Effects of serta and sertb knockout on aggression in zebrafish (Danio rerio).

Zebrafish (Danio rerio) are unusual in having two paralogues of the serotonin re-uptake transporter (Sert), slc6a4a (serta) and slc6a4b (sertb), the transporter that serves in serotonin re-uptake from a synapse into the pre-synaptic cell or in serotonin uptake from the extracellular milieu into cells in the peripheral tissues. To address a knowledge gap concerning the specific roles of these paralogues, we used CRISPR/Cas9 technology to generate zebrafish knockout lines predicted to lack functional expression of Serta or Sertb. The consequences of loss-of-function of Serta or Sertb were assessed at the gene expression level, focusing on the serotonergic signalling pathway, and at the behaviour level, focusing on aggression. Whereas serta mRNA was expressed in all tissues examined, with high expression in the heart, gill and brain, only the brain displayed substantial sertb mRNA expression. In both serta-/- and sertb-/- fish, changes in transcript abundances of multiple components of the serotonin signalling pathway were detected, including proteins involved in serotonin synthesis (tph1a, tph1b, tph2, ddc), packaging (vmat2) and degradation (mao), and serotonin receptors (htr1aa, htr1ab). Using a mirror aggression test, serta-/- male but not female fish exhibited greater aggression than wildtype fish. However, both male and female sertb-/- fish displayed less aggression than their wildtype counterparts. These differences in behaviour between serta-/- and sertb-/- individuals hold promise for increasing our understanding of the neurophysiological basis of aggression in zebrafish.

Evolution and divergence of teleost adrenergic receptors: why sometimes 'the drugs don't work' in fish.

Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and ß-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts.

A Qualitative Exploration of Patient and Staff Experiences of the Receipt and Delivery of Specialist Weight Management Services in the UK.

BACKGROUND: Addressing the increasing prevalence of obesity is a global public health priority. Severe obesity (body mass index > 40) reduces life expectancy, due to its association with people developing complications (e.g. diabetes, cancer, cardiovascular disease), and greatly impairs quality of life. The National Health Service (NHS) in the UK provides specialist weight management services (SWMS) for people with severe obesity, but key uncertainties remain around patient access to and engagement with weight management services, as well as pathways beyond the service. METHODS: In this multiple methods study, using online forum data and semi-structured interviews, stakeholders' experiences of delivering and receiving SWMS were explored. Using the web search engine Google with keywords and web address (URL) identifiers, relevant public online platforms were sourced with snowball sampling and search strings used to identify threads related to people's experiences of accessing SWMS (n = 57). Interviews were conducted with 24 participants (nine patients, 15 staff), and data from all sources were analysed thematically using the framework approach. RESULTS: Six themes related to access to and engagement with SWMS emerged during data analysis: (1) making the first move, (2) uncertainty and confusion, (3) resource issues, (4) respect and understanding, (5) mode of delivery, and (6) desire for ongoing support. CONCLUSION: There is a mixed and varied picture of SWMS provision across the UK. The service offered is based on local clinical decision making and available resources, resulting in a range of patient experiences and perspectives. Whilst service capacity issues and patient anxiety were seen as barriers to accessing care, peer support and positive clinical and group interactions (connectedness between individuals) were considered to increase engagement.

The control of breathing in fishes - historical perspectives and the path ahead.

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing.

Insights into the control and consequences of breathing adjustments in fishes-from larvae to adults.

Adjustments of ventilation in fishes to regulate the volume of water flowing over the gills are critically important responses to match branchial gas transfer with metabolic needs and to defend homeostasis during environmental fluctuations in O2 and/or CO2 levels. In this focused review, we discuss the control and consequences of ventilatory adjustments in fish, briefly summarizing ventilatory responses to hypoxia and hypercapnia before describing the current state of knowledge of the chemoreceptor cells and molecular mechanisms involved in sensing O2 and CO2. We emphasize, where possible, insights gained from studies on early developmental stages. In particular, zebrafish (Danio rerio) larvae have emerged as an important model for investigating the molecular mechanisms of O2 and CO2 chemosensing as well as the central integration of chemosensory information. Their value stems, in part, from their amenability to genetic manipulation, which enables the creation of loss-of-function mutants, optogenetic manipulation, and the production of transgenic fish with specific genes linked to fluorescent reporters or biosensors.

