AFD Neurons Research Articles

Multisite regulation integrates multimodal context in sensory circuits to control persistent behavioral states in C. elegans

Maintaining or shifting between behavioral states according to context is essential for animals to implement fitness-promoting strategies. How the integration of internal state, past experience and sensory inputs orchestrates persistent multidimensional behavioral changes remains poorly understood. Here, we show that C. elegans integrates environmental temperature and food availability over different timescales to engage in persistent dwelling, scanning, global or glocal search strategies matching thermoregulatory and feeding needs. Transition between states, in each case, involves regulating multiple processes including AFD or FLP tonic sensory neurons activity, neuropeptide expression and downstream circuit responsiveness. State-specific FLP-6 or FLP-5 neuropeptide signaling acts on a distributed set of inhibitory GPCR(s) to promote scanning or glocal search, respectively, bypassing dopamine and glutamate-dependent behavioral state control. Integration of multimodal context via multisite regulation in sensory circuits might represent a conserved regulatory logic for a flexible prioritization on the valence of multiple inputs when operating persistent behavioral state transitions.

Human FAM3C restores memory-based thermotaxis of Caenorhabditis elegans famp-1/m70.4 loss-of-function mutants.

The family with sequence similarity 3 (FAM3) superfamily represents a distinct class of signaling molecules that share a characteristic structural feature. Mammalian FAM3 member C (FAM3C) is abundantly expressed in neuronal cells and released from the synaptic vesicle to the extracellular milieu in an activity-dependent manner. However, the neural function of FAM3C has yet to be fully clarified. We found that the protein sequence of human FAM3C is similar to that of the N-terminal tandem domains of Caenorhabditis elegans FAMP-1 (formerly named M70.4), which has been recognized as a tentative ortholog of mammalian FAM3 members or protein-O-mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1). Missense mutations in the N-terminal domain, named Fam3L2, caused defects in memory-based thermotaxis but not in chemotaxis behaviors; these defects could be restored by AFD neuron-specific exogenous expression of a polypeptide corresponding to the Fam3L2 domain but not that corresponding to the Fam3L1. Moreover, human FAM3C could also rescue defective thermotaxis behavior in famp-1 mutant worms. An in vitro assay revealed that the Fam3L2 and FAM3C can bind with carbohydrates, similar to the stem domain of POMGnT1. The athermotactic mutations in the Fam3L2 domain caused a partial loss-of-function of FAMP-1, whereas the C-terminal truncation mutations led to more severe neural dysfunction that reduced locomotor activity. Overall, we show that the Fam3L2 domain-dependent function of FAMP-1 in AFD neurons is required for the thermotaxis migration of C. elegans and that human FAM3C can act as a substitute for the Fam3L2 domain in thermotaxis behaviors.

Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling

Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.

Temperature-Dependent Regulation of Proteostasis and Longevity.

Temperature is an important environmental condition that determines the physiology and behavior of all organisms. Animals use different response strategies to adapt and survive fluctuations in ambient temperature. The hermaphrodite Caenorhabditis elegans has a well-studied neuronal network consisting of 302 neurons. The bilateral AFD neurons are the primary thermosensory neurons in the nematode. In addition to regulating thermosensitivity, AFD neurons also coordinate cellular stress responses through systemic mechanisms involving neuroendocrine signaling. Recent studies have examined the effects of temperature on altering various signaling pathways through specific gene expression programs that promote stress resistance and longevity. These studies challenge the proposed theories of temperature-dependent regulation of aging as a passive thermodynamic process. Instead, they provide evidence that aging is a well-defined genetic program. Loss of protein homeostasis (proteostasis) is one of the key hallmarks of aging. Indeed, proteostasis pathways, such as the heat shock response and aggregation of metastable proteins, are also controlled by thermosensory neurons in C. elegans. Prolonged heat stress is thought to play a critical role in the development of neurodegenerative protein misfolding diseases in humans. This review presents the latest evidence on how temperature coordinates proteostasis and aging. It also discusses how studies of poikilothermic organisms can be applied to vertebrates and provides new therapeutic strategies for human disease.

Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.

On the Modeling of the Three Types of Non-spiking Neurons of the Caenorhabditis elegans.

The nematode Caenorhabditis elegans (C. elegans) is a well-known model organism in neuroscience. The relative simplicity of its nervous system, made up of few hundred neurons, shares some essential features with more sophisticated nervous systems, including the human one. If we are able to fully characterize the nervous system of this organism, we will be one step closer to understanding the mechanisms underlying the behavior of living things. Following a recently conducted electrophysiological survey on different C. elegans neurons, this paper aims at modeling the three non-spiking RIM, AIY and AFD neurons (arbitrarily named with three upper case letters by convention). To date, they represent the three possible forms of non-spiking neuronal responses of the C. elegans. To achieve this objective, we propose a conductance-based neuron model adapted to the electrophysiological features of each neuron. These features are based on current biological research and a series of in-silico experiments which use differential evolution to fit the model to experimental data. From the obtained results, we formulate a series of biological hypotheses regarding currents involved in the neuron dynamics. These models reproduce experimental data with a high degree of accuracy while being biologically consistent with state-of-the-art research.

Age‐dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans

Age‐dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin‐based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory‐evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD‐specific guanylyl cyclase, GCY‐8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY‐8 cyclase domain reduces these age‐dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy‐8 animals. The lack of apparent correlation between age‐dependent changes in the morphology or stimuli‐evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age‐related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature‐evoked AFD responses, and experience‐based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.

Neural Coding of Thermal Preferences in the Nematode Caenorhabditis elegans.

Animals are capable to modify sensory preferences according to past experiences. Surrounded by ever-changing environments, they continue assigning a hedonic value to a sensory stimulus. It remains to be elucidated however how such alteration of sensory preference is encoded in the nervous system. Here we show that past experiences alter temporal interaction between the calcium responses of sensory neurons and their postsynaptic interneurons in the nematode Caenorhabditis elegans. C. elegans exhibits thermotaxis, in which its temperature preference is modified by the past feeding experience: well-fed animals are attracted toward their past cultivation temperature on a thermal gradient, whereas starved animals lose that attraction. By monitoring calcium responses simultaneously from both AFD thermosensory neurons and their postsynaptic AIY interneurons in well-fed and starved animals under time-varying thermal stimuli, we found that past feeding experiences alter phase shift between AFD and AIY calcium responses. Furthermore, the difference in neuronal activities between well-fed and starved animals observed here are able to explain the difference in the behavioral output on a thermal gradient between well-fed and starved animals. Although previous studies have shown that C. elegans executes thermotaxis by regulating amplitude or frequency of the AIY response, our results proposed a new mechanism by which thermal preference is encoded by phase shift between AFD and AIY activities. Given these observations, thermal preference is likely to be computed on synapses between AFD and AIY neurons. Such a neural strategy may enable animals to enrich information processing within defined connectivity via dynamic alterations of synaptic communication.

The Caenorhabditis elegans INX-4/Innexin is required for the fine-tuning of temperature orientation in thermotaxis behavior.

Innexins in invertebrates are considered to play roles similar to those of connexins and pannexins in vertebrates. However, it remains poorly understood how innexins function in biological phenomena including their function in the nervous systems. Here, we identified inx‐4, a member of the innexin family in C. elegans, by a forward screening of thermotaxis‐defective mutants. The inx‐4 mutants exhibited abnormal migration to a temperature slightly higher than the cultivation temperature, called mild thermophilic behavior. Rescue experiments revealed that INX‐4 acts in the major thermosensory neuron AFD to regulate thermotaxis behavior. INX‐4::GFP fusion protein localized exclusively along axons in AFD neurons. In addition, over‐expression of INX‐4 in AFD neurons induced a cryophilic behavior, which is opposite to inx-4 mutants. Our findings suggest that INX‐4/Innexin in AFD may fine‐tune the execution of thermotaxis behavior when moving to desired temperatures.

Graphene oxide-induced neurotoxicity on neurotransmitters, AFD neurons and locomotive behavior in Caenorhabditis elegans

Graphene oxide (GO) and graphene-based nanomaterials have been widely applied in recent years, but their potential health risk and neurotoxic potentials remain poorly understood. In this study, neurotoxic potential of GO and its underlying molecular and cellular mechanism were investigated using the nematode, Caenorhabditis elegans. Deposition of GO in the head region and increased reactive oxygen species (ROS) was observed in C. elegans after exposure to GO. The neurotoxic potential of GO was then investigated, focusing on neurotransmitters contents and neuronal activity using AFD sensory neurons. The contents of all neurotransmitters, such as, tyrosine, tryptophan, dopamine, tyramine, and GABA, decreased significantly by GO exposure. Decreased fluorescence of Pgcy-8:GFP, a marker of AFD sensory neuron, by GO exposure suggested GO could cause neuronal damage on AFD neuron. GO exposure led decreased expression of ttx-1 and ceh-14, genes required for the function of AFD neurons also confirmed possible detrimental effect of GO to AFD neuron. To understand physiological meaning of AFD neuronal damage by GO exposure, locomotive behavior was then investigated in wild-type as well as in loss-of-function mutants of ttx-1 and ceh-14. GO exposure significantly altered locomotor behavior markers, such as, speed, acceleration, stop time, etc., in wild-type C. elegans, which were mostly rescued in AFD neuron mutants. The present study suggested the GO possesses neurotoxic potential, especially on neurotransmitters and AFD neuron in C. elegans. These findings provide useful information to understand the neurotoxic potential of GO and other graphene-based nanomaterials, which will guide their safe application.

A Calcium- and Diacylglycerol-Stimulated Protein Kinase C (PKC), Caenorhabditis elegans PKC-2, Links Thermal Signals to Learned Behavior by Acting in Sensory Neurons and Intestinal Cells.

Ca2+- and diacylglycerol (DAG)-activated protein kinase C (cPKC) promotes learning and behavioral plasticity. However, knowledge of in vivo regulation and exact functions of cPKCs that affect behavior is limited. We show that PKC-2, a Caenorhabditis elegans cPKC, is essential for a complex behavior, thermotaxis. C. elegans memorizes a nutrient-associated cultivation temperature (Tc ) and migrates along the Tc within a 17 to 25°C gradient. pkc-2 gene disruption abrogated thermotaxis; a PKC-2 transgene, driven by endogenous pkc-2 promoters, restored thermotaxis behavior in pkc-2-/- animals. Cell-specific manipulation of PKC-2 activity revealed that thermotaxis is controlled by cooperative PKC-2-mediated signaling in both AFD sensory neurons and intestinal cells. Cold-directed migration (cryophilic drive) precedes Tc tracking during thermotaxis. Analysis of temperature-directed behaviors elicited by persistent PKC-2 activation or inhibition in AFD (or intestine) disclosed that PKC-2 regulates initiation and duration of cryophilic drive. In AFD neurons, PKC-2 is a Ca2+ sensor and signal amplifier that operates downstream from cyclic GMP-gated cation channels and distal guanylate cyclases. UNC-18, which regulates neurotransmitter and neuropeptide release from synaptic vesicles, is a critical PKC-2 effector in AFD. UNC-18 variants, created by mutating Ser311 or Ser322, disrupt thermotaxis and suppress PKC-2-dependent cryophilic migration.

Magnetic orientation in C. elegans relies on the integrity of the villi of the AFD magnetosensory neurons

The magnetic field of the earth provides many organisms with sufficient information to successfully navigate through their environments. While evidence suggests the widespread use of this sensory modality across many taxa, it remains an understudied sensory modality. We have recently showed that the nematode C. elegans orients to earth-strength magnetic fields using the first pair of described magnetosensory neurons, AFDs. The AFD cells are a pair of ciliated sensory neurons crowned by fifty villi known to be implicated in temperature sensation. We investigated the potential importance of these subcellular structures for the performance of magnetic orientation. We show that ciliary integrity and villi number are essential for magnetic orientation. Mutants with impairments AFD cilia or villi structure failed to orient to magnetic fields. Similarly, C. elegans larvae possessing immature AFD neurons with fewer villi were also unable to orient to magnetic fields. Larvae of every stage however retained the ability to orient to thermal gradients. To our knowledge, this is the first behavioral separation of magnetic and thermal orientation in C. elegans. We conclude that magnetic orientation relies on the function of both cilia and villi in the AFD neurons. The role of villi in multiple sensory transduction pathways involved in the sensory transduction of vectorial stimuli further supports the likely role of the villi of the AFD neurons as the site for magnetic field transduction. The genetic and behavioral tractability of C. elegans make it a promising system for uncovering potentially conserved molecular mechanisms by which animals across taxa detect and orient to magnetic fields.

A C. elegans Thermosensory Circuit Regulates Longevity through crh-1/CREB-Dependent flp-6 Neuropeptide Signaling

Sensory perception, including thermosensation, shapes longevity in diverse organisms, but longevity-modulating signals from the sensory neurons are largely obscure. Here we show that CRH-1/CREB activation by CMK-1/CaMKI in the AFD thermosensory neuron is a key mechanism that maintains lifespan at warm temperatures in C.elegans. In response to temperature rise and crh-1 activation, the AFD neurons produce and secrete the FMRFamide neuropeptide FLP-6. Both CRH-1 and FLP-6 are necessary and sufficient for longevity at warm temperatures. Our data suggest that FLP-6 targets the AIY interneurons and engages DAF-9 sterol hormone signaling. Moreover, we show that FLP-6 signaling downregulates ins-7/insulin-like peptide and several insulin pathway genes, whose activity compromises lifespan. Our work illustrates how temperature experience is integrated by the thermosensory circuit to generate neuropeptide signals that remodel insulin and sterol hormone signaling and reveals a neuronal-endocrine circuit driven by thermosensation to promote temperature-specific longevity.

Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans.

Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.

Regulator of Calcineurin (RCAN-1) Regulates Thermotaxis Behavior in Caenorhabditiselegans

Regulator of calcineurin (RCAN) is a calcineurin-interacting protein that inhibits calcineurin phosphatase when overexpressed, often upregulated under neuropathological conditions with impaired learning and memory processes, such as Down syndrome or Alzheimer's disease. Thermotactic behavior in the nematode Caenorhabditis elegans is a form of memory in which calcineurin signaling plays a pivotal role in the thermosensation of AFD neurons. In this study, we found that rcan-1 deletion mutants exhibited cryophilic behavior dependent on tax-6, which was rescued by expressing rcan-1 in AFD neurons. Interaction between RCAN-1 and TAX-6 requires the conserved PxIxIT motif of RCAN-1, without which thermotactic behavior could not be fully rescued. In addition, the loss of crh-1/CREB suppressed the thermotaxis phenotypes of rcan-1 and tax-6 mutants, indicating that crh-1 is crucial in thermotaxis memory in these mutants. Taken together, our results suggest that rcan-1 is an inhibitory regulator of tax-6 and that it acts in the formation of thermosensory behavioral memory in C. elegans.

Pharyngeal pumping inhibition and avoidance by acute exposure to high CO2 levels are both regulated by the BAG neurons via different molecular pathways.

Carbon dioxide (CO2) is a key molecule in many biological processes. Studies in humans, mice, D. melanogaster, C. elegans, unicellular organisms and plants have shed light on the molecular pathways activated by elevated levels of CO2. However, the mechanisms that organisms use to sense and respond to high CO2 levels remain largely unknown. Previous work has shown that C. elegans quickly avoid elevated CO2 levels using mechanisms that involve the BAG, ASE and AFD neurons via cGMP- and calcium- signaling pathways. Here, we discuss our recent finding that exposure of C. elegans to high CO2 levels leads to a very rapid cessation in the contraction of the pharynx muscles. Surprisingly, none of the tested CO2 avoidance mutants affected the rapid pumping inhibition response to elevated CO2 levels. A forward genetic screen identified that the hid-1-mediated pathway of dense core vesicle maturation regulates the pumping inhibition, probably through affecting neuropeptide secretion. Genetic studies and laser ablation experiments showed that the CO2 response of the pharyngeal muscle pumping is regulated by the BAG neurons, the same neurons that mediate CO2 avoidance.

Ciliopathy proteins establish a bipartite signaling compartment in a C. elegans thermosensory neuron

How signaling domains form is an important, yet largely unexplored question. Here, we show that ciliary proteins help establish two contiguous, yet distinct cyclic GMP (cGMP) signaling compartments in Caenorhabditis elegans thermosensory AFD neurons. One compartment, a bona fide cilium, is delineated by proteins associated with Bardet–Biedl syndrome (BBS), Meckel syndrome and nephronophthisis at its base, and requires NPHP-2 (known as inversin in mammals) to anchor a cGMP-gated ion channel within the proximal ciliary region. The other, a subcompartment with profuse microvilli and a different lipid environment, is separated from the dendrite by a cellular junction and requires BBS-8 and DAF-25 (known as Ankmy2 in mammals) for correct localization of guanylyl cyclases needed for thermosensation. Consistent with a requirement for a membrane diffusion barrier at the subcompartment base, we reveal the unexpected presence of ciliary transition zone proteins where no canonical transition zone ultrastructure exists. We propose that differential compartmentalization of signal transduction components by ciliary proteins is important for the functions of ciliated sensory neurons.

Arsenite induces neurotoxic effects on AFD neurons via oxidative stress in Caenorhabditis elegans.

Arsenic permeates our environment. As a result, humans are continually exposed to it. This study investigates the possible roles of oxidative stress in arsenite (As(III))-induced neurotoxicity in Caenorhabditis elegans. Exposure to As(III), at the concentrations examined, caused a decrease in locomotor behaviors (frequencies of body bends, head thrashes, and reversals) of C. elegans. In addition, As(III) exposure (100 μM) decreased thermotactic behaviors, and induced severe deficits in the structural properties of AFD sensory neurons. Exposure to As(III) (100 μM) also caused an elevated production of intracellular reactive oxygen species (ROS) in wild-type C. elegans. Pretreatment with the antioxidant curcumin ameliorated the decrease in locomotor and thermotactic behavior, the formation of deficits in the structural properties of AFD sensory neurons, and intracellular ROS in As(III)-exposed nematodes. Our study suggests that oxidative stress plays a crucial role in the As(III)-induced neurotoxic effects on locomotor behavior and the structures and function of AFD sensory neurons in As(III)-exposed nematodes.

Correction: Phthalates Induce Neurotoxicity Affecting Locomotor and Thermotactic Behaviors and AFD Neurons through Oxidative Stress in Caenorhabditis elegans

The authors would like to acknowledge that the article contains overlap in text with that from our previous publication [1] (reference 43 in the original article) and fragments in the text that overlap word-for-word with text from our previous publication [2] and a publication in Environ Toxicol Pharmacol [3] (reference 42 in the original article). The authors apologize for the overlap, for omitting to cite the publication [2] and for not properly quoting the text from [1, 2, 3]. The overlap in text relates to some of the information described in the Introduction section and to the description of the methodology under the Materials and Methods section. However, it should be noted that the identified issues have no bearing on the results and conclusions of the study.

Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons

The nematode Caenorhabditis elegans navigates toward a preferred temperature setpoint (Ts) determined by long-term temperature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative thermotaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differences in the strategies for positive and negative thermotaxis. Negative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temperatures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsynaptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor transformations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.