campaigns, we applied MANOVA to examine the differences in the NFA and NFC scores between Chinese seniors with and without hearing loss. Moreover, correlation analysis was conducted to explore the relationship between individuals’ NFA and NFC and the tendency toward early hearing intervention seeking. These analyses were performed using SW 24 .r = .943, p < .001, ICC = .942, p < .001, and Cronbach’s α = .829, F  = 54.580, p < .001. Consistent with Kuang, Shi [F  = 99.992, p < .001. The relationship between NFA and NFC was assessed as an index of convergent validity [p < .001), which was comparable with that reported by Appel et al. [r = .170, p < .001), Z = 1.19, p = .234.The short 10-item Chinese scale based on Appel et al.  showed aang, Shi , the Chivalidity . The corl et al.  , Z = 18.08, p<. 001 , Z = 19.79, p < .001, German adults , Z = 18.11, p<. 001, Austrian couples , Z = 10.23, p<. 001, and UK adults , Z = 13.18, p<. 001. There was no significant difference in the approach-avoidance correlation between the American sample and the German/Austrian students  or Austrian couples . However, the correlation was significantly smaller in magnitude in the American sample than in the German adults, Z = 2.23, p=. 030, or the UK adults, Z = 2.25, p=. 025.Consistent with the finding of the noninvariant construct covariance shown in Table M = −.57, SD = 1.28) than in American seniors , F  = 167.33, p < .001, ηp2 = .22, and a significantly higher avoidance score in Chinese seniors  than in American seniors , F  = 16.93, p < .001, ηp2 = .03.The difference in NFA observed between the Chinese and American general public samples was also present in the older participants (≥60 yrs), with a significantly lower approach score in Chinese seniors  = 9.00, p < .01, ηp2 = .05, significantly lower avoidance scores, F  = 26.13, p < .01, ηp2 = .05, and significantly lower NFC scores, F  = 14.66, p < .01, ηp2 = .08. There was a small group of individuals (n = 34) who reported having normal hearing but failed the pure tone audiometric test and who had similar levels of NFA and NFC  = 1.01, p = .39, ηp2 = .01. Regardless of hearing status, participants tended to choose “when I have severe hearing loss and have difficulty communicating with others” in response to the question “When do you think you will consider wearing hearing aids and take serious action?” Of the 486 community-based participants, only 6.8 and 13.8% reported willingness to seek early intervention when they had mild and moderate hearing loss, respectively. Early hearing intervention intention was significantly predicted by NFC, B = .174, t = 2.662, p = .008, but not by the NFA approach score, B = −.052, t = −.721, p = .471, or by the NFA avoidance score, B = .025, t = .339, p = .735.Intention to avail themselves of early hearing intervention did not differ between groups, Considering target audiences’ needs for affect and cognition in contexts involving persuasive communication, such as global public health campaigns, is important because matching messages with receivers’ affective and cognitive orientation can significantly improve the effectiveness of persuasive communication . CulturaWe first developed a Chinese translation of the original English NFA scale , which dOur second question regarded the presence of differences in affective and cognitive intrinsic motivation among the Chinese, American and European samples. The Chinese participants reported lower motivation to approach emotional events and activities and a greater tendency to avoid emotion-inducing events and activities than their American and European counterparts Fig. . This fiA key notion underlying the NFA is that it subsumes both a motivation to approach emotions and a motivation to avoid them . These tIn the present study, whereas NFA approach and avoidance were negatively correlated in the American and European samples, they were positively correlated in the Chinese sample. This counterintuitive motivation to approach emotions might be explained by observations regarding differential reactions to emotions  across cultures . AccordiThe low correlations between NFC and NFA within the samples ~.2) but the reliable sample-related differences could also suggest that cultural factors are responsible for differences in NFC. When people value harmonious social relationships, they tend to put less effort into information search and deliberation and into the utilization of issue-relevant information to think, reason and form attitudes and behaviors different from the mainstream . These s but the Our third objective was to explore whether the intrinsic motivation level of community-based seniors in China with a specific highly prevalent public health condition  is high, which would warrant affectively motivated health campaigns. Given the reduction in NFA and NFC in Chinese seniors relative to American seniors, Chinese seniors with hearing loss showed even lower NFA and NFC than those without hearing loss. This result differed from our hypothesis that NFA and NFC would be similar in seniors with and without hearing loss. The particularly low level of NFA in seniors with hearing loss, might be associated with the link between hearing loss and late-life depression , the latEarly hearing intervention intention was low regardless of hearing status; the majority of older Chinese people preferred to wait until their hearing condition became difficult to cope with. Our community-based geriatric sample was representative of the urban older population targeted by hearing health campaigns in China. It had a hearing loss prevalence of 62.6%, which is consistent with a previous report . Low levAs the attempt to establish health goals by affective means has a relatively short history in China, the lack of a relationship between NFA (approach or avoidance) and intention to engage in early hearing intervention was not surprising. Given the numerous cultural differences associated with emotion , exploriχ2(1) = 1.782, p = .182), only 55% of the age data were available in the American sample,. Other demographic differences , as well as variables associated in the literature with affective style  [Our study has multiple limitations. First, limited demographic information was collected from the samples. Although the Chinese and American samples were comparable in terms of sex distribution (enomena) –69, mighenomena) , which ienomena)  scale scenomena)  sample aThese limitations notwithstanding, our results demonstrate a lack of affective and cognitive intrinsic motivation in Chinese individuals compared with the American public. These differences point to a potential challenge in framing effective messages for some cultures. Ideally, recognizing and understanding this challenge will inspire the consideration of novel persuasive strategies for these audiences. For example, using our example of public campaigns targeting hearing loss, instead of replicating the Western hearing healthcare campaign model, messages could target Asian values more specifically, such as by emphasizing a person’s ability to be in harmony with nature  and society  rather than individual affective and cognitive goals. Another direction might be strengthening the influence of messages using newly available technology. Breves and Heber  reportedWe conclude that the need for affect (NFA) construct may transcend its Western origins. Assessing NFA and NFC could inform global public health campaigns by helping them frame messages consistent with audiences’ culture-dependent intrinsic motivation.Additional file 1: Appendix The Chinese version of the Need for Affect-Short Scale. The Chinese translation of the 10-item NFA scale.Additional file 2: Supplement Normative NFA values of the general Chinese and American public. The Chinese and American norms of Need for Affect approach and avoidance subscale scores by age and gender in 7-point and 5-point rating scales.


Multiple mutants exhibiting increased O2 responsiveness concomitantly show defects in other sensory responses. One mutant, qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+ responses in the ADL pheromone-sensing neurons. At the same time, ADL responsiveness to pre-synaptic input from O2-sensing neurons is heightened in qui-1, and other sensory defective mutants, resulting in enhanced neurosecretion although not increased Ca2+ responses. Expressing qui-1 selectively in ADL rescues both the qui-1 ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling ADL neurons in qui-1 mutants highlights extensive changes in gene expression, notably of many neuropeptide receptors. We show that elevated ADL expression of the conserved neuropeptide receptor NPR-22 is necessary for enhanced ADL neurosecretion in qui-1 mutants, and is sufficient to confer increased ADL neurosecretion in control animals. Sensory loss can thus confer cross-modal plasticity by changing the peptidergic connectome.Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. We probe such mechanisms by performing a forward genetic screen for mutants with enhanced O Animals that lose a sensory modality often show increased sensitivity to other sensory inputs. This change can involve repurposing neurons or brain areas that normally mediate responses to the lost modality such that they process other sensory inputs. For example, in blind people the absence of visual stimulation leads to rewiring of inputs into primary visual cortex (V1), so that V1 becomes responsive to tactile stimuli, a characteristic absent in sighted individuals . The molCaenorhabditis elegans provides a favourable model to study cross-modal interactions in neural circuits, and how these connections may be altered by neural plasticity, in particular because of the careful reconstruction of its complete wiring diagram of chemical and electrical synapses  levels  levels . Instantto 7% O2 . The wirrneurons . Severalrneurons . The URXrneurons . These O and ADL . Recipro stimuli . HoweverC. elegans arousal in response to 21% O2 by using genetic backgrounds that reduce signalling from RMG. We then seek mutants that restore O2 responsiveness; such mutants are likely to reprogram sensory information processing across the hub-and-spoke circuit to circumvent RMG inhibition. We identify several sensory defective mutants that increase ADL’s ability to relay information from pre-synaptic neurons, including from URX O2 sensors and RMG interneurons. Specifically, these mutants show increased O2-evoked secretion of neuropeptides from ADL. Using RNA sequencing (RNAseq), we profile ADL neurons in wild-type control and one enhancer mutant, qui-1. We discover extensive remodelling of ADL’s peptidergic properties and find that increased expression of the neuropeptide receptor NPR-22 is necessary and sufficient to increase neurosecretion from ADL. Our data suggest that defects in sensory perception by the ADL pheromone sensors can increase ADL’s responsiveness to input from the O2 circuit by reconfiguring its sensitivity to neuropeptides. Changes in the peptidergic connectome may be an unappreciated mechanism by which loss of one sensory modality alters responsiveness to another.Here, we employ forward genetics to identify mechanisms that alter information