Advancement in the miniaturization of high-density power sources, electronic circuits, and communication technologies enabled the construction of miniaturized electronic devices, implanted directly in the heart. These include pacing devices to prevent low heart rates or terminate heart rhythm abnormalities (‘arrhythmias’), long-term rhythm monitoring devices for arrhythmia detection in unexplained syncope cases, and heart failure (HF) hemodynamic monitoring devices, enabling the real-time monitoring of cardiac pressures to detect and alert for early fluid overload. These devices were shown to prevent HF hospitalizations and improve HF patients’ life quality. Pacing devices include permanent pacemakers (PPM) that maintain normal heart rates, defibrillators that are capable of fast detection and the termination of life-threatening arrhythmias, and cardiac re-synchronization devices that improve cardiac function and the survival of HF patients. Traditionally, these devices are implanted via the venous system (‘endovascular’) using conductors (‘endovascular leads/electrodes’) that connect the subcutaneous device battery to the appropriate cardiac chamber. These leads are a potential source of multiple problems, including lead-failure and systemic infection resulting from the lifelong exposure of these leads to bacteria within the venous system. One of the important cardiac innovations in the last decade was the development of a leadless PPM functioning without venous leads, thus circumventing most endovascular PPM-related problems. Leadless PPM’s consist of a single device, including a miniaturized power source, electronic chips, and fixating mechanism, directly implanted into the cardiac muscle. Only rare device-related problems and almost no systemic infections occur with these devices. Current leadless PPM’s sense and pace only the ventricle. However, a novel leadless device that is capable of sensing both atrium and ventricle was recently FDA approved and miniaturized devices that are designed to synchronize right and left ventricles, using novel intra-body inner-device communication technologies, are under final experiments. This review will cover these novel implantable miniaturized cardiac devices and the basic algorithms and technologies that underlie their development. Advancement in the miniaturization of high-density power sources, electronic circuits, and communication technologies enabled the construction of miniaturized electronic devices, implanted directly in the heart. These include pacing devices to prevent low heart rates or terminate heart rhythm abnormalities (‘arrhythmias’), long-term rhythm monitoring devices for arrhythmia detection in unexplained syncope cases, and heart failure (HF) hemodynamic monitoring devices, enabling the real-time monitoring of cardiac pressures to detect and alert early fluid overload. These devices were shown to prevent HF hospitalizations and improve HF patients’ life quality. Pacing devices include permanent pacemakers (PPM) that maintain normal heart rates, defibrillators that are capable of fast detection and termination of life-threatening arrhythmias, and cardiac re-synchronization devices that improve cardiac function and survival of HF patients. Traditionally, these devices are implanted via the venous system (‘endovascular’) using conductors (‘endovascular leads/electrodes’) that connect the subcutaneous device battery to the appropriate cardiac chamber. These leads are a potential source of multiple problems, including lead-failure and systemic infection that result from the lifelong exposure of these leads to bacteria within the venous system. The development of a leadless PPM functioning without venous leads was one of the important cardiac innovations in the last decade, thus circumventing most endovascular PPM-related problems. Leadless PPM’s consist of a single device, including a miniaturized power source, electronic chips, and fixating mechanism, implanted directly into the cardiac muscle. Only rare device-related problems and almost no systemic infections occur with these devices. Current leadless PPM’s sense and pace only the ventricle. However, a novel leadless device that is capable of sensing both atrium and ventricle was recently FDA approved and miniaturized devices designed to synchronize right and left ventricles, using novel intra-body inner-device communication technologies, are under final experiments. This review will cover these novel implantable miniaturized cardiac devices and the basic algorithms and technologies that underlie their development. Advancement in the miniaturization of high-density power sources, electronic circuits, and communication technologies enabled the construction of miniaturized electronic devices, implanted directly in the heart. These include pacing devices to prevent low heart rates or terminate rapid heart rhythm abnormalities (‘tachyarrhythmias’), long-term rhythm monitoring devices for arrhythmia detection in unexplained syncope cases, and heart failure (HF) hemodynamic monitoring devices, enabling the real-time monitoring of cardiac pressures to detect and alert for early fluid overload. These devices were shown to prevent HF hospitalizations and improve HF patients’ life quality. Pacing devices include permanent pacemakers (PPM) that maintain normal heart rates, defibrillators that are capable of fast detection and the termination of life-threatening arrhythmias, and cardiac resynchronization devices that may improve cardiac function and survival in appropriately selected HF patients. Traditionally, these devices are implanted via the venous system (‘endovascular’), while using conductors (‘endovascular leads/electrodes’) that connect the subcutaneous device battery to the appropriate cardiac chamber. These leads are a potential source of multiple problems, including lead-failure and systemic infection that result from the lifelong exposure to bacteria within the venous system. One of the important cardiac innovations in the last decade was the development of a leadless PPM functioning without venous leads, thus circumventing most endovascular PPM-related problems. Leadless PPMs consist of a single device, including a miniaturized power source, electronic chips, and fixating mechanism, implanted directly into the cardiac muscle. Only rare device-related problems and almost no systemic infections occur with these devices. Current leadless PPMs sense and pace only the ventricle. However, a novel leadless device capable of sensing both atrium and ventricle was recently FDA approved and miniaturized devices that were designed to synchronize right and left ventricles, using novel intra-body inner-device communication technologies, are under final experiments. This review will cover these novel implantable miniaturized cardiac devices and the basic algorithms and technologies underlying their development.Coaxial leads have an inner conductor that extends down the length of the lead to the tip electrode, the cathode, arranged in a coil configuration with a central lumen to allow for the passage of a stylet at implantation. This coil is covered by a cylindrical length of inner insulation, which, in turn, is wrapped by another coil conductor that also runs down the lead to the ring electrode, the anode. A second outer insulation layer protects the ring conductor from the outside environment  have become a pivot treatment for various cardiac patients, including: (a) permanent pacemakers (PPM) aimed for pacing the heart at programmed rates, for patients with symptomatic low heart rates (‘bradycardia’). (b) Implantable cardiac defibrillators (ICD), which can detect and terminate life-threatening fast ventricular arrhythmias via anti-tachycardia pacing or high-energy shocks (‘direct current cardioversion/defibrillation’). These ICDs were shown to improve the survival of HF patients with impaired cardiac function, who are at increased risk for sudden arrhythmic death. ICD is also recommended for patients surviving prior arrhythmic cardiac death, considered to be high risk for recurrent ventricular arrhythmias ,2,3,4,5.ironment B. In corironment . Althougead bulk ,11.drawn filled tube composite-wire conductor strand consisting of a thick strong body of MP-35N filled with a central core of softer low-resistance silver metal, often encased in a further outer shell of platinum alloy. An alternative conductor design is the drawn brazed strand design, consisting of few strands of high-resistance MP-35N material tightly molded over a central silver strand. Thus, the inner low-resistance metal is forced between and around the strong outer strands. The functional purpose of these designs is to create conductors that are both resistant to corrosion and have low electrical resistance , or via a screw into the heart muscle . The mai3 weighting ~2 g), which is implanted as one piece directly into the appropriate cardiac chamber  technology that is extremely compact and operates at low energy cost , bottom. chamber . Notably3 vs. 1 cm3). Micra’s fixation is based on nitinol tines at its distal end, which are pushed into to myocardium, while Nanostim’s fixation is based on an active helix that is screwed into the cardiac muscle. The communication with remote programmer ‘telemetry’ is based on radiofrequency signals for Micra versus conductive communication through skin electrodes for Nanostim [The leadless PPMs differ in size, fixation mechanism, communication, rate response mechanism, and power source. Micra is smaller as compared with Nanostim  construction of efficient low-power electrical circuits; (2) absence of long high-impedance leads, saving energy needed for current conduction along the long conventional leads; (3) automatic capture management algorithm , processed on an hourly basis to ensure minimal but safe pacing outputs; and, (4) optimized battery longevity by setting the nominal pulse width duration to chronaxie (0.24 ms) . The preThe implantation of leadless PPMs was shown to be successful in the vast majority of cases (~95%) and free from serious complications ,34,35,36Despite this early success of leadless PPM, the clinical use of Nanostim was halted by the end of 2017 due to two safety issues: (1) premature battery depletion that was initially attributed to “lithium clusters” bridging the cathode and anode, causing electrical short-circuiting. A high percent of device malfunction attributed to early battery depletion was reported in a single center study . NotablyThe main limitation of leadless PPM results from the fact that they could pace and sense the right ventricle (RV) only without Atrio-Ventricular (AV) synchronous pacing, which is considered to be beneficial in most patients. Thus, their use is restricted to symptomatic bradycardia patients without the need for atrial sensing or pacing as patients with slow chronic atrial fibrillation, some patients with alternating fast atrial fibrillation and sinus bradycardia where no significant atrial pacing is needed, and elderly sedentary patients with multiple co-morbidities in whom ventricular pacing is sufficient. A second limitation results from device encapsulation into the myocardial tissue, occurring as soon as one year post-implantation, as described in a few autopsy studies ,43. ThisRecently, effort was made to expand current leadless PPM use by developing a novel atrial sensing algorithm for enabling AV synchronous pacing, which is the “physiological” optimal pacing method for most patients suffering from low heart rates. This algorithm is based on a complex analysis of Micra’s accelerometer signals, distinguishing intracardiac signals that are related to atrial contractions from those related to ventricular movement . This noBasically, PPMs are used to treat slow heart rhythms, while fast potentially life-threatening ventricular arrhythmias, which could result in sudden death if untreated, are terminated by electrical shocks or anti-tachycardia pacing (ATP). Current implantable cardiac defibrillators (ICD) are utilized for the rapid detection of fast ventricular arrhythmias and their termination by automatic DC shocks or ATP. Conventional ICDs are composed of a high-energy battery, trans-venous leads containing coils (capacitors), which are charged to high voltage, enabling the delivery of high-energy DC shocks through the leads into the heart muscle when needed. Similar to PPMs, these devices are prone to all endovascular-related complications. As noted earlier, cardiac defibrillation depends on high-voltage shocking coils, which limits (for the time being) the development of miniaturized ICDs due to these high-energy requirements that are beyond the capability of current miniaturized batteries. Nevertheless, a significant reduction of ICD volume occurred over the years that was mainly due to a reduction in its capacitor size.A subcutaneous ICD (S-ICD) was developed in order to circumvent endovascular-related complications  function was shown to improve cardiac function, prevent hospitalizations, and prolong survival in a selected subpopulation of HF patients with an electrical conduction disease, having asynchronized ventricular contraction. Conventional cardiac resynchronization therapy (CRT) devices are based on multiple transvenous leads, implanted within the right atrium, right ventricle, and a cardiac venous branch