Catecholamines modulate the hypoxic ventilatory response of larval zebrafish (Danio rerio).

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent ß-adrenoreceptor (AR)-mediated increase in fV that does not require expression of ß1-ARs, as the hyperventilatory response to ß-AR stimulation was unaltered in adrb1-/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min-1 without propranolol to 68±17 breaths min-1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1-/- mutants. Thus, ß1-AR is not required for the HVR and other ß-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.

A group-based behavioural intervention for weight management (PROGROUP) versus usual care in adults with severe obesity: a feasibility randomised controlled trial protocol.

BACKGROUND: Approximately 15 million people in the UK live with obesity, around 5 million of whom have severe obesity (body mass index (BMI) ≥35kg/m2). Having severe obesity markedly compromises health, well-being and quality of life, and substantially reduces life expectancy. These adverse outcomes are prevented or ameliorated by weight loss, for which sustained behavioural change is the cornerstone of treatment. Although NHS specialist 'Tier 3' Weight Management Services (T3WMS) support people with severe obesity, using individual and group-based treatment, the current evidence on optimal intervention design and outcomes is limited. Due to heterogeneity of severe obesity, there is a need to tailor treatment to address individual needs. Despite this heterogeneity, there are good reasons to suspect that a structured group-based behavioural intervention may be more effective and cost-effective for the treatment of severe obesity compared to usual care. The aims of this study are to test the feasibility of establishing and delivering a multi-centre randomised controlled clinical trial to compare a group-based behavioural intervention versus usual care in people with severe obesity. METHODS: This feasibility randomised controlled study is a partially clustered multi-centre trial of PROGROUP (a novel group-based behavioural intervention) versus usual care. Adults ≥18 years of age who have been newly referred to and accepted by NHS T3WMS will be eligible if they have a BMI ≥40, or ≥35 kg/m2 with comorbidity, are suitable for group-based care and are willing to be randomised. Exclusion criteria are participation in another weight management study, planned bariatric surgery during the trial, and unwillingness or inability to attend group sessions. Outcome assessors will be blinded to treatment allocation and success of blinding will be evaluated. Clinical measures will be collected at baseline, 6 and 12 months post-randomisation. Secondary outcome measures will be self-reported and collected remotely. Process and economic evaluations will be conducted. DISCUSSION: This randomised feasibility study has been designed to test all the required research procedures and additionally explore three key issues; the feasibility of implementing a complex trial at participating NHS T3WMS, training the multidisciplinary healthcare teams in a standard intervention, and the acceptability of a group intervention for these particularly complex patients. TRIAL REGISTRATION: ISRCTN number 22088800.

Characterization of two novel ammonia transporters, Hiat1a and Hiat1b, in the teleost model system Danio rerio.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.

Control of Breathing in Ectothermic Vertebrates.

The ectothermic vertebrates are a diverse group that includes the Fishes (Agnatha, Chondrichthyes, and Osteichthyes), and the stem Tetrapods (Amphibians and Reptiles). From an evolutionary perspective, it is within this group that we see the origin of air-breathing and the transition from the use of water to air as a respiratory medium. This is accompanied by a switch from gills to lungs as the major respiratory organ and from oxygen to carbon dioxide as the primary respiratory stimulant. This transition first required the evolution of bimodal breathing (gas exchange with both water and air), the differential regulation of O2 and CO2 at multiple sites, periodic or intermittent ventilation, and unsteady states with wide oscillations in arterial blood gases. It also required changes in respiratory pump muscles (from buccopharyngeal muscles innervated by cranial nerves to axial muscles innervated by spinal nerves). The question of the extent to which common mechanisms of respiratory control accompany this progression is an intriguing one. While the ventilatory control systems seen in all extant vertebrates have been derived from common ancestors, the trends seen in respiratory control in the living members of each vertebrate class reflect both shared-derived features (ancestral traits) as well as unique specializations. In this overview article, we provide a comprehensive survey of the diversity that is seen in the afferent inputs (chemo and mechanoreceptor), the central respiratory rhythm generators, and the efferent outputs (drive to the respiratory pumps and valves) in this group. © 2022 American Physiological Society. Compr Physiol 12: 1-120, 2022.