processing across the RMG hub-and-spoke circuit. We suppress C. elegans avoid and escape 21% O2 , referred to here as wild-type (WT), is not aroused by 21% O2 and does not accumulate on thick bacteria. This is due to a gain-of-function mutation in the neuropeptide receptor NPR-1, npr-1 215V  placed in the middle of a thin lawn (see Methods)   – Step 1controls  – Step 2at 7% O2 . To capt2-escape behaviour in these mutants we used a Deep Sequence Mapping strategy  mutants isolated in our screen displayed both aggregation and O2-escape behaviour   O2-escape phenotype (To identify the genetic defects causing increased Ostrategy . A list l strain . Previouthickest . qui-1(dehaviour . We next(ok3571) . These s(ok3571) , furtherhenotype .2 sensors are tonically activated by 21% O2 and in turn tonically activate the RMG hub interneurons  and pheromones (ADL). We used cell-specific rescue of qui-1 mutants to ask if QUI-1 acts in ASH and/or ADL neurons to inhibit O2-evoked escape behaviour. Expressing qui-1 selectively in ASH neurons reduced the O2-escape response of qui-1 mutants compared to wild-type animals  and accumulate in scavenger cells called coelomocytes  and fig-1  (wrt-6 (db102) allele substituted a conserved threonine residue (T460I) (fig-1 (db1239) allele changed a cysteine in a C6 domain into a tyrosine (C1951Y) . Appropriate protein levels may be necessary for correct fig-1 function.A search of the sequencing data from our mutant collection revealed two mutant strains carrying missense mutations in genes previously associated with impaired sensory perception, In Glia) . The wrt (T460I)  essentia(C1951Y) . A wild-function . To test alleles . All figat 7% O2 , suggest2 input from the hub-and-spoke circuit. To test this, we measured ADL neurosecretion in wrt-6 and fig-1 mutants and observed a robust increase in both mutants , used fluorescence-activated cell sorting (FACS) to sort ADL from freshly dissociated wild-type controls and qui-1 mutants, and then profiled the ADL transcriptome using RNAseq. Enrichment analysis highlighted ADL as the most enriched neural class in our dataset  in our experiments, giving us confidence the contribution of background mutations should be minimal.Principal component analysis confirmed that we could robustly differentiate  samples . We nexts in ADL . The majs in ADL . When wes in ADL  that wer mutants . qui-1 megulated . These dqui-1 mutants ADL responds more strongly to O2 input from the hub-and-spoke circuit by releasing more DCVs. The absence of increased O2-evoked Ca2+ responses in ADL in qui-1 animals , can strongly stimulate DCVs release , which was one of the most highly upregulated genes in qui-1 mutants compared to control  in qui-1 mutants. TeTX is predicted to cleave SNB-1, the main synaptobrevin expressed in ADL neurons. The O2-escape behaviour of qui-1 animals expressing TeTX in ADL was similar to that of qui-1 mutants  sensory cue. Several of the mutants we identify simultaneously increase responsiveness to O2 while disrupting other sensory responses – a hallmark of cross-modal plasticity. We analyse one of these mutants, qui-1, which is defective in the ortholog of mammalian NWD1, in depth. We show that loss of QUI-1 prevents the ADL sensory neurons from responding to pheromone, but increases ADL neurosecretion in response to input from upstream O2-sensing neurons. Loss of qui-1 thus recruits ADL sensory neurons more strongly into the O2-sensing circuit. We observe a similar change in additional sensory defective animals as well as in animals with ADL-specific impairment of the intraflagellar transport protein OSM-6. Loss of qui-1 is associated with extensive changes in gene expression in ADL neurons, notably induced expression of neuropeptide receptors, including NPR-22 and TKR-1. Elevated expression of NPR-22 and TKR-1 increase the coupling of ADL neurosecretion to O2 input in qui-1 mutants. We propose that impairing sensory perception can sensitize sensory neurons to other sensory modalities by reconfiguring peptidergic circuits.Cross-modal plasticity is thought to involve recruitment of impaired neurons to process additional sensory modalities. The molecular details of such rearrangement are not yet clear. Here, we use forward genetics as an entry point to seek mechanisms that increase qui-1 rescues both the increased O2-escape phenotype of qui-1 mutants and enhanced neurosecretion from ADL  or high (21%) O2 concentrations modulates ADL neurosecretion , the egl-46 zinc-finger protein , suggesting impairment in ADL’s ability to sense external cues. These data taken together suggest that the enhanced responsiveness to O2 input we observe in sensory defective mutants, characterized by increased O2-evoked neurosecretion from ADL, is consistent with cell-specific defects in ADL sensory perception. Enhanced ADL neuroendocrine output may modulate aspects of the animal’s physiology and behaviour we have not probed here.We identify several sensory defective mutants that display both enhanced neurosecretion from ADL and increased O filling , a proxy filling . Cell-specretion . It is id by ADL . wrt-6 mC. elegans, the defective ADL sensory neurons becomes sensitized to pre-synaptic input associated with a different modality, O2 sensing  using a Grasshopper camera (Point Grey) mounted on a stereomicroscope (Leica MZ6 and MZ7.5). Videos were analysed and animal speed calculated using a custom-written MATLAB software . Average speed values were extracted using Metaverage, a custom-written MATLAB software. Bar graphs show a ratio of the average speed 30s before the end of the first 21% O2 stimulus and the average speed 30-s before O2 levels were first switched from 7% to 21% O2. This ratio was computed for all biological replicates (independent assays) and entered into Prism for statistical analysis.Assays were performed as described previously . Low pep2, because it is a good proxy for the functionality of the O2-sensing circuit in regulating locomotion. Animals that respond strongly to O2, including natural wild isolates, npr-1 null mutants, and the mutant strains we study here, differ from strains that respond poorly to O2 such as N2 and npr-1;gcy-35 in their locomotory responses to both 21% and 7% O2. Animals with a functional O2-sensing circuit become highly active at 21% O2 and strongly quiescent at 7% O2. Animals with a defective O2 circuit move at intermediate speeds, with little change in their locomotory activity when O2 levels change from 7% to 21%. The higher baseline speed at 7% O2 of strains with a defective O2-sensing circuit reflects these animals being adapted to the low activity of this circuit. npr-1 animals kept for long periods at 7% O2 also gradually begin to move faster at this O2 concentration, and in fact respond more strongly than npr-1 animals kept at 21% O2 if O2 levels rise.We plot the ratio of animal speed at 21% and 7% OGenomic DNA for whole-genome sequencing was isolated from 5 to 10 crowded 5-cm NGM plates. Animals were washed off plates in M9 buffer, rinsed 2× in M9 buffer to remove OP50, and frozen at −80°C. Genomic DNA was extracted from thawed samples using the DNeasy Blood and Tissue Kit (Qiagen). Samples were left in Lysis buffer (Buffer AL) for 3 hr at 56°C and DNA isolated following the manufacturer’s instructions.Libraries for whole-genome sequencing were prepared using the Nextera XT DNA Library kit (Illumina) following the manufacturer’s instructions. Library quality was checked on a Bioanalyzer using Agilent High Sensitivity Gel. Library concentration was assessed using KAPA Library Quantification Kits for Illumina (KAPA Biosystems) prior to sequencing on the Illumina HiSeq 4000 platform.RNAseq of isolated neurons was adapted from 2+ sensors were glued to agarose pads  using Dermabond tissue adhesive, with their body immersed in OP50 washed off from a seeded plate using M9. The animals were quickly covered with a PDMS microfluidic chamber and 7% O2 pumped into the chamber for 2 min before imaging, to allow animals to adjust to the new conditions. Neural activity was recorded for 6 min with switches in O2 concentration every 2 min. Imaging was on an AZ100 microscope (Nikon) equipped with a TwinCam adaptor (Cairn Research), two ORCAFlash4.0 V2 digital cameras (Hamamatsu), and an AZ Plan Fluor 2x objective with 2x zoom. Recordings were at 2 frame-per-second (fps) with a 500ms exposure time. Excitation light from a C-HGFI Intensilight lamp (Nikon) was passed through a 438/24 nm filter and an FF458-DiO2 dichroic (Semrock). Emitted light was passed to a DC/T510LPXRXTUf2 dichroic filter in the TwinCam adaptor cube and then through 483/32 nm (CFP) or 542/27 nm (YFP) filters before collection on the cameras.Five to ten young adult transgenic animals (<24 hr old) expressing the YC2.60 (URX) or the YC3.60 (AQR and PQR) Cadb104 mutants and matched controls were imaged on an Axiovert 200 microscope (Zeiss) with a 40x NA 1.2 C-Apochromat objective using an EMCCD Evolve 512 Delta camera (Photometrics), which gave higher signal-to-noise than the Nikon AZ100.The imaging protocol was performed as reported for URX, except that 2+ sensor specifically in ADL, as previously described (2+ imaging used a 40x NA 1.2 C-Apochromat lens on an Axiovert 200 microscope (Zeiss) equipped with a Dual View emission splitter (Photometrics) and an Evolve 512 Delta EMCCD camera (Photometrics). Acquisition was at 2 frame-per-second (fps) with a 100ms exposure. Excitation light was from a Lambda DG-4 (Sutter Instruments) and was passed through an excitation filter , and a dichroic filter for GCaMP and RFP. A beam splitter  was used to separate the GCaMP and RFP signal using a dichroic filter 514/30–25 nm (GFP) and 641 nm (RFP) (Semrock).We used olfactory chips  to image young transgenic adults (<24 hr old) expressing the GCaMP3 Caescribed . Animalssrh-220p, in a bi-cistronic construct. Animals were immobilized with Dermabond glue, placed under a PDMS chamber and imaged on the same microscope and imaging setup used to image pheromone-evoked Ca2+ in ADL. Prior to recording activity, animals were pre-stimulated for 3 min to extinguish light-evoked ADL responses. Acquisition was at 2 frame-per-second (fps) with a 100ms exposure. O2 concentration was switched between 7% and 21% O2 every 2 min.We imaged young transgenic adults (<24 hr old) co-expressing GCaMP6s and mKate2 from the ADL-specific https://github.com/neuronanalyser/neuronanalyser .All recordings were analysed using Neuron Analyser, a custom-written MATLAB program available at 2 taken 30-s before the first 21% O2 stimulus (baseline) were subtracted from YFP/CFP values 30-s before the end of the first 21% O2 stimulus (stimulus). This ratio was normalized by dividing with the baseline. Values calculated for each biological replicate were entered into Prism for statistical analysis. For ADL pheromone responses, average GCaMP3 intensity was calculated 5-s before the C9 stimulus was removed (F) and 5s before the C9 stimulus was presented (F0). ADL pheromone-evoked Ca2+ responses were calculated as 2-evoked responses, a pseudo-ratio of GCaMP6s over mKate2 signal was computed to account for changes in GCaMP6s intensity due to animal movement. O2-evoked Ca2+ responses were calculated using the average GCaMP6s/mKate2 signal for a 15-s window centered around the peak of the response and a 30-s window before stimulation with 21% O2 (R0) and expressed as Bar graphs showing srh-220p:mKate2 were washed 5× in M9 buffer to remove bacteria and then dissociated as described  together with a coelomocyte marker (unc-122p:gfp). To assay the effects of O2 levels on ADL neurosecretion we grew animals form egg to young adult either at ambient O2 (21% O2) or at 7% O2 using a hypoxic chamber . To quantify neurosecretion, we imaged the most anterior pair of coelomocytes on a TE-2000 (Nikon) or a Ti2 (Nikon) wide-field microscope using a 10x air lens. Z-stack images were taken at subsaturating exposure for both GFP and mCherry intensities and analysed using ImageJ. Coelomocytes were delineated using the GFP signal and the mCherry signal measured in the same area. Values were plotted as arbitrary units of intensity.We quantified neurosecretion in young transgenic adults (<24 hr old) expressing DAF-28::mCherry specifically in ADL neurons (srh-220 promoter construct we imaged wild-type and qui-1 mutants carrying a transgene expressing mKate under the control of the srh-220 promoter (srh-220p:mKate). We imaged the ADL cell body using a Ti2 (Nikon) wide-field microscope using a 40x air lens. Z-stacks were taken without saturating the mKate signal. The boundary of the ADL cell body was taken and intensities extracted using ImageJ. Data were plotted using Prism.To validate the srh-220p (srh-220p:ida-1::gfp). Simultaneously, we specifically highlighted ADL by expressing cytosolic mKate (srh-220p:mKate2). Young adult double transgenic animals were imaged on a Ti2 (Nikon) wide-field microscope using a 40x air lens. Z-stack images were taken at subsaturating exposure for both GFP and mKate. We delineated the boundaries for the cell body and axon of ADL using the mKate signal, and measured signals in mKate+ pixels in the GFP channel using ImageJ. Values were plotted in Prism, in arbitrary units.To image DCVs the coding sequence of IDA-1, a DCV marker, was fused to GFP and expressed in the ADL pair of neurons using the ADL-specific promoter qui-1 we knocked in DNA encoding mNeonGreen in frame just upstream of the qui-1 initiation codon. We imaged young adult hermaphrodites using a Ti2 (Nikon) microscope equipped with a DragonFly (Andor) spinning disk module and an EMCCD camera  with 40x or 60x objectives. Z-stacks of images acquired with subsaturating exposure times were analysed using ImageJ.To examine the expression and subcellular localization of srh-220p and DiO intensities extracted using ImageJ. Data were plotted as arbitrary units (A.U.) using Prism.The ability of amphid neurons, including ADL, to take up the lipophilic dye DiO, was used as a proxy to monitor OSM-6::AID functionality. Briefly, worms were incubated with the DiO dye (10 μg/ml in M9 buffer) for 3 hr, and then transferred to a fresh plate for 1 hr to remove excess dye. To monitor amphid neuron dye filling, animals were inspected under a stereomicroscope . To monitor ADL dye filling in detail, animals were imaged on a Ti2 (Nikon) wide-field microscope using a 40x objective. Z-stack images were taken at subsaturating exposure for both DiO, imaged with a standard GFP filter, and tagBFP. To quantify ADL dye filling, the boundary of ADL cell body was taken using the tagBFP marker driven but the ADL-specific promoter https://github.com/lmb-seq/cross_filter; RRID SCR_022008 v1). Briefly, reads were checked for quality and aligned to the C. elegans reference genome. Lists of mutations for each sequenced strain were then cross-referenced with a compiled list of background mutations to generate a list of strain-specific mutations.Whole-genome sequence data were analysed using a custom Python script, Cross_filter (C. elegans transcriptome (EnsemblMetazoa: release 46) and C. elegans genome (WBcel235) as decoy. We performed differential gene expression analysis using tximport 1.14.2 and DEseq2 1.26.0. Output from these programs was imported into a custom-made R program  to visualize differentially expressed genes across genotypes. EnrichmentBrowser 2.16.1 was used to aggregate the enrichment of gene ontology (GO) terms from the following algorithms: Overrepresentation Analysis (ORA), Gene Set Enrichment Analysis (GSEA), and Gene Set Analysis (GSA). Beside the classic GO term annotation, we functionally annotated C. elegans neural genes using annotations from previously published reviews . When speed plots or Ca In this study Valperga and de Bono make the intriguing observation that interfering with the sensory function of a nociceptive neuron, termed ADL, alters its gene expression programs causing a reconfiguration of its functions. Upon loss of its properties as a primary sensor ADL gets repurposed by oxygen sensory circuits to enhance neurosecretion in an environmental oxygen dependent manner; thereby it adopts some interneuron like properties. The study is an interesting example of cross modal plasticity in neuronal circuits. It enables future studies on the ethological function of this phenomenon. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:Caenorhabditis elegans" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Piali Sengupta as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ilona C Grunwald Kadow (Reviewer #2).Thank you for submitting your article "Impairing one sensory modality enhances another by reprogramming peptidergic circuits in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:(1) The reviewers are not convinced that the results should be interpreted as evidence for cross modal plasticity (see public review of reviewer #3). The main concern is that the qui-1 mutant background does not distinguish between a specific effect on ADL's sensory properties and a more general switch that concomitantly upregulates neurosecretion and thereby elevated behavioural responses to oxygen. If the authors insist in their interpretation, an orthogonal approach should be taken. A cell specific knockdown of ocr-2 in ADL, either via RNAi or a transgenic Cre-lox strategy, would be a more specific way to disrupt the sensory properties of ADL. Such a manipulation should lead to the same effect, i.e. up-regulation of neurosecretion and elevated behavioural responses to 21% oxygen.(2) The authors did not exclude the possibility that other oxygen sensory neurons, e.g. AQR, PQR or BAG show altered responses in qui-1 mutants and could thereby explain the enhanced behavioral responses. In addition to URX and RMG, they should record AQR, PQR and BAG activity in wild-type and qui-1 mutants.(3) The authors suggest a rather detailed model while providing only partial evidence for it. The authors convincingly show that neurosecretion from ADL is up-regulated in qui-1 mutants, but this could be a sheer correlation with the behavioral phenotype.They should perform ADL cell ablations and in addition interfere with ADL neurosecretion, e.g. by expressing tetanus toxin, both in in qui-1 mutant background. The model predicts that these manipulations will suppress the enhanced behavioral responses to high oxygen.In this vein, the behavioral responses to 21% O2 of the strains qui1;npr-22, qui;npr-22 + ADLp:npr22 rescue, ADLp:npr-22 OE, and qui-1 tkr-1 should be analyzed to further support the model.(4) There is a large variability in the behavioral assay. For example, in Figure 1D qui-1 mutants show an elevated 21% O2 evoked response, however in Figure 1-S1B the mutants' major phenotype seems to be reduced speed at 7% O2. This would have a strong implication in how to interpret the data and requires further discussion. Related to this, quantifying locomotion speed responses as fold change with respect to the 7% condition cannot distinguish between effects on baseline speed at 7% and effects on 21% evoked response magnitude. Additional quantifications of absolute speed should be provided.(5) The authors start their introduction by explaining how the loss of vision, for instance, can lead to the repurposing of neurons and even the rewiring of neural circuits. The presented data, however, describes a cell-autonomous mechanism that increases neuropeptide receptor expression and thereby increases peptide release. The exact relationship between (1) increased O2 sensitivity, (2) increased receptor expression and (3) increased peptide release is not fully explained by the data. This is also evidenced by their model in Figure 6. I suggest that the authors still integrate all of their findings in their model but indicate what remains not fully elucidated.For instance, we suggest including URX etc. into your model, because this is the gained sensory modality that makes the whole paper more interesting.(6) The issue of possible background mutations influencing the ADL transcriptomic analysis should be addressed. Were the mutants sufficiently outcrossed? This point could be addressed by providing additional transcriptome data from the strains generated in response to (1), different alleles or fully outcrossed lines.(7) It should be stated whether the qui-1 mutant was derived from an N2 or flp-21 background, or in other words, what is the wild type.The label wild type (WT) is applied at least to 4 different genomic strains . This can be confusing for the audience and it is not specified in the methods section. A table of the strains used in the study would be much appreciated. Should be also mentioned that N2 is not a 'wild' strain.Related: The label wild type (WT) is applied at least to 4 other different genomic strains . This can be confusing for the audience and it is not specified in the methods section. A table of the strains used in the study would be much appreciated. Should be also mentioned that N2 is not a 'wild' strain.(8) It would be helpful if the authors could comment about why they think that ADL O2 responses disappear in qui-1 mutants, and how O2 information received through NPR-22 directly induces enhanced neuropeptide release without involving any detectable calcium signals.(9) In a previous research article of the lab, they showed strong responses in ADL to 21% oxygen in npr-1 background, and that ADL is unresponsive in N2 background. Contrarily, there seems to be a significant response in Figure 2E. This should be at least mentioned and discussed.(10) we am not a fan of the work 'reprogram'. We implicates an epigenetic mechanism, and we don't think you've shown that. We suggest finding another expression.(11) Two of the presented mutants are loss-of-functions of genes expressed in glia cells. I actually found this aspect very interesting given that these mutants phenocopy qui-1 in several aspects. We would like to hear more in the discussion about how the authors interpret these data.(12) Figure 1: the panels don't correspond to the text (13) The term hyperpolarization could be misleading in the context of calcium imaging .(14) Would a single copy fig-1 gDNA rescue fig-1?(15) "These data suggest that ADL neurons release more DCVs in bbs, wrt-6, and fig-1 mutants than wild type animals in response to input from the O2 circuit." The response to the O2 circuit was directly shown only for bbs-7 (in addition to qui-1).(16) Figure 5A: we assume each column is an independent repetition?Also, perhaps we got this wrong, but the scales in Figure 5 and S5 show a negative log2 fold change when the expression level had actually increased from WT to qui-1? Essential revisions:(1) The reviewers are not convinced that the results should be interpreted as evidence for cross modal plasticity (see public review of reviewer #3). The main concern is that the qui-1 mutant background does not distinguish between a specific effect on ADL's sensory properties and a more general switch that concomitantly upregulates neurosecretion and thereby elevated behavioural responses to oxygen. If the authors insist in their interpretation, an orthogonal approach should be taken. A cell specific knockdown of ocr-2 in ADL, either via RNAi or a transgenic Cre-lox strategy, would be a more specific way to disrupt the sensory properties of ADL. Such a manipulation should lead to the same effect, i.e. up-regulation of neurosecretion and elevated behavioural responses to 21% oxygen.2-escape behaviour . This may reflect a broader role for OCR-2 in ADL than sensory transduction. Disrupting OSM-6 truncates sensory cilia and severely compromises many chemosensory responses, but only weakly reduces aggregation and O2 responses.We thank our reviewers for their comments and suggestions. We have sought to test our model with a functional experiment that selectively disrupts sensory input into the ADL neurons. To achieve this, we decided to knock down a protein required for intraflagellar transport, OSM-6, rather than the OCR-2 TRP channel subunit. OCR-2 mediates not only pheromone responses in ADL, but also Oosm-6 locus, and expressed TIR1 in ADL to achieve cell-specificity. TIR1 is required for AID. We have added the new data to Figure 4F–G and Figure 4—figure supplement 2. We show that expressing TIR1 in ADL disrupts OSM-6::AID function both in the presence and absence of Auxin. This agrees with recent work that tested the efficiency and specificity of the AID system . A partial OSM-6::AID reduction in ADL recapitulates many of the phenotypes of qui-1 mutants, including increased neurosecretion from ADL, heightened ADL responses to O2 inputs and a small but significant enhancement of the O2-escape response. We think these new data support our interpretation that a change in ADL’s sensory properties leads to heightened response of ADL neurons to O2 inputs, a phenotype observed in qui-1 and multiple other sensory defective mutants and a hallmark of cross-modal plasticity. However, the effects of knocking down osm-6 on ADL function also appear to be complex, as the stronger osm-6 knockdown achieved by adding auxin to the osm-6::AID knockin animals expressing TIR1 in ADL, unexpectedly gives weaker phenotypes than when auxin is absent.To target OSM-6 degradation specifically to the ADL neurons we knocked in DNA encoding an Auxin Inducible Degron (AID) into the (2) The authors did not exclude the possibility that other oxygen sensory neurons, e.g. AQR, PQR or BAG show altered responses in qui-1 mutants and could thereby explain the enhanced behavioral responses. In addition to URX and RMG, they should record AQR, PQR and BAG activity in wild-type and qui-1 mutants.2 sensors such as AQR, PQR and BAG. We focused on URX and RMG since they are connected with ASH and ADL neurons in a hub-and-spoke circuit. We have now imaged O2-evoked Ca2+ responses in AQR and PQR in qui-1 mutants using the Ca2+ sensor YC3.60, and incorporated the data in Figure 1—figure supplement 1D and 1E. In both neurons, the responses of qui-1 and wild-type animals are comparable. This suggests the enhanced O2-escape behaviour of qui-1 mutants is not explained by increased activity of URX, AQR, PQR or RMG O2-sensing neurons.Our reviewers are right that we did not probe the contribution of other O2-evoked Ca2+ responses. These responses are smaller than in other O2 sensors and require a different microscope set-up capable of higher magnification, and we have encountered technical difficulties in setting up this system.We regret we have not been able to image BAG’s O(3) The authors suggest a rather detailed model while providing only partial evidence for it. The authors convincingly show that neurosecretion from ADL is up-regulated in qui-1 mutants, but this could be a sheer correlation with the behavioral phenotype.They should perform ADL cell ablations and in addition interfere with ADL neurosecretion, e.g. by expressing tetanus toxin, both in in qui-1 mutant background. The model predicts that these manipulations will suppress the enhanced behavioral responses to high oxygen.In this vein, the behavioral responses to 21% O2 of the strains qui1;npr-22, qui;npr-22 + ADLp:npr22 rescue, ADLp:npr-22 OE, and qui-1 tkr-1 should be analyzed to further support the model.qui-1 mutants display enhanced O2-escape behaviour. We have now updated the Discussion to clarify that this mechanism remains unclear, especially in the light of further experiments suggested by our reviewers.Our reviewers are correct that in our model we speculated as to why 2-evoked DCV release from ADL neurons and enhanced O2-escape behaviour . Follow up experiments with osm-6::AID knockin animals that selectively perturb ADL cilia function by expressing TIR1 in this neuron recapitulate these phenotypes (See Point 1). As suggested by our reviewers, we probed the molecular mechanism linking ADL output to O2-escape behaviour. We disrupted ADL neurosecretion in qui-1 mutants by selectively expressing tetanus toxin (TeTx) in this neuron, and assayed O2-evoked escape behaviour . The TeTx expressing qui-1 animals retained elevated O2-escape; this suggests that elevated neurosecretion does not, on its own, explain their altered behaviour. We previously showed that ablating ADL and ASH neurons disrupts aggregation in npr-1 animals , confirming that these neurons are important for this behaviour. We had also shown that expressing TeTx in ASH and ADL neurons in npr-1 mutants has only a small effect on O2-escape response, although it reduced by about half the reversal response evoked by 3 mM Cu2+ .Our work identified multiple mutants that concomitantly exhibit O2-escape behaviour in qui-1;npr-22 or qui-1;tkr-1 double mutants compared to qui-1 controls, or in transgenic animals over-expressing NPR-22 compared to wild-type . Together, these data suggest removing npr-22, tkr-1 or blocking ADL’s synaptic output, is not sufficient to account for the O2-responses of qui-1 mutants. Most likely, communication through gap junctions is also important for the O2-escape response of qui-1 mutants. Further experiments are clearly required to test this model.We also did not observe any change in O(4) There is a large variability in the behavioral assay. For example, in Figure 1D qui-1 mutants show an elevated 21% O2 evoked response, however in Figure 1-S1B the mutants' major phenotype seems to be reduced speed at 7% O2. This would have a strong implication in how to interpret the data and requires further discussion. Related to this, quantifying locomotion speed responses as fold change with respect to the 7% condition cannot distinguish between effects on baseline speed at 7% and effects on 21% evoked response magnitude. Additional quantifications of absolute speed should be provided.2-escape assays. This is something we have observed before. The biggest source of variability appears to be the thickness of the bacterial lawn on which animals are either grown or assayed. For this reason, all our assays are day-matched – different genotypes are grown together and assayed on the same day, on plates seeded at the same time. All our Figures also include data obtained on at least three different days.Our reviewers are right in pointing out that there is some variability in animals’ speed during Oqui-1 mutants and N2 animals differ in their responses to both 21% and 7% O2: not only are qui-1 animals more strongly aroused by 21% O2 but they also move more slowly than WT at 7% O2. Other aggregation mutants, including npr-1, show these same phenotypic differences from N2. Disrupting the O2 responses of npr-1 animals, for example by deleting genes encoding the molecular O2 sensors GCY-35 and GCY-36, confers N2-like behavior: npr-1;gcy-35 and npr-1;gcy-36 animals move slower than npr-1 at 21% O2 but faster at 7% O2. We think the intermediate speed of N2 animals  at 7% O2 reflects circuit plasticity; the persistent low activity of the O2-sensing circuit in these animals renders the locomotory circuit more sensitive to input from other circuits. By contrast, in npr-1 and qui-1 mutants kept at 21% O2 the locomotory circuit is adapted to persistent high input form the O2 sensing neurons; when this input is removed, by shifting animals to 7% O2, the locomotory circuit is less sensitive to input from other circuits – leading to animals moving slowly. Consistent with this interpretation, npr-1 animals kept for long periods at 7% O2 gradually begin to move faster at this O2 concentration. Moreover, when such animals are stimulated by 21% O2, they reach significantly higher speeds than animals habituated to 21% O2.The reviewers also correctly point out that C. elegans’ speed at 21% O2 and 7% O2 is a good proxy for the functionality of the O2 circuit in regulating the locomotory circuit.This is why we think the ratio of 2. However, we think the observations described above justify using the change in the animals’ speed as we switch them from low to high O2 as a proxy for changes in the activity of the O2 circuit. We have outlined this reasoning in the Method Section.We acknowledge that the circuits controlling locomotory activity are complex, and regulated by sensory modalities other than O(5) The authors start their introduction by explaining how the loss of vision, for instance, can lead to the repurposing of neurons and even the rewiring of neural circuits. The presented data, however, describes a cell-autonomous mechanism that increases neuropeptide receptor expression and thereby increases peptide release. The exact relationship between (1) increased O2 sensitivity, (2) increased receptor expression and (3) increased peptide release is not fully explained by the data. This is also evidenced by their model in Figure 6. I suggest that the authors still integrate all of their findings in their model but indicate what remains not fully elucidated.For instance, we suggest including URX etc. into your model, because this is the gained sensory modality that makes the whole paper more interesting.2-sensory neurons (including URX). We have also added question marks to our model to highlight unknown molecular mechanisms that need to be investigated in future work. Namely, the molecular mechanisms that reconfigures the expression profile of ADL, and the signal from O2-sensing neurons that supports ADL’s O2-evoked neurosecretion after sensory impairment.We thank the reviewers for their comment. We agree that our model suggests that cell-autonomous changes in ADL alters ADL responsiveness to input from other neurons, notably URX. We have updated our model in Figure 6 to indicate O(6) The issue of possible background mutations influencing the ADL transcriptomic analysis should be addressed. Were the mutants sufficiently outcrossed? This point could be addressed by providing additional transcriptome data from the strains generated in response to (1), different alleles or fully outcrossed lines.qui-1 related phenotypes. We regret we did not explicitly address this point in our initial submission. To remove background mutations, mutants isolated in our screen, including qui-1, were backcrossed with the N2 laboratory strain a minimum of four times. These qui-1 animals were further crossed into a 5 times outcrossed line that expresses the fluorescent protein mKate specifically in ADL, to generate the strains from which we sorted ADL neurons by FACS. Mutant and transgenic strains were outcrossed using the N2 laboratory strain. We explain this in the Methods section of the revised manuscript.We worried about unspecific effects of background mutations both on the ADL transcriptome and on other qui-1 samples in ADL are due to the absence of qui-1. Supporting this, both mutations in neuropeptide receptors identified by our profiling, npr-22 and tkr-1, suppress ADL’s elevated neurosecretion. Nevertheless, we have added a note to explicitly bring up the concern raised by our reviewers, that some transcriptional differences could be the result of background mutations.The extensive outcrossing make us confident that the large majority of differentially regulated genes between wild-type and (7) It should be stated whether the qui-1 mutant was derived from an N2 or flp-21 background, or in other words, what is the wild type.The label wild type (WT) is applied at least to 4 different genomic strains . This can be confusing for the audience and it is not specified in the methods section. A table of the strains used in the study would be much appreciated. Should be also mentioned that N2 is not a 'wild' strain.Related: The label wild type (WT) is applied at least to 4 other different genomic strains . This can be confusing for the audience and it is not specified in the methods section. A table of the strains used in the study would be much appreciated. Should be also mentioned that N2 is not a 'wild' strain.qui-1 mutant from the N2 parental strain. We have now inserted a line in the Results section of the revised manuscript to say this.We isolated the C. elegans genetic nomenclature can be confusing to non-specialist readers. To address this issue and ensure we do not mislead our readers, we opted for the term “Control” in Text and Figures while referring to strains we used to image Ca2+ responses, ADL neurosecretion, IDA-1 localisation and analyses of ADL profiling, while also reporting the genotype of each control line in the respective Figure legends. We have also compiled a Strain List (Supplementary File 6) listing the genotype of all strains used in the paper and detailing the figure in which they have been used, to help readers track different control strains.We agree that our use of “WT” is misleading. We used this term in an attempt to keep figures accessible since We also highlight in the revised version of the Results section that the N2 laboratory strain has accumulated mutations since the original wild strain was domesticated more than 60 years ago.(8) It would be helpful if the authors could comment about why they think that ADL O2 responses disappear in qui-1 mutants, and how O2 information received through NPR-22 directly induces enhanced neuropeptide release without involving any detectable calcium signals.2-evoked responses in ADL disappear in qui-1 mutants. One possibility is that ADL becomes less excitable due to the reconfigured gene expression associated with loss of qui-1 in ADL. This model would predict that selectively knocking down qui-1 in ADL would confer the same Ca2+ response phenotype. Blocking ADL neurosecretion with TeTx in qui-1 mutants would test if the increased ADL neurosecretion we describe feeds back to reduce the O2-evoked Ca2+ response in ADL. An alternative hypothesis is that the effect of disrupting qui-1 is non-cell-autonomous, altering excitatory or inhibitory input to ADL from other qui-1 expressing neurons. We have not tested if neurosecretion from other qui-1-expressing neurons is altered in qui-1 mutants.We can only speculate why Oqui-1 leads to loss of a measurable O2-evoked Ca2+ response in ADL, these neurons display elevated O2-evoked neurosecretion in qui-1 mutants. This implies that some O2-evoked Ca2+ responses are retained in ADL’s axons in qui-1 mutants. It also suggests that other second messengers upregulate neurosecretion. Elevating cAMP, for example, can promote dense-core vesicle release more efficiently than increasing Ca2+ levels . Altered G-protein coupled receptor signalling could lead to elevated cAMP levels and increased neurosecretion in qui-1 mutants. It is worth noting that in N2 controls, ADL does not display O2-evoked neurosecretion despite showing measurable Ca2+ responses.Strikingly, while disrupting (9) In a previous research article of the lab, they showed strong responses in ADL to 21% oxygen in npr-1 background, and that ADL is unresponsive in N2 background. Contrarily, there seems to be a significant response in Figure 2E. This should be at least mentioned and discussed.2+ sensors. Fenk et al. imaged ADL’s O2-evoked Ca2+ response in the N2 laboratory strain and npr-1 mutants using GCaMP3; in this paper we use GCaMP6s an improved version of GCaMP3 . The 10-fold increase in signal intensity given by GCaMP6s compared to GCaMP3 likely explains why our imaging experiments detect Ca2+ responses not observed by Fenk et al. We have added a note to say this.Our reviewers are correct to note the difference. This likely reflects use of improved Ca(10) we am not a fan of the work 'reprogram'. We implicates an epigenetic mechanism, and we don't think you've shown that. We suggest finding another expression.We borrowed the term “reprogramming” to denote a change in ADL’s proprieties, but did not wish to imply that epigenetic mechanisms were involved. Given that what we report changes in gene expression, we can see that such a misunderstanding could easily arise. To avoid confusion, we have removed the word “reprogramming” from the revised manuscript, and simply refer to changes in gene expression.(11) Two of the presented mutants are loss-of-functions of genes expressed in glia cells. I actually found this aspect very interesting given that these mutants phenocopy qui-1 in several aspects. We would like to hear more in the discussion about how the authors interpret these data.wrt-6 and fig-1, phenocopy qui-1. We have revised the Discussion to expand on this observation.We agree that it is interesting that mutants for two genes expressed in glial cells, (12) Figure 1: the panels don't correspond to the text We apologise for this oversight. We have corrected the text.(13) The term hyperpolarization could be misleading in the context of calcium imaging .2 stimulation increases ADL’s Ca2+ levels, in qui-1 mutants the same stimulus seems to reduce Ca2+ levels.We have corrected the text. We now state that while in wild-type animals O(14) Would a single copy fig-1 gDNA rescue fig-1?fig-1 gene would rescue phenotypes associated with this mutant. In the absence of this rescue experiment, our analysis of multiple fig-1 alleles gives us confidence that impairing fig-1 increases O2-escape behaviour.Yes, our prediction would be that a single copy insertion of the wild-type (15) "These data suggest that ADL neurons release more DCVs in bbs, wrt-6, and fig-1 mutants than wild type animals in response to input from the O2 circuit." The response to the O2 circuit was directly shown only for bbs-7 (in addition to qui-1).bbs-7 and qui-1 mutants increased ADL neurosecretion reflects increased responsiveness to O2 input. We also suggest that enhanced neurosecretion from ADL in other sensory defective mutants characterised in this study may similarly reflect increased responsiveness to O2-input. We believe data from our OSM-6::AID knockdown experiments support this interpretation.Thank you for pointing out this misleading statement. We have reworded this statement to say that (16) Figure 5A: we assume each column is an independent repetition?Also, perhaps we got this wrong, but the scales in Figure 5 and S5 show a negative log2 fold change when the expression level had actually increased from WT to qui-1?That is correct. We apologise for the oversight. Figure legends now state that each column in heat maps represents a biological replicate.References:Caenorhabditis elegans is induced by neurons that detect aversive stimuli. Nature 419:899–903. doi:10.1038/nature01169de Bono M, Tobin DM, Davis MW, Avery L, Bargmann CI. 2002. Social feeding in Steuer Costa W, Yu S, Liewald JF, Gottschalk A. 2017. Fast cAMP Modulation of Neurotransmission via Neuropeptide Signals and Vesicle Loading. Curr Biol 27:495–507. doi:10.1016/j.cub.2016.12.055Caenorhabditis elegans. Biorxiv 2021.08.06.455414. doi:10.1101/2021.08.06.455414Hills-Muckey K, Martinez MAQ, Stec N, Hebbar S, Saldanha J, Medwig-Kinney TN, Moore FEQ, Ivanova M, Morao A, Ward JD, Moss EG, Ercan S, Zinovyeva AY, Matus DQ, Hammell CM. 2021. An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in C. elegans. eLife 4:e1004156. doi:10.7554/eLife.04241Laurent P, Soltesz Z, Nelson GM, Chen C, Arellano-Carbajal F, Levy E, de Bono M. 2015. Decoding a neural circuit controlling global animal state in Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL. 2009. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods 6:875–881. doi:10.1038/nmeth.1398


The patients were evaluated using speech perception (SP) measured in quiet  and noise . The subjective hearing ability was assessed using Oldenburg inventory (OI). The Fazekas PVWM score predicted postoperative speech perception two years after CI in the mid-age population. The periventricular white matter lesions (PVWM) could explain 27.4% of the speech perception (FMT) variance. Our findings support the hypothesis about the influence of pre-existing WMLs on CI outcome. We recommend the evaluation of Fazekas score as a predictive factor for post-implantation hearing ability.The presented prospective study investigated whether structural brain damage, measured with the Fazekas score, could predict hearing rehabilitation outcomes with cochlear implantation (CI). With a follow-up period of 24 months, this study included 49 bilaterally, postlingually hearing impaired CI candidates for unilateral CI  older than 50 at the time of implantation. The differences in the predictive value between two age groups, 50–70 year-olds (mid-age;  The socio-demographic challenge of the 21st century includes the ongoing growth of the aging population. Unfortunately, extended life expectancy correlates with an increased prevalence of dementia and hearing impairment, the latter affecting 66% of people over 70 . On the In 2017, the Lancet Commission on Dementia Prevention, Intervention and Care performed an extensive investigation of risk factors associated with dementia . That woIn light of current research findings, auditory rehabilitation is gaining importance in medical care, but despite that, according to the WHO , only arLin et al.  demonstrInterestingly, not only the primary auditory cortex in the temporal lobe is affected by auditory deprivation but also the associated brain regions such as speech memory ,24. In eMagnetic resonance imaging (MRI) can be used to evaluate brain volume and brain aging. This approach was used in current research evaluating hearing impairment and cognitive dysfunction ,28,29,30One particular cause of structural brain damage can be a small lesion of cerebral blood vessels , frequenThe present study aimed to determine a predictive link between the Fazekas score and the outcome of hearing rehabilitation with a cochlear implant. The second aim was to determine possible differences in the Fazekas score predictive value between the mid-life and elderly populations.The local Ethics Committee of Charité—Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health approved this prospective, non-interventional, longitudinal study (permit number EA2/030/13). All investigations were conducted according to the principles expressed in the Declaration of Helsinki. All patients gave their informed written consent. Forty-nine patients aged 50 years or older, 20 male and 29 female, were included in our study. The entire study population was divided into 50 to 70 years (mid-age), including 10 males and 16 females, and the second group of 70 years and older (elderly), including 10 males and 13 females. Detailed age-related patient characteristics are presented in diagnosis of bilateral severe or profound hearing loss; speech perception ≤ 40% in the Freiburg monosyllabic test in quiet at 65 dB sound pressure level (SPL) with hearing aids of individually verified settingsage over 18 yearsmother tongue GermanMRI-scan of the head and inner ear before surgery○post-lingual deafness (onset of hearing impairment after speech acquisition)○exclusion of disorders affecting the vestibulocochlear nerve ○unremarkable cochlear anatomy○possibility to use general anesthesia○the motivation for postoperative audiological rehabilitationmeeting of the clinical criteria for cochlear implantation:Patients were consecutively, prospectively enrolled in the study based on the following inclusion criteria:The data were collected between April 2009 and March 2016. The patients were referred to the hospital between April 2009 and March 2014 for unilateral cochlear implantation with a multichannel cochlear implant produced either by MED-El  or by Cochlear . Before surgery, all patients underwent computed tomography (CT) and cranial magnetic resonance imaging (cMRI) of the temporal bone and the auditory nerve and central nervous system to exclude retro-cochlear disorders.Routine cMRI examinations before cochlear implantation were performed using a 1.5-T imaging system . A specific MRI imaging protocol for neurodegenerative disorders was applied. In detail, the MRI-sequences used for WML assessment consisted of axial PD/T2-weighted imaging , and coronal FLAIR imaging .To assess possible structural brain damage, a routine scoring of T2 hyper-intense white matter lesions visualized by MRI was conducted according to the Fazekas score . The assPeriventricular white matter lesions (PVWM)0 = absent1 = “caps” or pencil-thin lining2 = smooth “halo”3 = irregular periventricular signal extending into the deep white matterDeep white matter lesions (DWM)0 = absent1 = punctate foci2 = beginning confluence3 = large confluent areasThe Fazekas total score is a sum of the PVWM score and the DWM score . The MRI scanning was performed before cochlear implantation. During the follow-up examinations at 6, 12, and 24 months post-surgery, audiological tests and subjective audiological assessment were conducted in parallel.After the cochlear implantation, MRI was not performed because the implants were not suitable for magnetic resonance imaging, or the presence of an implant would result in an extinction artifact in the MRI image.The speech perception in quiet was assessed with the Freiburg monosyllabic test (FMT) at 65 dB SPL (sound pressure level). Before surgery, the test was performed with an individually fitted, conventional hearing aid, whereas after surgery, with the cochlear implant speech processor. The higher the measured value between 0% and 100%, the better the speech perception.S/N), at which the percentage of correct word score is 50% . In detail, twenty test sentences per list were presented in a random combination with a fixed scheme . Two test runs were performed before the evaluation run. With a constant background noise level of 65 dB, the sound level was adapted stepwise to the number of correctly understood words. For sentences 2–5, the sound pressure level was adjusted by +3 and −3 dB depending on the percentage of words understood (0 to 5). For sentences 6 to 21, the sound pressure level was modulated by +/−2 dB. For the evaluation, the SPL used for sentences 12 to 21 were added and divided by 10. Finally, the background noise level was subtracted from this result in order to obtain the presented result (SVS).The Oldenburg sentence test (OLSA) measures the speech intelligibility threshold in background noise at 65 dB SPL. The standardized, broadband “Oldenburg noise” was used as background noise during the measurement, following previously described procedure . Each se0N0 (speech and noise from the front) was used. The OLSA was used postoperatively with CI, as it enables an excellent approximation of everyday situations in background noise in rehabilitation. The reference value for persons with normal hearing is −7.1 ± 1.1 dB S/N (65 dB SPL), with a lower value indicating better speech perception [The configuration Srception .For the evaluation of the subjective hearing ability, the OI questionnaire was used. The OI comprises a total score and three subcategories “hearing in quiet”, “hearing with background noise”, and “localization hearing”. There are 12 questions regarding everyday situations with scores from 1 to 5. The higher the score, the better the subjective hearing .Sp). The predictors of speech perception were identified using multiple linear regression with backward elimination. The requirements for implementation were checked. The test for the normal distribution of the residuals was performed using SPSS.For the statistical analyses, we used software SPSS version 25.0 . The results are shown as a mean ± standard deviation. The level of significance was 5%. Because of a lack of normal distribution in most of the dataset, Wilcoxon signed-rank test was used to compare the data before and after cochlear implantation. The statistical comparisons were made using the Mann-Whitney U-Test. Correlations were performed by computing the Spearman’s rank correlation coefficient (rp < 0.01) to 36.1% ± 31.3% in the entire cohort . Six months after implantation, it improved significantly  and two years  after cochlear implantation. The comparison between the groups, mid-age and elderly, indicated lack of significant difference . There was a significant difference in the OLSA scores obtained by the elderly group between 6 months and one year. However, the absolute difference between one year and two years was significantly greater. The value after two years was also associated with a significantly larger SD.Six months after implantation, the mean OLSA score in the entire cohort was 5.7 ± 5.00 dB Comparing the mid-age and elderly groups revealed a lack of significant differences .An age-group-specific rank correlation analysis was carried out to determine the degree of association between the participants’ age and speech perception. For 50 to 70-year-olds, there is no significant correlation  between Sp = 0.666; p < 0.01).In the elderly group, no association between age and FMT could be determined . In contp < 0.01). When compared to the value before surgery, the improvement remained significant one year  and two years after implantation . A significant change in OI’s total score between half a year, one year, and two years after implantation was not found. The mid-age and elderly groups’ analysis also demonstrated significant improvement within all Oldenburg inventory domains six months after implantation (p < 0.01). Results remained stable over the whole follow-up period.The total score OI is a subjective assessment of the auditory abilities regarding hearing in quiet, hearing in noise, and sound localization. In the entire cohort, six months after implantation, the total score and the three subscales of OI improved significantly , despite absolute differences in the mean values. In the entire study population, the Fazekas total score  and the PVWM score  correlate significantly with the patients’ age. No correlations were found between Fazekas scores and age in the mid-age and elderly groups .In the entire cohort, the mean Fazekas total score in the cMRI before surgery was 1.2 ± 1.5, the mean PVWM score was 0.7 ± 0.9, and the mean DWM score was 0.5 ± 0.8. In the mid-age group, the Fazekas total score was 0.8 ± 1.2, whereas, in the elderly group, it was 1.7 ± 1.8 . The difMultiple linear regression with backward elimination was performed to examine the predictive value of Fazekas score concerning the post-implantation speech perception. When checking the basic requirements, the Fazekas total (the sum of PVWM and DWM), was excluded from further consideration due to a collinearity conflict. Thus, in addition to speech perception as a dependent variable, the variables PVWM, DWM, and age were examined as possible predictors for further consideration.The distribution pattern was determined using a histogram of the residuals. There was a normal distribution of the predictor PVWM in the mid-age group concerning speech comprehension two years after implantation. A borderline normal distribution was determined for the OLSA two years after implantation in the elderly group concerning age. In contrast, the distribution of PVWM values regarding OLSA and FMT half a year after implantation was not normal in the elderly group. Alternative models were examined but could not be reliably calculated due to the small study population. Therefore, PVWM was excluded from the modeling regarding OLSA and FMT values half a year after implantation.The regression analysis demonstrated that PVWM is a significant predictor for speech perception in quiet two years after CI for the mid-age group . The PVWFor the speech perception in noise (OLSA) in the elderly group, two years after implantation, the study participants’ age was a significant predictor . The patThe present work examined whether structural brain damage measured with the Fazekas score is related to bilateral speech understanding of severely hearing-impaired adult individuals who underwent unilateral cochlear implantation. It was further investigated whether Fazekas score could be used as a factor predicting postoperative speech understanding.To our best knowledge, this study demonstrates for the first time the age-dependent relationship between the Fazekas score PVWM and the speech perception in quiet after cochlear implantation in adults. White matter lesions measured with the Fazekas score were identified as predictors of postoperative speech perception in the mid-age group two years after cochlear implantation. The detailed analysis demonstrated that two years after CI, 27.4% of the speech perception in quiet (FMT) variance could be explained by the PVWM in the mid-age but not in the elderly. However, in the elderly group, a close relationship was seen between the patients’ age and the speech perception in noise. The age was analyzed in the multiple linear regression as a predictor for the OLSA. However, in the mid-age group, the age was not significantly associated with speech perception for either the FMT or the OLSA test scores.Speech perception improved significantly after cochlear implantation in both age groups after just six months, corroborating the already published reports ,16,37. AIn addition to the audiological assessment, structural brain damage was measured with the Fazekas score. The regression analysis included the PVWM, DWM, and age as parameters potentially controlling speech perception. Fazekas et al.  observedThe cohort division into a mid-age and the elderly population has become an established procedure in recent research. Mid-life is usually defined as beginning around the age of 50. The transition to the elderly cluster occurs between 65 and 70 ,40. The Various scoring systems are rating the WML, and the Fazekas scoring system is one of the commonly used ones. The neuroradiological Fazekas score was introduced for the MRI-based assessment of dementia and not exclusively to the potential damage to auditory brain regions. Consecutive neurological studies classified other cognitive functional disabilities and cerebral anomalies using MRI and the Fazekas score ,42.n = 672) was not adjusted for specific diseases, and vascular risk factors were reported in 49% and a stroke in approximately 14% of the participants. Another cohort of 639 patients aged 65 to 84 years has been described in the LADIS study [There are no published data concerning persons with bilateral, long-term, severe hearing impairment unilaterally implanted with CI and evaluated with the Fazekas score for white matter lesions to the best of our knowledge, making comparative analyses difficult. Valdés Hernández et al.  reportedIS study , evaluatRecent studies show that microstructural changes are already visible in the mid-life phase of life  and thatThe connection between hearing disorders and functional cognitive disorders was established in 1989 by Uhlmann et al. . CurrentAlthough complex medical and biomedical examinations became a part of adult hearing disorders’ elucidation, they mainly rule out other diseases and conditions. Predictors for hearing rehabilitation are not yet used in current clinical practice. Cochlear implantation’s clinical success is based on speech perception measured with various international tests lacking generally applicable criteria. Moreover, different parameters influencing the outcome of CI were reported. In addition to procedures presented in our study, using functional MRI has been suggested as an essential measure of the integrity of the central auditory pathway before implantation, predicting the CI outcome .To date, apart from the acknowledged negative association between audiological performance and duration of deafness, no other standardized, recognized predictors are in clinical use ,22. At tOur study’s significant drawbacks are the small size of the study sample and the variation in the cohort, which makes it difficult to compare the age groups or stratify the results based on gender. Another limitation is an unequal distribution of patients regarding gender. Both of the limitations could be addressed in a more extensive, better-powered study, in which patients equally representing both genders and different age groups would be included. The study population itself introduces another limitation. A CI allows only a limited postoperative MRI examination. Frequent MRI monitoring to assess the progression of structural brain damage is therefore not possible or very limited.The present study indicates the relationship between white matter lesions, hearing impairment, and WML’s possible effects on speech perception following cochlear implantation. The results also imply the influence of age or the limitations caused by aging with the consequence of structural brain damage in the elderly. However, in the mid-life phase, the connection between hearing impairment and speech perception seems to be partially accounted for by WML. Assuming the avoidable sensory deprivation as a trigger for WML, hearing rehabilitation is recommended for people with profound, bilateral hearing loss. With the CI, we have a tried and tested, and established method at our disposal. The extent to which the WML or its progress can be influenced by hearing rehabilitation remains withheld from future research. Further studies should confirm whether the Fazekas score can be used as a routine diagnostic marker to predict the outcome of hearing rehabilitation after cochlear implantation.


There is a lack of studies assessing how hearing impairment relates to reproductive outcomes. We examined whether childhood hearing impairment (HI) affects reproductive patterns based on longitudinal Norwegian population level data for birth cohorts 1940–1980. We used Poisson regression to estimate the association between the number of children ever born and HI. The association with childlessness is estimated by a logit model. As a robustness check, we also estimated family fixed effects Poisson and logit models. Hearing was assessed at ages 7, 10 and 13, and reproduction was observed at adult ages until 2014. Air conduction hearing threshold levels were obtained by pure-tone audiometry at eight frequencies from 0.25 to 8 kHz. Fertility data were collected from Norwegian administrative registers. The combined dataset size was N = 50,022. Our analyses reveal that HI in childhood is associated with lower fertility in adulthood, especially for men. The proportion of childless individuals among those with childhood HI was almost twice as large as that of individuals with normal childhood hearing (20.8% vs. 10.7%). The negative association is robust to the inclusion of family fixed effects in the model that allow to control for the unobserved heterogeneity that are shared between siblings, including factors related to the upbringing and parent characteristics. Less family support in later life could add to the health challenges faced by those with HI. More attention should be given to how fertility relates to HI. There is a scarcity of studies assessing how hearing impairment relates to reproductive outcomes. The effects of early life hearing impairment (HI) on fertility patterns has to our knowledge, never been analyzed using longitudinal population level data with objective measures of hearing. Longitudinal data with hearing observed early in life are necessary to study whether HI influences fertility in adult life. We use unique new data from the School Hearing Investigation in Nord-Trøndelag (SHINT), an audiometric screening of all schoolchildren attending regular schools in the County of Nord-Trøndelag from 1954 to 1986. Following the children for 28–60 years allow us to study how childhood HI relates to male and female childbearing patterns.1. Another US based study (N = 682 deaf adults and 602 hearing siblings) found that although marital rates were similar by hearing status, married deaf individuals had a lower number of children than married normal hearing individuals (2.1 vs. 2.3 children)2. The lack of representative population level studies calls for more inclusive assessments.The relatively few existing studies of hearing and fertility tend to use relatively small samples. E.g., one regional study in Sweden identified that cohort fertility was only 1.3 children among the deaf population compared to 1.6 children per woman for the general population—this was driven by very high levels of childlessness among the deaf women (affecting more than 4 out of 10)3. In children, most HI cases are due to congenital and neonatal conditions5. Hearing impairment and deafness have been found to be linked to specific genotypes6. Infections can also cause hearing impairment8; hearing loss among children is frequently a result of meningitis, measles, mumps or prenatal conditions including maternal rubella9. HI can be a side-effect resulting from other causes certain cancers11; injuries and traumas to the head8 as well as noise exposures, both temporary and long-term13.Global prevalence estimates of disabling hearing loss in children has been estimated to be 1.7% and in high income countries 0.5%15, and HI may through these factors reduce partnering opportunities for those who are deaf or hearing impaired16. The hearing impaired have a greater disease burden and higher mortality18. Education, income, communication skills, and health affects the likelihood of entering a stable partnerships21. A stable partnership remains one of the strongest predictors of childbearing in advanced societies19.Hearing impairment has been found to be associated socioeconomic factors, including school attainment and income22.This study investigates whether those with a particular hearing loss during childhood (i.e. prior fertility decisions) have fewer children. If so, we will examine if this is mediated by partnership status or education, and if it has changed over time. A major advantage of our study is that we do not rely on self-assessed hearing. Most existing datasets use subjective HI, which is often a difficult measure with low comparability. Subjective measures of hearing could be poorly related to actual hearing, but rather influenced by peer groups, contextual factors, personality, degree of optimism as well as socioeconomic standardsThe relationship between fertility and hearing has not yet been studied using population level data incorporating objective hearing assessments. We combine data from the School Hearing Investigation in Nord-Trøndelag (SHINT), data from the Trøndelag Health Study (HUNT), and population registry data  which allow these relations to be studied.https://www.ntnu.edu/hunt). All adult participants included in the survey have been informed and given written consent to use data from the child, main and follow-up studies as well as to link data to other registries including patient registries for research purposes. The Norwegian Data Protection Authority has licensed HUNT Research Centre to store and link data collected in all HUNT surveys. All HUNT surveys, and the present nonparticipant study that includes a linkage to patient and population registry data, are approved by The Norwegian Regional Committee for Medical and Health Research Ethics.Our data comes from the HUNT survey 24 which was a necessary criterion to link the SHINT data with the National Population Registry.The base for our study is a population cohort with participation in two temporally different health examinations. We included participants from SHINT . In short, nearly all 7-, 10- and 13-years old school children in the entire Nord-Trøndelag County underwent screening with pure-tone audiometry from 1954 to 1986. Children with hearing loss at the screening (thresholds ≥ 20 decibel hearing level (dB HL) at three or more frequencies, or ≥ 30 dB HL at one or more frequencies) were invited to an otorhinolaryngologist (ORL) examination and most attended. For instance, from 1954 to 1962, average attendance at the ORL examinations for children with positive screening was 97%23. Hence, our analyses are based on the approximation that children with no notification of hearing impairment in the SHINT survey were recorded as normal hearing. We include all HUNT participants who were born in the same period as the SHINT participants as our study group and observe these at HUNT 1, 2 or 3  . For subjects born 1954 and later we have information of residence during primary school age, and for subjects born before 1954 we have information of residence at birth. We excluded those born 1954 and later with confirmed residence outside Nord-Trøndelag during primary school age (n = 5671) and those born before 1953 with confirmed residence outside Nord-Trøndelag at birth (n = 4568). This provides us with a dataset of n = 50,022 participants. A limitation with this procedure is that SHINT participants who died, emigrated or refused to participate in HUNT will be excluded and this which may potentially cause selection bias. However, this problem is reduced by participation rates in HUNT .An extensive effort was made with the aim to include all children born 1940–1980 residing in Nord-Trøndelag in the SHINT survey, though only children who had hearing problems had records included in the datasetData from SHINT and HUNT were linked with individual level data from the National Population Registry . The Regional Committees for Medical and Health Research Ethics approved the study (REK 23178 “HUNT hørsel”). The study met all requirements in accordance with the General Data Protection Regulation (GDPR) and a Data Protection Impact Assessment (DPIA) was conducted.25. Hearing thresholds were defined as binaural pure-tone average (PTA) of four frequencies  measured in dB HL. Hearing loss was defined as PTA greater than 25 dB HL. SHINT included several follow-ups, and data from the last audiometric test was used including only permanent hearing losses diagnosed with either sensorineural hearing loss, otosclerosis, chronic suppurative otitis media, or permanent hearing loss after recurrent acute otitis media. Subjects with diagnosis yielding temporary hearing loss such as secretory- and acute otitis media was regarded as normal hearing in childhood with hearing threshold equal to zero. We further considered all subjects born between 1940 and 1980 (being in primary school age during SHINT) and not registered with a hearing loss in SHINT as normal hearing in childhood.Air conduction hearing threshold levels were obtained by pure-tone audiometry at eight frequencies from 0.25 to 8 kHz according to the Norwegian standards at the timeData on number of children ever born was obtained for the year 2014 from the National Population Registry.As mentioned earlier, we investigate whether partnership status is a mediator in the relationship between childhood HI and fertility. First by using a dummy variable that is equal to one if the individual has ever been married and second by using a dummy variable for if the individual has ever been cohabiting. This information is obtained based on the records from the National Population Registry between 1975 and 2014. Cohabiting status was supplied with self-reported data on cohabiting status obtained from HUNT2 (1996–1998).We considered birth cohort (year of birth) as possible factors modifying the association between adult hearing loss and fertility.From national education registers, we included information on level of highest education .For adjusted models we selected covariates that might confound the effect of hearing loss on fertility. We collected information on covariates from national registers and from the HUNT 1, 2 and 3 questionnaires.The adjusted models first include as additional covariates the educational level of the parents. We then include several health-related variables that may be correlated to both fertility outcomes and HI. Missing data of the parent’s education amounted to 9.6% for the mother and 13.3% for the father. This probably because these parents are deceased and thus had no records. These missing values were imputed using median values according to birth year and sex. Missing data on any of the other covariates (9%) were handled by listwise deletion.Poisson regression models estimate the association between the number of children ever born and HI, expressed as incidence rate ratio (IRR). We use Poisson rather than negative binomial models because we did not find evidence of overdispersion . We have also estimated the models by Ordinary Least Squares as a sensitivity analysis estimated with heteroskedasticity-robust error variance and we obtained similar results. The association with childlessness is estimated by a logit model and expressed as odds ratio (OR). The latter was estimated with robust error variance.As a robustness check, we also estimated family fixed effect (FFE) Poisson and logit models. FFE models analyze fertility as a function of features that vary between siblings. The models control for all observed and non-observed confounders that are shared between siblings, including factors related to upbringing and parent characteristics. The drawback is that this analysis is restricted to individuals with at least one sibling (of any sex). The family fixed effects were compared with estimates from ordinary Poisson and logit models in the same restricted sample of siblings. We further tested if the effect differed according to severity of hearing loss by dividing childhood HI into three categories: moderate-severe (> 40 dB HL) (n = 161), mild (26–40 dB HL) (n = 410) and slight hearing loss (16–25 dB HL) (n = 489).All analyzes were done separately for men and women adjusting for birth year and subsequently also accounting for other covariates.26. The proportion mediated was calculated as ORNDE(ORNIE − 1)/(ORNDE × ORNIE − 1) in which ORNDE is the odds ratio for the natural direct effect and ORNIE is the risk ratio for the natural indirect effect.In order to study if the associations were mediated by cohabiting status or education, we applied a counterfactual approach allowing us to estimate mediation effects in a non-linear model when exposure-mediator interaction is presentPotential birth cohort trends were estimated by stratifying on birth cohorts.All methods were performed in accordance with relevant regulations from the data owners.Table Table Table The results of the mediation analysis  Poisson and logit models that allow us to further control for family-invariant unobserved heterogeneity for individuals with at least one sibling. The results regarding the number of children are displayed in Supplementary Table The current study discussed how fertility relates to HI using population level longitudinal data with objective hearing assessments. Our results suggest that (i) individuals with HI have fewer children than their normal hearing peers, and that this relationship is only observed for men, and (ii) this is to a large extent explained by a lower likelihood of having ever been married or cohabited.Our thresholds analysis for childhood HI shows that it does not only affect individuals with severe childhood HI but also those with mild childhood HI and is therefore relevant for a broader section of the population rather than only those with severe HI or deafness, although the effects are stronger among those with more severe HI.This study is conducted using larger longitudinal data than what has previously been available. Our findings remain significant also when controlling for family fixed effects. Moreover, many earlier studies have been based on self-reported hearing, which is a measure that can be difficult to interpret . We use audiometrically based measures of hearing function, which gives objective assessments.27. Further, we lack information on the cognitive ability of the participants in our study—and as a substantial share of Norwegian children with hearing loss may have cognitive challenges28, which could be a source of bias. Nevertheless, the model using family fixed effects allows to control for a large share of the unobserved heterogeneity. Moreover, thanks to the longitudinal nature of the data, reverse causality cannot explain the observed association.While this study provides strong evidence suggesting that HI affects fertility, we cannot rule out the possibility that our results are biased due to sample selection or unobserved confounders. Sample selection bias might be due to selected mortality or selected emigration. Assuming that mortality is positively correlated with childhood HI and negatively correlated with fertility, bias due to selected mortality would imply that our estimated association is underestimating the negative effect of childhood HI on fertility. The bias due to selected emigration is expected to overestimate the negative effect of childhood HI on fertility if emigration is negatively correlated with childhood HI and positively correlated with fertility. Nevertheless, these biases are likely to be very small given relatively few deaths or emigration occur before HUNT 1 (1986) in the relevant age groups in NorwayOther health characteristics may potentially help explain why the individuals with HI have lower fertility, such as possible social, psychological or cognitive characteristics that we do not observe in our data and therefore are unable to control for. Nevertheless, HUNT includes a very broad array of health, economic and sociodemographic information and linkage to national registers. Moreover, HUNT is one of the largest health surveys in the world—and has a relatively high response rate (ranging between 54 and 89%) compared to most of health survey of similar size . As we also have national register data, hence we are able to assess the representatively of the HUNT sample compared to the non-respondents.29 may affect childbearing outcomes between men and women with HI.The stronger negative effect of HI on male as opposed to female fertility could be linked to sex differences in partnering selection. The fact that male income remains a stronger determinant of partnership patterns while female income has little effectSocietal and technological developments have changed the life of many hearing impaired in recent decades, where several improvements in hearing aids or change in communication form (online text-based communications) benefit individuals with HI. In spite of this, our study shows that HI is strongly negative related to fertility outcomes.32, the high prevalence of low fertility and childlessness among the hearing impaired could adverse relate to health risk factors associated with greater risk of lifestyle related illnesses and shorter life expectancy. One may consider providing more emphasis to adverse lifestyles associated with a lack of family as a health challenge among the hearing impaired.This study has relevance to understanding health outcomes among those with hearing impairment. As parenthood, marital status and family support is a strong determinant of healthSupplementary Tables.