of the coronary sinus, which, due to its proximity to the LV, enables LV pacing. These leads pace the ventricles simultaneously, resulting in a synchronized improved cardiac contraction. Multi-component leadless PPM implanted directly within both RV and LV, enabling their synchronous pacing, are under development . One of Recently, a totally leadless CRT system was developed, which was based on three independent leadless PPM’s implanted directly into the right atrium, right ventricle, and left ventricle . Each ofThe novel CIC method is based on several very-high-frequency small alternating current pulses that were transmitted and received by the devices, whereby communication data are encoded in the time shifts between pulses. The alternating current communication signal propagates almost at the speed of light and allows for the transition of several bits of data per pulse. Using very high frequency signals has major advantages, which are prerequisite for long-term intra-body communication: (1) low signal absorption and attenuation within cardiac tissue, resulting in highly efficient energy communication; (2) does not interfere with intrinsic cardiac electrical activity used to both conduct biological electrical pulses throughout the cardiomyocytes and depolarize the cardiomyocytes’ membrane to enable cardiac contraction (known as ‘excitation-contraction coupling’), thus preventing inappropriate pacing with its potentially deleterious consequences; and, (3) low risk for external electromagnetic interference. A communication frequency of 1 MHz was found to be optimal, causing no cardiac intrinsic electrical interference and achieving a minimal (0.3 µW) power requirement. Notably, each device pacing action is triggered by a CIC message sent from another device containing sender and receiver address (which cardiac device chamber sends/receives data). As the CIC message travels through the heart, the received signal undergoes some attenuation and it is also affected by noise. Accordingly, the communication nodule within each device amplifies and filters the incoming signal in order to obtain the reconstructed messages, which can then trigger a pacing stimulus. The above experimental system was shown to successfully resynchronize cardiac function in porcine hearts and only recently tried in humans ,59,60. IImportantly, one should be aware of the embolic risk that is associated with any metal device implanted within the LV or left atrium (LA) . This may result from the detachment of the LV/LA device itself or from thrombus formation on these metal-based devices, which, due to the vigorous cardiac contractions and high pressure blood stream, may embolize into the aorta, causing stroke or systemic embolism. A continuous lifetime anticoagulation treatment is necessary to prevent a thrombus formation on these LV devices, although such treatment may lead to bleeding complications and, at times, needs to be stopped (before surgery or other invasive procedures). Trying to prevent the embolic detachment of LV device itself is a challenge, since it requires a firm screw or other fixation mechanism to firmly attach to the LV wall. However, this firm attachment may cause too much pressure on the LV wall, resulting in cardiac perforation, which is the second complication that is associated with these devices. Indeed, the main challenge at present is to find the appropriate fixation mechanism to ensure enough strength to prevent dislodgment, but without the risk of cardiac wall perforation. Notably, device emboli to lower extremities and a cerebral stoke occurred among 2/35 patients that were implanted with the SELECT-LV system . AdditioNoteworthy, a few novel ‘physiologic’ pacing paradigms were recently developed, as an alternative to conventional CRT pacing. These include His bundle and left bundle branch (LBBB) pacing, in which a conventional PPM device is connected to a special lead positioned in the vicinity of the His bundle or LBBB one, respectively ,70,71,72The development of a miniaturized implantable loop recorder (ILR) for long-term monitoring of arrhythmias made a revolution in the evaluation of patients suffering from recurrent fainting (‘syncope’) episodes ,74, stroHF is a chronic syndrome that is caused by inability of the heart to pump sufficient blood to the body tissues along with elevated cardiac filling pressures. These translate to fluid overload in the lungs and tissue hypoperfusion, which will eventually lead to pulmonary congestion and end-organ dysfunction, correspondingly. The clinical consequences of these changes include symptoms, such as shortness of breath due to pulmonary congestion, peripheral edema due to inadequate pumping of blood from the extremities, general weakness, attenuated exercise tolerance, and multiorgan dysfunction due to inadequate blood supply. Although some patients remain stable for years, most will suffer from exacerbations that may lead to hospitalizations, disability, and eventually death. The timely detection of exacerbations before overt clinical manifestations ensue will trigger identification of reversible causes and appropriate treatment to prevent hospitalization and further deterioration. To this end, several intracardiac microdevices have been developed and put into clinical use. The leading concept behind these intra-cardiac devices for HF monitoring is that cardiac filling pressure increases days to weeks before overt clinical exacerbation occurs ,87. SeveThe first of these devices that was clinically tested was the Chronicle implantable continuous hemodynamic monitor . This device resembles a PPM, with a programmable component that processes and stores information that is similar in appearance to the pulse generator of a conventional PPM, and a transvenous lead that has a sensor incorporated near its tip, which enables continuous intra-cardiac pressure measurements. The lead tip was inserted into the RV near the outflow tract and collected RV systolic and diastolic pressures. The leading clinical trial (COMPASS-HF), randomized HF patients to HF treatment with and without hemodynamic monitoring, revealing no significant change in HF-related adverse events apart from delaying the time to first HF-related hospitalization .A significant success with hemodynamic monitoring in HF was first demonstrated while using the CardioMEMS pulmonary artery (PA) pressure monitoring system . This novel wireless device with a battery free technology is powered and interrogated via an external antenna, using electromagnetic coupling. The device is implanted into a branch of the PA where pressure to its sensor causes the deflection of the pressure-sensitive surface, causing a shift in the resonant frequency. The device external antenna is usually embedded within a pillow on which the patient lies, so pressure transmissions occur while the patient is resting. Clinical utilization was tested in the CHAMPION trial that randomized 550 HF patients and demonstrated a significant reduction in the rate of HF hospitalizations . This piWhile pulmonary pressure monitoring was found to be successful, the direct measurement of left atrial pressure (corresponding to LV filling pressure) is thought to have further clinical advantages for HF monitoring. Left atrial pressure (LAP) is less influenced by pulmonary disease and pulmonary vascular remodeling, and it may be a better marker of response to HF medications. Furthermore, animal studies demonstrated a strong correlation between increases in LAP and pulmonary congestion. The utility of direct LAP monitoring was first evaluated using the HeartPOD system . This system includes an implantable sensor lead coupled to a subcutaneous antenna coil, a patient advisory module, and remote clinician access via secure computer-based data management. The tip of the sensor system lead was transvenously implanted into the left atrium via the atrial septum. The implant was powered and interrogated through the skin by wireless transmissions from the patient advisory module. The LAPTOP-HF trial was intended to evaluate the utility of this system, but it was stopped early due to a perceived excess of implant-related complications .Newer devices to measure and transmit LAP with promising initial results include the V-LAP system  . This isNotably, among HF patients implanted by an ICD or CRT devices, one could use these intracardiac devices to promote continuous monitoring of various hemodynamic parameters to facilitate diagnosis of HF exacerbations ,102,103.Major technological advances, including large-scale integration circuitry using complementary metallic oxide semiconductor (CMOS)-based electrical chips or advanced microelectronic chips (ASIC), which result in highly compact electric circuits with low-power consumption, underlie the development of various implantable miniaturized devices used for pacing and real-time monitoring of cardiac patients. Thereafter, use of novel communication technologies enable fast and reliable intracardiac inter-device communication with minimal interference from cardiac motion and external signals. These miniaturized leadless devices, which are directly implanted into the heart, overcome most endovascular-related complications that are associated with conventional pacing devices. Although many of these devices are still not micro-scale, current significant size reduction, along with novel medical engineering approaches, suggest real micro-scale devices are forthcoming.As of today, single chamber  leadless pacing systems are widely used and multiple leadless pacing systems, such as the leadless CRT, are under clinical trials. Initial studies on leadless CRT pacing systems reveal good implant efficacy, but with high complication rates. Should these multiple leadless device systems prove to be safe and feasible, this would enable the wide use of leadless pacing systems for all cardiac pacing and resynchronization purposes. Last and not least, new technology is needed in order to develop miniaturized high-energy power sources to enable the development of miniaturized ICDs. Until then, leadless pacing systems would need to interact with endovascular conventional ICD or subcutaneous ICD.Regarding real-time hemodynamic monitoring of HF patients, various implanted devices were developed in order to measure cardiac pressure and utilize it for clinical practice . The onl


Psychiatric disorders are a prevalent global health problem, over 900 million individuals affected by a continuum of mental and substance use disorders. Due to this high prevalence, and the substantial direct and indirect societal costs, it is essential to understand the underlying mechanisms of these disorders to facilitate development of new and more effective treatments. Since the advent of recombinant DNA technologies in the early 1980s, genetically modified rodent models have significantly contributed to the genetic and molecular basis of psychiatric disorders. Despite significant advancements, many challenges remain after unsuccessful drug development based on rodent models. Recent human genetics show the polygenetic nature of mental disorders, identifying hundreds of allelic variants that confer increased risk. However, given the complexity of the brain, with many unique cell types, gene expression profiles, and developmental trajectories, proper animal models are needed more than ever to dissect genes and circuits in a cell type-specific manner to advance our understanding and treatment of psychiatric disorders. In this mini-review, we highlight current challenges and promises of using rodent models in advancing science and drug development, focusing on advanced techniques, and their applications to rodent models of psychiatric disorders. An estimated 970 million people worldwide are affected by substance use or mental disorders. At the individual level, these psychiatric disorders were the leading cause of years lived with disability of any disease group, and were comparable to cardiovascular and circulatory diseases for disability-adjusted life years . In 2010C. elegans, Drosophila and zebrafish. Additionally, rodent models are cost- and time-effective compared to primate models for drug screening and development.These heterogeneous responses to typical treatments are only further hindered by diagnostic criteria based on symptomology, with reliable biomarkers for disease diagnosis and monitoring yet to be established. This emphasizes the importance of preclinical research and animal models of psychiatric disorders to fully characterize their underlying genetic and neural mechanisms, and facilitate the development of new treatments. In particular, rodent models have been particularly useful towards this end. Regarding genetic architecture, brain structures and behavioral phenotypes, rodent models are more similar to humans than other non-mammalian models such as Despite some instances of drugs showing promise in rodents and failing human trials, these model systems are needed to untangle the complexity of the brain and its vast array of cell-types, each with unique gene expression profiles and interconnections in distinct neural circuits, ultimately giving rise to behavioral states. In this review, we focus on newly available forward and reverse genetics models and how these models are useful for neural imaging and modulation techniques, which will give researchers an unprecedented ability to understand the connection between genes, circuits, and behavior, and facilitate the development of new biomarkers and therapies for individuals suffering from substance use and other mental disorders.The genetic and molecular characterization of psychiatric disorders has drastically improved over the last decade with the development of large-scale sequencing technologies. Advances in forward genetics have allowed scientists and physicians to examine the entire genome of patients more quickly and cost-effectively than ever before. Given the profound genetic and environmental interactions in the etiology of psychiatric disorders, other next-generation sequencing technologies are also important to characterize changes in gene expression profiles associated with allelic variants or from epigenetic modifications . Although blood driven DNA or RNA sequencing may provide correlational etiology, these techniques fundamentally require primary tissue from the brain, and have largely not been feasible outside of post-mortem brain samples in humans. Therefore, rodent models are particularly advantageous for measuring gene expression profiles in behavioral or genetic models of psychiatric disorders. Despite these advances, the brain is composed of thousands of different cell types with diverse gene expression profiles and developmental trajectories. Having considered these limitations, it would be particularly useful to further clarify the role of genetic risk loci for psychiatric disorders, by pinpointing unique changes in gene expression down to specific cell-types. Recently, single-cell based RNA sequencing (scRNA-seq) can more precisely address the molecular and biological basis of several psychiatric disorder phenotypes. With the recent advancement of scRNA-seq, several publically available datasets offer cell-specific profiles –5. FurthIn one exemplary study, the authors investigated the neural dynamics of mouse prefrontal cortex mainly comprising of anterior cingulate, prelimbic and infralimbic areas during adolescence and in a model of addiction . They seP-value across all tested cell types, 2) within the dataset, identify independent associations with step-wise conditional analysis, and 3) evaluate if the significant associations with cell-types from distinct datasets are driven by similar genetic signals. With these steps, the scRNA-seq data will be meaningfully integrated with clinical data as well.This new technique will reveal the molecular dynamics of many psychiatric disorders. However, scRNA-seq has several notable drawbacks. First, it is still expensive to carry out large scale sequencing, especially compared to tissue-based RNA sequencing. Second, like other -omics approaches, it requires optimization of the data analysis process. Recently, a study provided a promising data analysis platform for complex traits . The autMany of these ‘omics’ techniques have provided large datasets, documenting hundreds of risk-conferring genes and alleles in psychiatric disorders. However, given the complex polygenetic nature of these disorders, the connection between genotype and phenotype often remains obscured. This requires further loss and gain of function studies in animal models to determine how a change in gene expression or altered gene products may contribute to the development or expression of maladaptive behavior or cognition.Classical transgenic rodent models contribute to understanding loss of function for specific genes in psychiatric disorders using conventional KO mice. In addition, the advent of tissue-specific Cre-loxP system enables to investigate more region and temporal deletion and overexpression of gene. However, it has limitations to understand the cell-specific or circuit-specific function of certain genes. Further, the generation of lines can often be financially costly and time-consuming. Other methods such as short interfering RNA’s (siRNA’s) have also been successfully used to reduce expression of target genes, but have some limitations in its flexibility and degree of knockdown.Recently, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a powerful tool, allowing researchers to edit the genome of any organism with precision . GeneralWhile clinical trials utilizing CRISPR-based gene-editing have emerged in other fields, clinical applications for psychiatric disorders remain distant, and will require marked advancements in our understanding of how specific mutations may contribute to brain dysfunction. In rodent models, CRISPR can be used to develop knockout lines more efficiently than traditional methods by injecting single-cell embryos with Cas9 protein and guide RNA targeting the gene of interest, and re-implanting the mutant embryos back into the donor mouse. Alternatively, when packaged into a virus, CRISPR can be used to edit genomes in a region or cell type-specific fashion in vivo Fig. B. One adFor example, the 3q29 deletion is known to increase the risk of developing an intellectual disability, autism spectrum disorder, generalized anxiety disorder, and greater than the 40-fold increased risk for schizophrenia . UtiliziThere have been some trade-offs in editing efficiency and the degree of edit predictability between HDR and NHEJ and concerns of off-target effects and the generation of mutant proteins with unknown functions . More reWhile uncovering the role of genetic risk factors in the development of psychiatric disorders is now more possible than ever, altered gene expression and allelic variants ultimately have their effect through directly or indirectly altering the structure or function of neurons in the brain. Further, these neurons are wired together within and/or between brain regions producing complex neural circuits that ultimately drive behavior. Traditionally, neurophysiological techniques such as electrophysiology have been used to record neural activities. Currently, many techniques enable us to assess neural activity in a real-time manner, which come with various advantages and limitations. One promising advance is calcium imaging due to its improved feasibility in imaging freely-moving animals. Calcium imaging has been a reliable and well-established tool of directly recording neural activity for decades –21. ConsRecently, several applicable options of in vivo calcium imaging are available, according to the primary experimental goals. Largely, optic fiber-based and probe-based observations of GECI fluorescence such as fiber photometry and micro-endoscopy are currently available approaches. The fiber photometry is used to observe regional activity by exciting GECI and measuring the changes in fluorescence utilizing chronic implantation of optic fiber, ranging from 200 to 400 μm in diameter Fig. D. This aTo visualize individual cellular dynamics, the advent of head-mountable micro-endoscopic techniques has been quite useful. Observation of GECIs with a single-cell resolution has required a high enough resolution, a path of fluorescence light transfer from deep brain, and use of a full-size microscope, which traditionally prevents the use of free-moving animals. In addition to dramatic improvements in GECIs, new development of miniaturized micro-endoscopes are able to be mounted on the head of rodents with light-burden of weight (approximately 2 g) and allows for single-cell resolution in behavior-synchronized observation. This miniature microscope typically has fluorescence excitation light source and sensor within the head-mounted part and read the GECIs’ value via chronically implanted GRIN lenses (250 to 1000 μm in diameter). Since this head-mount micro-endoscopic approach can detect changes in GECIs at a single-cell resolution level with spatial information, it is possible to observe whether there is a specific colony of cells that is responsive to specific behavioral patterns or emotional states. In addition, monitoring the spatial location of individual cells makes it possible to trace an identified cell across multiple recording sessions. The main limitation of this approach so far is that the use of single-photon microscopy does not provide the rejection of fluorescence reflecting the outside of the focal plane. Thus, to observe the changes in detailed compartments of cells, further computational process is essential to reduce the noise in the data. To overcome the limitation of this head-mount single-photon micro-endoscopy, along with the development of two-photon head-mountable microscopes  new two-Although we focused on calcium imaging in this review to explain the approaches of measuring neuronal activity synchronized with preclinical animal behaviors, there are also several approaches to visualize spatiotemporal cellular dynamics using fluorescence indicators targeting a wide array of cellular activity such as sensing chloride ion and voltage changes –42.In conjunction with neural imaging, manipulation of specific neural circuits is necessary to establish a causal role of specific neural circuits resulting in behavior or symptoms related to a psychiatric disorder. Traditional methods in rodent models involved lesioning brain regions, electrical stimulation, or pharmacology. However, given the vast diversity of cell types and intricate neuronal connections within and between brain regions, these methods can often be crude, unable to pinpoint the exact neural structures involved. Recently, increasingly powerful tools such as chemogenetics and optogenetics have allowed for modulation of neural activity with high temporal and cell/circuit-specificity.αq protein) and GsD (coupled with Gαs protein) so that they increase intracellular Ca2+ level [αi protein) inhibits adenylyl cyclase [αi (KORD) by the inactive drug-like metabolite salvinorin B (SALB) also leads to inhibit adenylyl cyclase [+ (PSAM-serotonin receptor 5HT3 for activators) and Cl− (PSAM-glycine receptor GlyR for silencers) influx [Chemogenetics involves using chemically engineered ligands and genetically-modified receptors [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) or ligand-gated ion channels ] to control cell signaling Fig. a 43]. C. C43]. C2+ level  or activ2+ level , respect cyclase . Additio cyclase . As the  cyclase . When it) influx .Fig. 4C+ ion and cellular excitability [− ion for cellular inhibition [+ ion levels and reduces cellular activities [aq-coupled α1 adrenergic receptor), B2 (from Gas-coupled β2 adrenergic receptor), D1 (from Gas-coupled D1 dopamine receptor), and A2AR (from Gas-coupled adenosine A2A receptor) [Optogenetics is another powerful tool that is a light-related biological technique with invasive optical fibers implanted into the target brain region. At a specific time point, light stimulation dynamically changes the activities of cells which express light-sensitive receptors (opsin): channelrhodopsin (ChR), halorhodopsin (NpHR), archaerhodopsin (Arch), and OptoXR Fig. b. ChR, atability . On the hibition . Unlike hibition . Using dhibition , 53. Arctivities . OptoXR eceptor) –57.Interestingly, neuromodulation techniques such as deep brain stimulation (DBS) are already being used in clinics to treat a variety of disorders including Parkinson’s disease and Tourette syndrome, as well as depressive disorder and obsessive-compulsive disorder . OptogenWhile these new technologies have allowed us to rapidly advance our understanding of psychiatric disorders in rodent models at the cellular, molecular, and brain circuit levels as summarized in Table While this review primarily focused on recent molecular profiling, and neural imaging and modulation techniques, the importance of representative behavioral paradigms and animal models cannot be overemphasized for the validity of translational psychiatric research. Given the often comprehensive classifications of psychiatric disorders solely based on symptomology, rodent models have often been limited to representing certain aspects of mental disorders based on relatively subjective inferences about the emotional state of animals and their likeness to human conditions. Having said that, a new emphasis on advancing traditional behavioral models will be required to further gleam the emotional and cognitive state of animals. While rodents show common behavioral patterns with other mammals, including humans, it is also important to consider species-specific behaviors, as more natural selective pressures may be optimal for understanding unique behaviors for each species. This will be helpful for generalizing animal behavior and translating it to decipher the molecular basis of human psychiatric disorders. Further, improved nosology for clear behavior outcome measures and discovery of new biomarkers will also greatly advance our ability to create valid and representative rodent models, while simultaneously improving criteria for diagnosis and treatment evaluation.Altogether, despite numerous examples of failed treatments that showed promise in preclinical studies, rodent models will be essential to advance both the understanding and treatment of psychiatric disorders. Used in conjunction with advancing technologies and improved clinical practice, these models will accelerate innovation in the field and have the potential to help improve the lives of the almost one billion individuals across the world, living with mental or substance use disorders.