Regulation of heart rate following genetic deletion of the ß1 adrenergic receptor in larval zebrafish.

AIM: Although zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1-adrenergic receptor (AR) in the regulation of heart rate in zebrafish. METHODS: CRISPR-Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1-/- line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR. RESULTS: Compared with genetically matched wild-types (adrb1+/+ ), adrb1-/- larvae exhibited ~20 beats min-1 lower heart rate, measured from 2 to 21 days post-fertilization (dpf). Nevertheless, adrb1-/- larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß-AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO2 ) at 5 dpf, but tachycardia was blunted in adrb1-/- larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1-/- larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1-/- larval hearts. CONCLUSION: Despite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1-/- line. The chronotropic effects of adrenergic stimulation persist in adrb1-/- zebrafish, likely due to the upregulation of other ß-AR subtypes.

Aquatic surface respiration improves survival during hypoxia in zebrafish (Danio rerio) lacking hypoxia-inducible factor 1-α.

Hypoxia-inducible factor 1-α (Hif-1α), an important transcription factor regulating cellular responses to reductions in O2, previously was shown to improve hypoxia tolerance in zebrafish (Danio rerio). Here, we examined the contribution of Hif-1α to hypoxic survival, focusing on the benefit of aquatic surface respiration (ASR). Wild-type and Hif-1α knockout lines of adult zebrafish were exposed to two levels (moderate or severe) of intermittent hypoxia. Survival was significantly compromised in Hif-1α knockout zebrafish prevented from accessing the surface during severe (16 mmHg) but not moderate (23 mmHg) hypoxia. When allowed access to the surface in severe hypoxia, survival times did not differ between wild-type and Hif-1α knockouts. Performing ASR mitigated the negative effects of the loss of Hif-1α with the knockouts initiating ASR at a higher PO2 threshold and performing ASR for longer than wild-types. The loss of Hif-1α had little impact on survival in fish between 1 and 5 days post-fertilization, but as the larvae aged, their reliance on Hif-1α increased. Similar to adult fish, ASR compensated for the loss of Hif-1α on survival. Together, these results demonstrate that age, hypoxia severity and, in particular, the ability to perform ASR significantly modulate the impact of Hif-1α on survival in hypoxic zebrafish.

The evolutionary and physiological significance of the Hif pathway in teleost fishes.

The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems.

Hypoxia inducible factor 1-α is minimally involved in determining the time domains of the hypoxic ventilatory response in adult zebrafish (Danio rerio).

In the current study, adult zebrafish (Danio rerio) were exposed to 72 h hypoxia (90 mmHg) to assess the time domains of the hypoxia ventilatory response (HVR) and the consequence on a subsequent more severe (40 mmHg) bout of acute hypoxia. Experiments were performed on wild-type fish and mutants in which one or both paralogs of hypoxia inducible factor-1α (hif-1α) were knocked out. Although there were subtle differences among the wild-type and knockout fish, resting fV was reestablished after 2-8 h of continuous hypoxia in both groups, a striking example of hypoxic ventilatory decline (HVD). When fish were subsequently exposed to more severe hypoxia, a rapid increase in fV was observed, the magnitude of which was independent of genotype or prior exposure history. During recovery, fish that had been exposed to 72 h of 90 mmHg hypoxia exhibited a pronounced undershoot in fV, which was absent in the hif-1α double knockouts. Overall, the results revealed distinct time domains of the HVR in zebrafish that were largely Hif-1α-independent.

The effects of dissolved organic carbon on the reflex ventilatory responses of the neotropical teleost (Colossoma macropomum) to hypoxia or hypercapnia.

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O2 and/or CO2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l-1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l-1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro.

Hif-1α is not required for the development of cardiac adrenergic control in zebrafish (Danio rerio).