Across sleep and wakefulness, brain function requires inter-neuronal interactions lasting beyond seconds. Yet, most studies of neural circuit connectivity focus on millisecond-scale interactions mediated by the classic fast transmitters, GABA and glutamate. In contrast, neural circuit roles of the largest transmitter family in the brain–the slow-acting peptide transmitters–remain relatively overlooked, or described as “modulatory.” Neuropeptides may efficiently implement sustained neural circuit connectivity, since they are not rapidly removed from the extracellular space, and their prolonged action does not require continuous presynaptic firing. From this perspective, we review actions of evolutionarily-conserved neuropeptides made by brain-wide-projecting hypothalamic neurons, focusing on lateral hypothalamus (LH) neuropeptides essential for stable consciousness: the orexins/hypocretins. Action potential-dependent orexin release inside and outside the hypothalamus evokes slow postsynaptic excitation. This excitation does not arise from modulation of classic neurotransmission, but involves direct action of orexins on their specific G-protein coupled receptors (GPCRs) coupled to ion channels. While millisecond-scale, GABA/glutamate connectivity within the LH may not be strong, re-assessing LH microcircuits from the peptidergic viewpoint is consistent with slow local microcircuits. The sustained actions of neuropeptides on neuronal membrane potential may enable core brain functions, such as temporal integration and the creation of lasting permissive signals that act as “eligibility traces” for context-dependent information routing and plasticity. The slowness of neuropeptides has unique advantages for efficient neuronal processing and feedback control of consciousness. In modern neuroscience textbooks, coverage of functional interactions between neurons and their postsynaptic targets remains biased toward fast (millisecond-level on/off) interactions mediated by small-molecule neurotransmitters such as ACh, GABA, and glutamate . This isWhile these fast interactions are undoubtedly one fundamental aspect of brain function, most neurotransmitters in the brain do not operate on these rapid timescales. For example, neuropeptides–which are the largest known class of neurotransmitters (>100)–alter postsynaptic neuronal activity at timescales more similar to typical behaviors and important sensory associations, typically seconds to minutes .These slower actions of neuropeptide transmitters are often described as “modulatory.” This term is not clearly defined, but implies a qualitative difference, or that the main role of the peptides is to change the action of fast neurotransmitters . HoweverThe role of neuropeptides in behavioral control, and more specifically, in sleep regulation and vigilance state switching, and their impact on the sleep electroencephalogram has been long known . Insteadvia cytosolic messengers, to the opening of ion channels in the postsynaptic membrane . Most of the non-neuroendocrine hypothalamic neurons examined so far appear to synthesize and/or use classic neurotransmitters such as GABA and glutamate . In addivia the histaminergic system  or slowly (minutes to hours)  . The sloo hours) . LH gluco hours) . The LH o hours) . In addi control . These i control . In conthe brain .Lateral hypothalamus orexin cell firing may thus communicate a combined representation of fast and slow sensory variables. The consequent control of fast and slow behaviors and brain state transitions by orexin neurons has been recently reviewed elsewhere . Below, Orexin neurons co-express several neurotransmitters in addition to orexins, such as the fast transmitter glutamate and the neuropeptide dynorphin . Upon seInside the hypothalamus, the neural circuit between LH orexin and tuberomammillary histamine neurons has been examined. During constant-frequency optogenetic stimulation of orexin cells, histamine cell firing did not follow the temporal pattern of orexin cell stimulation , but responded with a temporal dynamics reminiscent of the sum of a first order derivative and an integral of the presynaptic orexin cell activity . Pharmacin vivo  that exert well-characterized long-lasting effects on neuronal function . Non-neuThe resources invested by the brain into making neuropeptides  are presumably not trivial. Thus, it is tempting to speculate that the 100 + neuropeptides in the brain play some unique functions, and did not evolve solely to serve as a “redundant safeguard” should something go awry with the other transmitters (or as convenient molecular markers for neuroscientists). We speculate that their unique function does not arise at the receptor level, since, as alluded above, other transmitters have similar types of receptors, namely GPCRs linked to long-lasting postsynaptic effects. It could, instead, stem from what happens to neuropeptides in the extracellular space, during the time between release and receptor binding. Unlike other neurotransmitters, which are rapidly cleared from the extracellular space by uptake into neurons and glia by specialized membrane transporters , neuropeIn order to gain a broader understanding of functional neural circuits, we propose that neuropeptide-mediated postsynaptic signals should be routinely analyzed alongside the classic small-molecule neurotransmitters in circuit connectivity screens. While such broader analysis is becoming commonplace in functional dissections of hypothalamic circuits, it is still relatively unusual in other neural circuits, e.g., in cerebral cortex where neuropeptides such as NPY and somatostatin are also abundantly expressed . Thanks in vivo), and to allow sufficient time for the postsynaptic response to appear. Third, while the above-chosen example of orexin illustrates how it directly creates postsynaptic excitation, this is not the case of all neuropeptides. Even within the LH, the MCH neuropeptide, which is made by neurons intermixed with (but distinct from) orexin neurons, does not appear to have direct effects on postsynaptic membrane potential but rather seems to act by altering GABA or glutamate signaling, which could supply a lasting permissive signal  for crety trace . Thus, “In summary, the ability of neuropeptidergic postsynaptic effects to substantially outlast presynaptic firing may bind together fast and slow brain functions in a way that cannot be achieved by fast transmitters alone. Slow on/off neuropeptide signals may thus enable core brain functions, such as creating temporal eligibility traces for memory formation and information routing. Neuropeptides that directly affect the firing of postsynaptic neurons can, in addition, create functional neural circuits able to perform control-relevant computations such as signal integration. This gives peptidergic neural circuits unique advantages for efficient neuronal processing and feedback control of consciousness.DB and MG wrote this review. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WW declared a past co-authorship with one of the authors, DB, to the handling editor.


Bacteroides and Clostridium), meaning industrial microbiomes have low resilience potential. Additionally, database bias obfuscates resilience estimates, as we were 2–5 times more likely to identify SCFA-encoding species in industrial microbiomes compared to non-industrial. Overall, we find high phylogenetic diversity, richness, and evenness of bacteria encoding SCFAs in non-industrial gut microbiomes, signaling high potential for resilience in SCFA production, despite database biases that limit metagenomic analysis of non-industrial populations.High taxonomic diversity in non-industrial human gut microbiomes is often interpreted as beneficial; however, it is unclear if taxonomic diversity engenders ecological resilience (i.e. community stability and metabolic continuity). We estimate resilience through genus and species-level richness, phylogenetic diversity, and evenness in short-chain fatty acid (SCFA) production among a global gut metagenome panel of 12 populations (n = 451) representing industrial and non-industrial lifestyles, including novel metagenomic data from Burkina Faso (n = 90). We observe significantly higher genus-level resilience in non-industrial populations, while SCFA production in industrial populations is driven by a few phylogenetically closely related species (belonging to  Compared to industrial human gut microbiomes, non-industrial gut microbiomes have higher genus and species richness, functional enrichment of amino acid metabolism, greater diversity of genes involved in complex carbohydrate metabolism, and higher amounts of short chain fatty acids (SCFAs) in stool4. These trends have been linked to plant-based diets rich in fibers, infrequent consumption of highly processed foods, and low exposure to pharmaceutical drugs, such as antibiotics, in non-industrial populations5.Lifestyle alterations have repeatedly coincided with biological changes throughout the human past2, which would imply that a non-industrial gut is healthier than the industrial gut, in the absence of confounding variables, such as bacterial pathogens6, eukaryotic parasites7, and malnutrition8. Yet, commonly used diversity statistics oversimplify more complex microbial associations. Ecological approaches that provide context for microbe-microbe interactions, and present insights into how taxonomic shifts influence microbial and host metabolic processes, are making progress towards mitigating this issue. Taxa-gene relationships are at the heart of deeper ecological understandings of human microbiomes and can be assessed through functional diversity and redundancy10. Functional diversity, which is similar to the macroecological concept of response diversity11, refers to the abundance and phylogenetic diversity (PD) of taxa that encode specific genes. It conceptualizes structure–function relationships in the microbiome by tying together taxonomic and metagenomic gene abundance data. Similarly, redundancy can be thought of as the total number of taxa encoding a function, as well as how evenly the production of any given protein is spread amongst taxa.Higher diversity in the gut microbiome is typically considered healthy, all other factors being equalFunctional diversity can be multi-layered, ranging from a fine-tuned focus on individual genes to a broad genome-wide approach. Gene-centric approaches present the opportunity for niche-specific interpretations, while a broader approach allows for study of how entire microbiomes may shift in the face of outside perturbations. No matter the depth and focus of study, high functional diversity is found in microbiomes where phylogenetically diverse bacteria encode the same functions. Under an idealized model, phylogenetically diverse taxa will have an equal contribution to gene production, leading to high redundancy. Functional diversity and redundancy are intertwined and together estimate microbiome resilience. Shifts in taxonomic abundance are less likely to alter the functional potential of a resilient community because any given function is encoded by a wide range of bacteria and production is distributed between these diverse taxa. Consequently, the loss of one phylogenetic branch of bacteria within the ecosystem will not cause a loss of function that those bacteria encode; however, communities with low functional diversity and redundancy may suffer ecosystem-wide functional changes during minor taxonomic perturbations. Accurately quantifying functional diversity is therefore a necessary part of ecologically minded microbiome research because it more deeply describes how structure–function relationships influence resilience in a microbiome.13. Unsurprisingly, variation in SCFA abundance is a classic link to human health. For example, high butyrate levels are found to decrease diastolic blood pressure via regulating inflammation, and acetate abundance is tied to appetite, thus impacting metabolic regulation13.SCFA synthesis is the most intuitive area of study for understanding ecological differences between industrial and non-industrial gut microbiomes, given the trends attributed to high-fiber diets among non-industrial populations. SCFAs are important byproducts of microbial metabolism and dietary fiber fermentation in the human gut. The three most prominent SCFAs in the human gut  are vital for maintenance of tight junction integrity between epithelial cells in the gastrointestinal (GI) tract, serve as an energy source for colonocytes, and signal immune cells, amongst a number of other functions5. It is assumed that non-industrial gut microbiomes bear an ecology that is resilient for SCFA production due to high overall taxonomic diversity and high SCFA levels in stool, but this has not been demonstrated. We address this gap by analyzing functional diversity and redundancy of SCFA synthesis genes in metagenomic datasets from 12 different populations , pastoral, rural agricultural, and hunter-gatherer populations. Industrial populations were split between North America/Europe and Central/East because previous work has suggested variation in gut microbiome composition between these regions15, although more work needs to be done in this area. We chose to evaluate SCFA genes that are involved in end-stage synthesis in different pathways for each SCFA: acetate kinase (ackA) for acetate16, butyrate kinase (buk) and butyryl-CoA: acetate CoA transferase (but) for butyrate18, and methylmalonyl-CoA decarboxylase (mmdA), lactoyl-CoA dehydratase (lcdA), and CoA-dependent propionaldehyde dehydrogenase (pduP) for propionate19. These genes are known to be encoded by a diverse range of bacteria . We functionally profiled these metagenomes using HUMAnN228.Studying SCFA functional diversity is particularly intriguing as it provides a line of evidence as to whether estimates of taxa/gene diversity and ecological resilience are concordant. Research suggests that non-industrial populations have high SCFA abundance, which is attributed to dietary compositionia Table , which p19. For analytical purposes, taxonomic-gene abundance for but and buk were combined for butyrate and data for mmdA, lcdA, and pduP were aggregated for propionate. Response diversity was estimated through genus and species richness and phylogenetic diversity, while the Gini-Simpson index and Hill Numbers were used to assess redundancy by documenting evenness in the community. For our purposes, the Gini-Simpson index29 represents the probability that two sequencing reads originate from different taxa, and therefore values close to 1 represent a community with a high diversity of taxa encoding the SCFA, while values close to 0 indicate that the SCFA is encoded almost entirely by one taxon. Hill numbers30 are a diversity measure that allows for interpretation of effective taxonomic richness at different levels: at diversity order 0 the Hill number value is equal to the total number of taxa (i.e. richness), at diversity order 1 the Hill number value is the effective number of commonly occurring taxa, and at diversity order 2 the Hill number value is the effective number of dominant taxa. A higher number of common and dominant taxa indicates resilience, as a community with minimal common and dominant taxa would be prone to function elimination if those few common/dominant taxa are removed from the community. Therefore, Gini-Simpson and Hill number investigations permit analyzing how evenly SCFA production is distributed between taxa and present opportunities for resilience estimation.Each gene we analyzed is involved in terminal or near-terminal steps of production of each respective SCFA–84) and butyrate was more abundant than propionate  and butyrate  synthesis genes were more abundant in each of the non-industrial populations  only had one or two species per each genus  for acetate and butyrate synthesis compared to the industrial populations  but lower diversity than the European/North American industrial population for acetate . These results demonstrate the industrial populations have only a few common and dominant genera encoding SCFAs, indicating that they are prone to loss in SCFA production if those common/dominant genera are lost. At the species level, evenness is significantly lower for each SCFA in every non-industrial population compared to the industrial groups  and diversity order 2 (number of dominant genera), tell a unique story for each SCFA. Hunter-gatherers and rural agriculturalists have higher effective number of common and dominant genera compared to the industrial populations for butyrate and propionate, but diversity is only greater in non-industrial populations at Hill number 1 for acetate when compared to the Central/East Asian population . The stratified HUMAnN2 output provides abundance of genes matched to bacteria at different taxonomic levels (‘classified’), as well as gene abundance not accounted for by any taxon (‘unclassified’). Across all genes identified in each metagenome, there is a significant decrease in the proportion of gene abundance that is classified to a bacterium at each taxonomic level from industrial to non-industrial populations  to have up to nine species encoding SCFAs, while highly abundant non-industrial genera only have one or two species. Therefore, what first appears to indicate high species-level ecological resilience in SCFA production in the industrial populations is actually the result of closely related species performing the same function. It follows that closely related species may be prone to changes in abundance or even elimination after certain types of ecosystem shift events. For example, narrow-spectrum antibiotics33 and exposure to various xenobiotic compounds that lead to variable bacterial metabolic responses34 are events that can affect a limited range of bacteria and lead to shifts in microbial abundance and metabolic activity. While this result has ecological implications, it is also likely the result of historical trends of microbiology research. Bacterial taxa at high abundance in non-industrial gut microbiomes have not been a focus of microbiological isolation and species identification until recently; therefore, we expect more species to be identified from non-industrial gut microbiomes in the future35. Additionally, classification of bacteria into distinct genera and species is undergoing a revolution in the genomic era36 meaning that the high number of species classified to Bacteroides and Clostridium may ultimately be reclassified to different genera. Nevertheless, the fact that we observe a large jump in species richness, but only a minor increase in species PD, in the industrial gut microbiomes suggests that the high industrial species richness is driven by closely related species and therefore, results in the same interpretation as the genus richness results: diversity is high in non-industrial populations.The increased species-level alpha diversity in industrial populations initially runs counter to the genus-level results but the genus and species level results ultimately yield similar interpretations after accounting for ecology and ascertainment bias, as discussed below. The substantially higher species richness in industrial populations is striking; however, the differences in PD between industrial and non-industrial populations are not nearly as extreme. This means that the high species richness in the industrial populations is driven by species that are closely phylogenetically related. Indeed, we observed SCFA producing genera found at high abundance in industrial populations  observed in industrial gut microbiomes may contribute to the increase in non-communicable chronic diseases found at higher prevalence in industrial populations. The root cause of MIS in industrial populations is undoubtedly multifactorial; however, diet is suggested to play a major role2. This syndrome is compelling and we postulate that this insufficiency precisely rests on the stability of functional capacity. Our findings of decreased resilience in industrial populations, as well as species-level diversity driven by a few closely related species, fits in well with MIS. Low resilience in SCFA production may ultimately manifest itself as altered colonocyte function and/or autoimmune disruptions (both symptoms of MIS) due to a decrease in SCFA bioavailability after a group SCFA-producing bacteria were wiped-out during an ecological shift, such as antibiotic or xenobiotic exposure. Similar to MIS, diet is likely to play an important role in SCFA resilience. The non-industrial populations studied in this paper consume much more fiber than industrial populations, on average26, and microbial fermentation of dietary fibers is a major source of SCFAs in the human digestive tract39. A diet poor in dietary fiber means less substrate for microbial fermentation and therefore less SCFA production and also higher competition for that fiber, potentially resulting in competitive exclusion and less microbial diversity. Nevertheless, if we are unable to fully characterize and annotate non-industrial gut microbiomes then we will be unable to paint a complete picture of MIS. Currently, we have confidence that there is a wealth of undiscovered resilience in non-industrial gut microbiomes. Once we describe the extent of this diversity/resilience, through increased sampling and focus on partnerships with research institutes in industrializing countries, we will have a more complete picture of MIS and possibly develop therapeutic approaches to combat non-communicable chronic diseases related to the human gut microbiome.Limitations in annotating the full extent of microbial diversity impacts health research. Recently proposed ‘Microbiota Insufficiency Syndrome (MIS)’32; however, future work will undoubtedly add to our findings. One avenue for future work is through analyzing SCFA molar concentrations in fecal samples in a longitudinal setting and comparing these results to predicted SCFA resilience from metagenome panels. Unlike genomic data, where we can infer about SCFA production potential via taxonomic diversity, one-time measures of fecal SCFA molar concentrations will not inform about future resilience because SCFA molar concentrations carry no information about which taxa produce each SCFA. Longitudinal SCFA concentration and metagenomic data from non-industrial populations, or animal models, is necessary to inform about SCFA resilience and production in diverse lifestyles. Another avenue for future work is to focus resilience analysis on other microbiome functions of interest, such as resilience of antibiotic resistance genes and amino acid biosynthetic pathways. These valuable studies would be valuable for comparing microbiome resilience dynamics for different functions, with the caveat that there is sufficient genomic annotation data to yield interpretable results.Improved sampling, metabolic profiling, and annotation will not only improve our understanding of SCFA resilience, but it will also permit more detailed picture microbiome wide resilience. Our work shows the value of focusing on specific SCFA genes, due to their importance in human biology and previously reported variation in SCFA molar abundance between industrial and non-industrial populations40. Thus, while there have been improvements, GWAS clearly fail to reflect the breadth of human diversity. Incorporating diverse populations in human genome and microbiome research has the potential to greatly benefit the scientific community’s understanding of human biology and develop treatments that are based on human diversity rather than European-ancestry genetics and microbiomes. A key component of increasing representation in genetics and microbiome studies is that these studies are designed as partnerships with minority and/or indigenous communities in a manner that builds both trust between the community and researchers, as well as facilitates the ability for the sample donors to exercise their rights on how data are treated and shared41.Lack of sample diversity is not unique to human microbiome research, as human genetics research has been grappling with this very issue for decades. In 2009, 96% of individuals included in human genome-wide association studies (GWAS) claimed European ancestry, as compared to 78% in 201942 showing that at least 20 samples per population are required for the types of ecological analyses pursued in this study. Similarly, 10 million reads was chosen as a threshold to allow for sufficient read depth to attain coverage of as many genes as possible from each metagenome. The number of reads generated for the Burkina Faso samples is available in Data File Datasets were chosen because they represent a wide diversity of lifestyles, have a minimum of 20 samples per population, and were sequenced to an average read depth of 10 million reads per sample. We used 20 samples as a threshold based on previous researchFor datasets with available metadata, we included only healthy adults  in the analysis; however, children were included in the Matses and Hadza datasets due to limitation in sample size.Outliers were included in all analysis.Our research objective was to assess resilience in SCFA production across different lifestyles. SCFAs are a key component of human-microbiome interaction and taxonomic diversity is higher in non-industrial populations. Our pre-specified hypothesis was that resilience would be higher in non-industrial populations. After our first phase of analysis, we uncovered the contradictory results between genus and species level resilience and we hypothesized this was due to reference database bias.25 , Oklahoma3 , Northern Europe27 (n = 43); Industrial Central/East Asia—China 21 , Tokyo 20 , Astana 24 ; Pastoral—Mongolia23 ; Rural Agriculturalist—Burkina Faso (n = 90), Madagascar (n = 50); Hunter Gatherer—Matses3 , Hadza22 .All participants from Burkina Faso (n = 90) were healthy volunteers. After filtering the datasets according to our inclusion/exclusion criteria, we analyzed following number of samples from each population: Industrial North American/European—Human Microbiome Project37. All experiments were performed in accordance with relevant guidelines and regulations, with the University of Oklahoma Institutional Biosafety Committee approving the chemical assays (IBC #1044).Human fecal samples were collected with informed consent from resident volunteers of a single village in central Burkina Faso under the ethics review committee of Centre MURAZ, a national health research institute in Burkina Faso (IRB ID No. 31/2016/CE-CM); the US-based University of Oklahoma (OU) researchers received these samples de-identified, omitting individual names, however, with consent, sex, age, education level, and residential area was recorded for each individual and is reported in Data File We randomly subsampled 50 individuals, in R, from each of the Madagascar, Human Microbiome Project, and Mongolian datasets, due to the much higher numbers of individuals in these studies as compared to the remaining datasets. We did not want to skew the different lifestyle groups with overrepresentation from a single dataset.3 and Hadza from Tanzania22), pastoralists , rural-agriculturalists (Madagascar26), industrial European/North American populations , and industrial Central/East Asian populations . Accession numbers can be found in Data File Metagenomic reads for the following datasets were downloaded from either the NCBI Sequence Read Archive or European Nucleotide Archive: hunter-gatherers . The resulting FASTQ files  were used as input for HUMAnN228 with default parameters and using the UniRef50 database44. Briefly, pangenomes are generated for each species identified from metagenomic reads using MetaPhlAn245. Metagenomic reads are mapped against those pangenomes to identify genes; reads not mapping to any pangenome are then mapped against the UniRef50 database44 to identify ‘unclassified’ genes. Reads not mapping to neither the pangenomes nor UniRef50 database are termed ‘UNAMAPPED’. Gene abundance is normalized to reads per kilobase (RPKs) to account for differences in reference database size. The gene family output at the species level from HUMAnN2 was used to perform downstream analysis. Each sample’s output was normalized to gene abundance RPKs per 1 million DNA kilobases and then merged into a single file with all samples. The RPK gene abundance is further stratified by the abundance of the gene that is mapped to a species. Phylum, Family, and Genus level tables were created using the humann2_infer_taxonomy script from HUMAnN2. Acetate, Butyrate, and Propionate gene family tables were generated by pulling out all lines that were annotated with the gene names listed above from the respective normalized phylum, family, genus, species normalized gene abundance tables.All metagenomic data (newly generated from Burkina Faso and downloaded) was processed as follows: AdapterRemoval v2ackA) is the primary end stage enzyme for acetate synthesis16. Butyrate kinase (buk) and butyryl-CoA:acetate CoA transferase (but) can both catalyze butyrate production from butyryl-CoA18. Propionate synthesis can follow three different biochemical pathways: succinate, acrylate, and propanediol depending on the initial substrate19. Methylmalonyl-CoA decarboxylase (mmdA) is a biomarker for the succinate pathway, lactoyl-CoA dehydratase (lcdA) for the acrylate, and CoA-dependent propionaldehyde dehydrogenase (pduP) for the propanediol pathway19. Taxonomic-gene abundance for but and buk were combined for butyrate and likewise data for mmdA, lcdA, and pduP combined for propionate to facilitate SCFA comparisons.The following gene names were used to identify SCFAs: acetate kinase, butyrate kinase, butyryl-CoA: acetate CoA transferase, methylmalonyl-CoA decarboxylase, lactoyl-CoA dehydratase, and CoA-dependent propionaldehyde dehydrogenase. Acetate kinase  was used to account for multiple testing and all p-values can be found in Data File 52.Richness, phylogenetic diversity (PD), Gini-Simpson, and Hill NumbersSupplementary Information.


Messaging through mobile apps and texts are a common feature of technology-based physical activity interventions. We aimed to examine perspectives of mid-life and older cancer survivors on message content, timing, and two-way communication. We conducted qualitative interviews with 14 participants  who completed a remotely delivered intervention that included text messages to support physical activity. After transcription, the interviews were coded and analyzed thematically using inductive, directed content analysis. Themes related to content preferences included personalization, accountability, perspective on the ‘bigger picture,’ and acknowledgement of PA-related achievements. Random timing for receipt was considered acceptable whereas interest in responding to messages was highly variable. Older cancer survivors’ preferences for highly personalized messages are an important consideration in designing technology-based physical activity interventions. A combination of accountability, relation to personal factors, and acknowledgement of goal attainment need to be considered in efforts to scale interventions. Part of a symposium sponsored by the Cancer and Aging Interest Group.


Polyglutamine (polyQ) spinocerebellar ataxias (SCAs) are the most prevalent subset of SCAs and share the aberrant expansion of Q-encoding CAG repeats within the coding sequences of disease-responsible genes as their common genetic cause. These polyQ SCAs  are inherited neurodegenerative diseases characterized by the progressive atrophy of the cerebellum and connected regions of the nervous system, which leads to loss of fine muscle movement coordination. Upon the expansion of polyQ repeats, the mutated proteins typically accumulate disproportionately in the neuronal nucleus, where they sequester various target molecules, including transcription factors and other nuclear proteins. However, it is not yet clearly understood how CAG repeat expansion takes place or how expanded polyQ proteins accumulate in the nucleus. In this article, we review the current knowledge on the molecular and cellular bases of nuclear proteotoxicity of polyQ proteins in SCAs and present our perspectives on the remaining issues surrounding these diseases. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases that cause progressive loss of cerebellar neurons and connected regions of the nervous system, resulting in unsteady gait, clumsiness, and dysarthria . With ovDiscrepancies in the affected neural regions, disease severity, and disease phenotype between the polyQ SCAs are well described in a previous review . What isDespite tremendous amounts of research that have directly linked the cause of polyQ SCAs to a single expansion pattern of genetic aberration, scientists have yet to produce drugs or therapeutic treatments that robustly ameliorate the deteriorating effects of these diseases. The current lack of effective treatment may be attributed to the historical approach of mitigating polyQ SCAs either by reducing the expression (or correcting the misfolding) of polyQ proteins or by enhancing their degradation. The theory behind such approaches is that a reduction in accumulated polyQ proteins lessens their proteotoxicity and provides therapeutic benefit to patients. Though this idea has been widely explored, it remains unclear whether strategies involving the reduction of toxic polyQ proteins are therapeutically feasible given the current technical limitations in gene therapy and in selective clearance of nuclear accumulated toxic polyQ proteins. Researchers now face the challenge of searching for an alternative curative approach to suppress the disease-causing features of nuclear-accumulated polyQ SCA proteins. Thus, it is essential to address fundamental questions, including how CAG repeat expansion takes place, how expanded polyQ SCA proteins accumulate in the nucleus, and how nuclear polyQ SCA proteins form insoluble aggregates that escape degradation. In this review, we summarize the currently available answers to these questions and provide our perspectives on the key issues surrounding polyQ SCAs to better handle the challenges that these diseases present.4C2 repeat expansion, amyotrophic lateral sclerosis), also exist  within the coding sequences of disease-associated genomic loci. The abnormal genetic expansion of repeated sequences as units is known as repeat instability, a unique form of mutation that is associated with more than 40 neurological, neuromuscular, and neurodegenerative diseases . Althougso exist . An impoThe mechanism of repeat expansion can be potentially inferred by the structural properties of DNA following expansion. Expanded TNRs are intrinsically able to form non-B DNA structural elements , which mThe mechanism of TNR expansion may be inferred from the genetic risk factors with which it is associated. In a search for these factors, analysis of single nucleotide polymorphisms (SNPs) in SCA patients showed a significant association between DNA repair genes, including FAN1, PMS2, and RRM2B, and the onset of SCAs . In addiin vivo polyQ SCA models shows the association between repeat instability, transcription-coupled nucleotide excision repair (TC-NER), and regulators of DNA repair. Interestingly, TC-NER, as opposed to mismatch repair, is shown to promote TNR expansion; the knockout of XPA, a component of TC-NER, in neuronal tissues of SCA1 model mice reduces CAG TNRs  modulates CAG repeat instability in a A3 model . FurtherA3 model ; this suAs mentioned earlier, the repeat instability is associated with more than 40 diseases, some of which likely arise from common genetic or molecular pathways. It is, therefore, reasonable to hypothesize that therapeutic measures that intervene the aberrant repeat expansion of disease-associated genes induced by repeat instability may have broad application across many of the abovementioned diseases, quite possibly beyond polyQ SCAs.Most proteins carry out specific functions at defined intracellular locations, signifying that an accurate knowledge of protein localization to and within subcellular compartments is essential to understand the function of proteins. In particular, the nuclear import of proteins affects several key cellular processes, including regulation of gene expression, signal transduction, and cell cycle . This inNuclear transport occurs through the nuclear pore complexes (NPCs), which are comprised of combinations of approximately 30 different nucleoporins (NUPs) . For proDrosophila and mouse models . In SCA3, for example, SCA3 proteins originally located at the cytoplasm are abnormally translocated into the neuronal nucleus upon the abnormal expansion of polyQ domain near the NLS . Highlige models . Althouge models .F-box gene FipoQ as a novel modifier of polyQ toxicity, whereby the protein interacts with Cul1, a scaffolding protein associated with the Cullin-RING ub ligase complexes, and expanded polyQ proteins to ubiquitinate and degrade the latter via the UPS pathway , and SCA7, where the expanded polyQ SCA proteins abnormally translocate into the nucleus, there are few known determinants that cause polyQ SCA proteins to translocate into the nucleus. However, several disease-associated features are suspected to be contributing factors. The first is, as can be expected, the expansion of Q-repeat itself. However, the fact that the translocation to the nucleus is not observed in SCA2 even with polyQ expansion (see below for discussion) implies that the expansion of CAG repeat is not sufficient for nuclear translocation. The second factor is the cleavage of proteins. It is known that polyQ proteins undergo proteolytic cleavage by specific proteases and that cleaved proteins are more toxic and more likely to form nuclear aggregates than full-length ones . This seRegarding the mechanism underpinning nuclear translocation of polyQ SCAs, two major hypotheses can be made . The firOnce expanded polyQ proteins accumulate in the nucleus, they sequester various nuclear proteins and alter RNA homeostasis . InteresAggregation itself, however, may not be the main contributing factor to polyQ toxicity. There are ongoing debates in the field of polyQ disease as to whether or not insoluble aggregates are, in fact, more toxic than soluble oligomers. In the case of Huntington’s disease, soluble oligomers of polyQ-expanded huntingtin engage more extensively with key proteins . This imSo far, we have reviewed current updates on the cell biological mechanisms underpinning the polyQ expansion and nuclear accumulation of disease-associated polyQ SCA proteins. However, these mark only the beginning stages of the polyQ SCA development as the accumulation of polyQ proteins in the nucleus subsequently leads to a multitude of cellular impairments, which ultimately cause neuronal cell death characteristic of polyQ SCAs. The nature of broad interactions of toxic proteins with various TFs implies a wide range of deleterious cellular defects, including dendrite defects , caused Due to the practical limitations surrounding the characterization of pathogenic mechanisms of polyQ SCAs in human patients, animal models overexpressing expanded polyQ proteins are often used. These animal models, including fruit fly , mouse , and nonHere, we present a model in which nuclear accumulation of polyQ SCA proteins may explain the subtype-specific characteristics of the diseases. Based on the fact that CAG expansion occurs in subtype-specific genetic loci for each subtype of polyQ SCA, our model involves each subtype of expanded polyQ proteins having an affinity toward subtype-specific nuclear proteins. Although all polyQ proteins have an expanded CAG domain, regions that surround the polyQ repeat regions contain subtype-specific flanking residues. These residues may also exert toxicity by abnormally trapping their interacting partners during multimerization of the mutated proteins. Therefore, the differences in protein residues that flank the polyQ expanded region might give rise to subtype-specific interactions with nuclear proteins. More research elucidating the interacting partners of nuclear polyQ proteins is needed to better understand polyQ SCA diseases.Although there are currently no known cures for polyQ SCAs, there are some promising avenues worth exploring. Therapies that target structural properties of polyQ SCAs may be useful in ameliorating toxicity of disease-associated proteins. For example, QBP1 is a peptide with selective affinity toward an expanded form of polyQ repeats containing β-sheet structures and acts as a structural inhibitor of polyQ protein multimerization . InterveDL, Y-IL, Y-SL, and SL conceptualized the theme of the review and wrote the manuscript together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including  Cyanobacteria are a group of photosynthetic prokaryotes that exist in a wide range of environments and temperatures. Several species of these cyanobacteria produce a variety of secondary metabolites that can adversely affect the surrounding flora and fauna. These secondary metabolites are called cyanotoxins and are classified based on their chemical structure  and toxicity based on their target organ system . Amongst all the cyanotoxins, microcystins are produced by several strains of cyanobacteria .Microcystin is produced by cyanobacteria, or blue-green algae, that contaminate freshwater environments around the world during harmful algal blooms (HABs) . These HThe effects of HAB toxins, such as microcystins, have been extensively studied in humans and have been recently reviewed . In humaThe effects of microcystins are not limited to humans but are a major, direct health threat to the aquatic species inhabiting contaminated waters and the surrounding fauna. HAB toxins such as microcystins have been found to have bioaccumulation within fish, frogs, mussels, water snails, and various other aquatic species . ExtensiHAB toxins such as microcystins also have detrimental effects on the reproductive system of many species and subsequently affect species survival. Microcystins can affect the quality of egg and sperm production and can cause developmental neuropathology within offspring . Within While some species are more prone to toxic effects of HAB toxins such as microcystins, other species have developed mechanisms of HAB toxin resistance, such as crayfish . Some spLithobates catesbeiana (American bullfrog) tadpoles. These tadpoles represent one of the populations that are frequently found to inhabit freshwater environments contaminated by HAB toxins.To date, most studies of HAB toxicity in aquatic wildlife have been done in fish, mollusks, and shellfish. One of the populations that remain to be thoroughly evaluated for HAB effects is amphibians. In order to address this, we have characterized the acute, short-term effects of HAB toxins, including microcystins , in the intestines and livers of p < 0.0001).Histopathological analysis of hematoxylin and eosin (H&E)-stained intestinal sections from tadpoles revealed visibly distended intestines in HAB-toxin-exposed tadpoles as compared with control tadpoles that were exposed to normal pond water A. FurtheHistopathological analysis of H&E-stained intestinal sections from tadpoles also revealed that intestinal folds were visibly shorter in height in the HAB-toxin-exposed tadpoles as compared with control tadpoles that were exposed to normal pond water A. FurtheThe total number of intestinal folds per tadpole was normalized to the total length of intestine per tadpole. Normalized intestinal fold numbers revealed no significant differences between HAB-toxin-exposed tadpoles and control tadpoles exposed to normal pond water .Histopathological analysis of H&E-stained liver sections from tadpoles revealed visibly larger hepatocytes in HAB-toxin-exposed tadpoles as compared with control tadpoles that were exposed to normal pond water A. FurtheHistopathological analysis of H&E-stained liver sections from tadpoles revealed a visibly greater number of binucleated hepatocytes in HAB-toxin-exposed tadpoles as compared with control tadpoles that were exposed to normal pond water A. FurtheIntestine and liver tissue sections were processed for immunohistochemical (IHC) analysis of protein carbonylation as a marker of oxidative stress. Positive staining for protein carbonylation is demonstrated by diffuse brown 3,3′-Diaminobenzidine (DAB) staining within the intestinal wall and liver tissue as pointed out by red arrows . AnalysiHAB toxins have become a growing global health concern, not only for humans but also for the fauna that inhabit environments surrounding contaminated water sources. We aimed to investigate the acute effects of short-term exposure to HAB toxins containing microcystins on tadpoles, knowing that most previous studies have observed deleterious effects with long-term exposure.In the current study, we have shown that just 7 days of HAB toxin exposure causes damaging effects in tadpole livers and intestines. Our results show that the intestines in HAB-toxin-exposed tadpoles were significantly more distended than in control tadpoles. The number of intestinal folds is similar between exposed and non-exposed tadpoles, and the actual intestinal fold heights are significantly shorter in HAB-toxin-exposed tadpoles compared with control tadpoles. Together, these suggest that there is intestinal distention in HAB-toxin-exposed tadpoles, with intestinal diameter expansion and flattening of intestinal fold heights.Changes in intestinal diameter, fold height, and fold number have been previously shown to indicate intestinal remodeling in thyroid hormone receptor knock-out tadpoles . IntestiOne of the most notable pathologies observed within the livers of exposed tadpoles was hepatocyte hypertrophy. Hepatocyte hypertrophy can be a result of numerous pathological processes . InteresAnother liver finding seen in HAB-toxin-exposed tadpoles was an increase in hepatocyte binucleation. Binucleation or polyploidization can be a result of cellular stress from toxins, metabolic overload, or oxidative stress, and has been linked to many liver diseases . It has To further characterize the oxidative stress response to HAB toxins, we examined protein carbonylation in both the intestines and the livers of HAB-toxin-exposed tadpoles and found that it was elevated in both organs. Protein carbonylation, as measured by immunohistochemistry, is a hallmark of oxidative damage . HAB toxLithobates catesbeiana tadpoles [There have recently been several studies that have also investigated HAB toxin toxicity in amphibians, as this is a quickly growing area of concern. Junior et al. recently studied microcystin toxicity at a long-term exposure level of 16 days within tadpoles . Junior tadpoles . HAB toxXenopus laevis (African clawed frog) tadpoles, a different species of tadpole [Xenopus laevis during their early life stages [Xenopus laevis tadpoles and early-stage Xenopus laevis, which contrasts with the significant hepatic and gastrointestinal toxicity we observed in Lithobates catesbeiana tadpoles. Our results importantly highlight that, while short-term exposure to HAB toxins such as microcystins to some species has limited effects, other species, such as Lithobates catesbeiana tadpoles may be more susceptible and vulnerable to HAB toxicity [Our findings are significant within the limited research currently published on HAB toxicity within amphibians, given that others have found HAB toxin, such as microcystins to have little to no effects in different amphibian species. Zikova et al. have previously investigated microcystins’ effects within  tadpole . They exe stages . They retoxicity .Microcystis by Rana grylio tadpoles in ponds within Guanqiao of Wuhan, China [Microcystis levels in ponds with tadpoles than ponds without tadpoles. Within a laboratory environment, Zhang et al. confirmed that, when tadpoles were exposed to Microcystis suspensions, there was a 73% decrease in Microcystis levels in just 24 h. In addition, it is well known that the early stages of amphibian development occur in the shallow littoral zones of surface waters, and this is exactly where high levels or HAB toxins such as microcystins co-occur [It is important to note that HAB toxicity is a very real environmental hazard for tadpoles as it is indeed a significant part of their oral intake. Zhang et al. have previously investigated the oral intake of n, China . Within co-occur . These cco-occur . We alsoco-occur ,10,46,47Microcystis cyanbacteria. Secondly, it is important to note that our HAB material exposure was adjusted based on total microcystin concentration  of the HAB material before addition to the terraria, and we did not measure dissolved microcsytins after addition of the HAB toxins to the terraria. Therefore, because a large percentage of the total microcystin was indeed intracellular (98.6%), the actual freely dissolved microcystin concentration to which the tadpoles were exposed may in fact be lower than the 1 μg/L total concentration stated. We also note that, while we and others have shown Microcystis to be the dominant species in our sample and, therefore, the focus of our investigation, there could also be potential effects from heterotrophic bacteria and cyanobacterial LPS. Though cyanobacterial LPS could contribute potential effects, previous studies have demonstrated that aquatic vertebrates such as amphibians and fish are resistant to LPS toxicity as many even lack a TLR4 ortholog and the costimulatory molecules for TLR4 activation by LPS [It is important to note some limitations within the current study. First, although we were able to accurately quantify the amount of extracellular, intracellular, and total microcystins, our study did not measure other potentially important parameters such as the total number of cyanobacterial cells present in the HAB material, chlorophyll content, or composition of minor species other than the dominant n by LPS . Others n by LPS . Wendel n by LPS .Lithobates catesbeiana tadpoles. While a recent report by Junior et al. showed that prolonged exposure of tadpoles (16 days) to microcystins induces toxic effects within the intestines and livers [Lithobates catesbeiana tadpoles, may be more vulnerable and susceptible to the toxic effects of HAB toxins than others. These findings highlight the need to further investigate acute, short-term HAB toxin effects within populations at risk of exposure of HAB-toxin-contaminated waters and to be cognizant that some species may be more vulnerable and susceptible to HAB toxins’ effects than others.Collectively, our results show that HAB toxins, such as microcystins, have significant toxic effects within the intestines and livers of d livers , our stuPrevious and concurrent studies have established that algal blooms in Lake Erie and the Maumee River are dominated by the genus Microcystis ,51,52,53The HAB toxin material used for the current study was obtained in September 2017 from site L1 of the Maumee Bay area of Western Lake Erie  as described in  during aImmediately after collection, we separated our algal sample into three aliquots and froze them. Total levels of total microcystins were established by passing the aliquot of unfiltered, concentrated algae through 3 freeze/thaw cycles in order to lyse cells, releasing all intracellular total microcystin. ELISA was then run on the sample, which accounts for the initially present, extracellular levels of total microcystin and the lysis-released, intracellular levels of total microcystin. The ELISA assay , which dLithobates catesbeiana) tadpoles were collected in August 2018 from Ottawa National Wildlife Refuge, Ottawa County, Ohio using a fyke net. Importantly, the collection pond had no direct contact or water exchange with Lake Erie and had not experienced harmful algal blooms. Therefore, the tadpoles were never exposed to HAB toxins. Once collected, the tadpoles were randomly divided into two experimental groups: control (n = 10) and HAB-toxin-exposed (n = 10). Tadpoles were housed in pairs in 5-gallon, plastic terraria filled with 2 gallons of pond water collected at the same time and locations as the tadpoles. We thawed the third aliquot of concentrated, unfiltered algae (collected as described above), re-froze and re-thawed it twice more for a total of 3 freeze–thaw cycles, and then, to each of the cages in the HAB-toxin-exposed group, we added 60 mL of concentrated, unfiltered algae to reach a final concentration of 1 μg/L of total microcystins in the terrarium water, the concentration at which detectable but sub-lethal effects have been observed in the congener Rana nigromaculata [All animals were captured and handled in accordance with Institutional Animal Care and Use Committee Protocol 108803 and Institutional Biosafety Committee Protocol 108801 (University of Toledo), Wild Animal Permit 21-016 , and Research Permit 2018010 (U.S. Fish and Wildlife Service), All animals were captured and handled in accordance with University of Toledo Institutional Animal Care and Use Committee Protocol 108803 (approved 6 March 2018), Institutional Biosafety Committee Protocol 108801, Wild Animal Permit 21-016 , and Research Permit 2018010 (U.S. Fish and Wildlife Service). Tadpole sampling and exposure were conducted as follows. Briefly, a total of 20 one-year-old American bullfrog . Five (5)-micron tissue sections were placed on glass slides and stained with H&E. Images of histology slides were taken using an Olympus VS120 Slide Scanner.Intestinal diameter, intestinal fold height, and intestinal fold number have been previously quantified in order to reflect gastrointestinal pathology . IntestiThe total number of intestinal folds per tadpole intestine was counted manually. The intestinal fold heights were measured from the outer edge of the intestinal wall to the peak of each fold using Olympus CellSens software. Twenty intestinal folds were measured randomly from each tadpole, intestine and averages were obtained per tadpole.For fecal content density analysis, 5 representative images from the lumen of the gut of each tadpole were evaluated by FIJI biological image analysis software   was measured using the Protein Carbonyls Immunohistochemical Staining Kit . A goat, anti-rabbit IgG primary antibody  was applied on each sample. Secondary solutions A and B (Vectastain Elite ABC HRP Immunodetection Kit) were subsequently applied. Slides were developed using HRP Color Development Reagent, DAB  and counterstained with hematoxylin .Photographs of whole tissue slides were taken at 20× magnification using a VS120 Virtual Slide Microscope . Images were converted from .vsi to .tif and compressed using the Fiji distribution of ImageJ 1.52p  . Percentt-test (two-tailed), as we have previously adhered to in previous investigations [p values were <0.05.Data presented are mean ± SD. Data obtained were first tested for normality via the Shapiro–Wilk and D’Agostino and Pearson normality tests. If the data did not pass the normality test, the Mann–Whitney Rank Sum test was used to compare the data. If the data did pass the normality test, parametric comparisons were performed using the unpaired Student’s igations ,60. Equa