Adrenergic regulation, acting via the sympathetic nervous system, provides a major mechanism to control cardiac function. It has recently been shown that hypoxia inducible factor-1α (Hif-1α) is necessary for normal development of sympathetic innervation and control of cardiac function in the mouse. To investigate whether this may represent a fundamental trait shared across vertebrates, we assessed adrenergic regulation of the heart in wild-type and Hif-1α knockout (hif-1α -/- ) zebrafish (Danio rerio). Wild-type and hif-1α -/- zebrafish larvae (aged 4 and 7 days postfertilisation) exhibited similar routine heart rates within a given age group, and ß-adrenergic receptor blockade with propranolol universally reduced heart rate to comparable levels, indicating similar adrenergic tone in both genotypes. In adult fish, in vivo heart rate measured during anaesthesia was identical between genotypes. Treatment of spontaneously beating hearts in vitro with adrenaline revealed a similar positive chronotropic effect and similar maximum heart rates in both genotypes. Tyrosine hydroxylase immunohistochemistry with confocal microscopy demonstrated that the bulbus arteriosus (outflow tract of the teleost heart) of adult fish was particularly well innervated by sympathetic nerves, and nerve density (as a percentage of bulbus arteriosus area) was similar between wild-types and hif-1α -/- mutants. In summary, we did not find any evidence that adrenergic cardiac control was perturbed in larval or adult zebrafish lacking Hif-1α. We conclude that Hif-1α is not essential for the normal development of cardiovascular control or adult sympathetic cardiac innervation in zebrafish, although it is possible that it plays a redundant or auxiliary role.

Does hypoxia-inducible factor 1α play a role in regulating cutaneous oxygen flux in larval zebrafish (Danio rerio)?

Previous studies have demonstrated that hypoxia tolerance is improved in zebrafish (Danio rerio) larvae after prior exposure to lowered ambient O2 levels. Such improved hypoxia performance was attributed in part, to increased levels of hypoxia-inducible factor 1α (Hif-1α) exerting downstream effects on various physiological processes including promotion of trunk skin angiogenesis. Since O2 uptake ([Formula: see text]) in larvae is facilitated largely by O2 diffusion across the skin, enhanced cutaneous vascularization is expected to enhance [Formula: see text] during hypoxia and thus contribute to improved hypoxia tolerance. In this study, we used the scanning micro-optrode technique together with quantification of cutaneous vascularity in wild types (WT) and Hif-1α knockouts (hif1aa-/-ab-/-) to test the hypothesis that improved hypoxia tolerance after hypoxia acclimation in larvae at 4 or 7 days post-fertilization (dpf) was associated with Hif-1α-dependent increases in skin vascularity and regional cutaneous O2 fluxes (JO2). Hypoxia tolerance, as determined by measurements of critical PO2 (Pcrit), was unaltered by hypoxia pre-exposure in larvae at 4 dpf and there were no significant differences in Pcrit between WT and hif1aa-/-ab-/- larvae at this developmental stage. However, at 7 dpf there was a significant effect of genotype with WT larvae showing a lower Pcrit than hif1aa-/-ab-/- larvae, an effect that was being driven by a reduced Pcrit in the WT larvae after hypoxia pre-exposure (19.2 ± 1.9 mmHg) compared to hif1aa-/-ab-/- fish (35.5 ± 3.5 mmHg). Regardless of genotype, pre-exposure status or developmental age, JO2 decreased along the body in the anterior-to-posterior direction. Neither hypoxia pre-exposure nor genotype affected JO2 at any region along the body. The lack of any effect of hypoxia pre-exposure or genotype on JO2 was consistent with the lack of any effect on skin vascularity as measured in Tg(fli1:EGFP)yl transgenic larvae. Thus, the decreased hypoxia performance (increased Pcrit) at 7 dpf in the hif1aa-/-ab-/- larvae did not appear to be reliant on changes in trunk vascularity or cutaneous O2 diffusion.

Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio).

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish (Danio rerio) is expressed abundantly in Na+-absorbing/H+-secreting H+-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a-/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a-/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na+ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na+ content remained constant. While Cl- uptake was significantly reduced in ca17a-/- mutants, Cl- content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl- uptake, implicating Ca17a in the mechanism of Cl- uptake by larval zebrafish. H+ secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a-/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

Disruption of tph1 genes demonstrates the importance of serotonin in regulating ventilation in larval zebrafish (Danio rerio).

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O2 chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a-/- mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b-/- mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR.