Diving deep: zebrafish models in motor neuron degeneration research.

In the dynamic landscape of biomedical science, the pursuit of effective treatments for motor neuron disorders like hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA) remains a key priority. Central to this endeavor is the development of robust animal models, with the zebrafish emerging as a prime candidate. Exhibiting embryonic transparency, a swift life cycle, and significant genetic and neuroanatomical congruencies with humans, zebrafish offer substantial potential for research. Despite the difference in locomotion-zebrafish undulate while humans use limbs, the zebrafish presents relevant phenotypic parallels to human motor control disorders, providing valuable insights into neurodegenerative diseases. This review explores the zebrafish's inherent traits and how they facilitate profound insights into the complex behavioral and cellular phenotypes associated with these disorders. Furthermore, we examine recent advancements in high-throughput drug screening using the zebrafish model, a promising avenue for identifying therapeutically potent compounds.

The caloric value of food intake structurally adjusts a neuronal mushroom body circuit mediating olfactory learning in Drosophila.

Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. In this study, we used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, we observed a decrease in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, we found that this rearrangement in synaptic connections influenced aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.

Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase.

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Flonicamid metabolite 4-trifluoromethylnicotinamide is a chordotonal organ modulator insecticide†.

BACKGROUND: The selective aphicide flonicamid is known to cause symptoms in aphids that are like those of chordotonal organ TRPV channel modulator insecticides such as pymetrozine, pyrifluquinazon and afidopyropen. Flonicamid is classified by the Insecticide Resistance Action Committee as a chordotonal organ modulator with an undefined target site. However, although it has been shown not to act on TRPV channels, flonicamid's action on chordotonal organs has not been documented in the literature. RESULTS: Flonicamid causes locusts to extend their hindlegs, indicating an action on the femoral chordotonal organ. In fruit flies, it abolishes negative gravitaxis behavior by disrupting transduction and mechanical amplification in antennal chordotonal neurons. Although flonicamid itself only weakly affects locust chordotonal organs, its major animal metabolite 4-trifluoromethylnicotinamide (TFNA-AM) potently stimulates both locust and fly chordotonal organs. Like pymetrozine, TFNA-AM rapidly increases Ca2+ in antennal chordotonal neurons in wild-type flies, but not iav1 mutants, yet the effect is nonadditive with the TRPV channel agonist. CONCLUSIONS: Flonicamid is a pro-insecticide form of TFNA-AM, a potent chordotonal organ modulator. The functional effects of TFNA-AM on chordotonal organs of locusts and flies are indistinguishable from those of the TRPV agonists pymetrozine, pyrifluquinazon and afidopyropen. Because our previous results indicate that TFNA-AM does not act directly on TRPV channels, we conclude that it acts upstream in a pathway that leads to TRPV channel activation. © 2022 Society of Chemical Industry.

A Markerless Pose Estimator Applicable to Limbless Animals.

The analysis of kinematics, locomotion, and spatial tasks relies on the accurate detection of animal positions and pose. Pose and position can be assessed with video analysis programs, the "trackers." Most available trackers represent animals as single points in space (no pose information available) or use markers to build a skeletal representation of pose. Markers are either physical objects attached to the body (white balls, stickers, or paint) or they are defined in silico using recognizable body structures (e.g., joints, limbs, color patterns). Physical markers often cannot be used if the animals are small, lack prominent body structures on which the markers can be placed, or live in environments such as aquatic ones that might detach the marker. Here, we introduce a marker-free pose-estimator (LACE Limbless Animal traCkEr) that builds the pose of the animal de novo from its contour. LACE detects the contour of the animal and derives the body mid-line, building a pseudo-skeleton by defining vertices and edges. By applying LACE to analyse the pose of larval Drosophila melanogaster and adult zebrafish, we illustrate that LACE allows to quantify, for example, genetic alterations of peristaltic movements and gender-specific locomotion patterns that are associated with different body shapes. As illustrated by these examples, LACE provides a versatile method for assessing position, pose and movement patterns, even in animals without limbs.

Visualization of naive and learned odor representations using in vivo calcium imaging and immunohistochemical bouton mapping of single Drosophila mushroom body neurons.

This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. We also present subsequent characterization of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz et al. (2020).

Visualization of a Distributed Synaptic Memory Code in the Drosophila Brain.

During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. We analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body γ lobe, a brain structure that mediates olfactory learning. A fluorescent Ca2+ sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca2+ could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, we show that odor-evoked Ca2+ dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter.

The Orphan Cytokine Receptor CRLF3 Emerged With the Origin of the Nervous System and Is a Neuroprotective Erythropoietin Receptor in Locusts.

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.

Strategies of locomotion composition.

This review aims to highlight the importance of saccades during locomotion as a strategy to reduce sensory information loss while the subject is moving. Acquiring sensory data from the environment during movement results in a temporal flow of information, as the sensory precept changes with the position of the observer. Accordingly, the movement pattern shapes the sensory flow. Therefore, the requirements of locomotion and sensation have to be balanced in the behaviour of the organism. Insect vision provides deep insight into the interplay between action and perception. Insects can shape their optic flow by reducing their rotational movements to fast and short saccades. This generates prolonged phases of translations which provide depth information. Extensive behavioural and physiological studies on insects show how shaping the optic flow facilitates the coding of motion vision. Indeed the saccadic strategy provides an elegant solution to optimise sensory flow. Complementary studies in other taxa reported similar locomotion strategies emphasising the crucial influence of sensory flow on locomotion.

Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Correcting locomotion dependent observation biases in thermal preference of Drosophila.

Sensing environmental temperatures is essential for the survival of ectothermic organisms. In Drosophila, two of the most used methodologies to study temperature preferences (TP) and the genes involved in thermosensation are two-choice assays and temperature gradients. Whereas two-choice assays reveal a relative TP, temperature gradients can identify the absolute Tp. One drawback of gradients is that small ectothermic animals are susceptible to cold-trapping: a physiological inability to move at the cold area of the gradient. Often cold-trapping cannot be avoided, biasing the resulting TP to lower temperatures. Two mathematical models were previously developed to correct for cold-trapping. These models, however, focus on group behaviour which can lead to overestimation of cold-trapping due to group aggregation. Here we present a mathematical model that simulates the behaviour of individual Drosophila in temperature gradients. The model takes the spatial dimension and temperature difference of the gradient into account, as well as the rearing temperature of the flies. Furthermore, it allows the quantification of cold-trapping and reveals unbiased TP. Additionally, our model reveals that flies have a range of tolerable temperatures, and this measure is more informative about the behaviour than commonly used TP. Online simulation is hosted at http://igloo.uni-goettingen.de . The code can be accessed at https://github.com/zerotonin/igloo .

Proprioceptive Opsin Functions in Drosophila Larval Locomotion.

Animals rely on mechanosensory feedback from proprioceptors to control locomotory body movements. Unexpectedly, we found that this movement control requires visual opsins. Disrupting the Drosophila opsins NINAE or Rh6 impaired larval locomotion and body contractions, independently of light and vision. Opsins were detected in chordotonal proprioceptors along the larval body, localizing to their ciliated dendrites. Loss of opsins impaired mechanically evoked proprioceptor spiking and cilium ultrastructure. Without NINAE or Rh6, NOMPC mechanotransduction channels leaked from proprioceptor cilia and ciliary Inactive (Iav) channels partly disappeared. Locomotion is shown to require opsins in proprioceptors, and the receptors are found to express the opsin gene Rh7, in addition to ninaE and Rh6. Besides implicating opsins in movement control, this documents roles of non-ciliary, rhabdomeric opsins in cilium organization, providing a model for a key transition in opsin evolution and suggesting that structural roles of rhabdomeric opsins preceded their use for light detection.

Neuroligins Nlg2 and Nlg4 Affect Social Behavior in Drosophila melanogaster.

The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, we, here, analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.

Neuroprotection and endocytosis: erythropoietin receptors in insect nervous systems.

Erythropoietin (Epo) plays a dual role as an erythropoiesis-stimulating hormone and a locally produced cytoprotectant in various vertebrate tissues. Splice variants and engineered derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis suggest that alternative receptors, different from the 'classical' homodimeric receptor involved in haematopoiesis, mediate neuroprotective Epo functions. Previous studies on grasshoppers demonstrated neuroprotective and neuroregenerative effects of Epo that involved similar transduction pathways as in mammals. To advance the characterization of yet unidentified neuroprotective Epo receptors, we studied the neuroprotective potency of the human non-erythropoietic Epo splice variant EV-3 in primary cultured locust brain neurons. We demonstrate that EV-3, like Epo, protects locust neurons from hypoxia-induced apoptotic death through activation of the Janus kinase/signal transducer and activator of transcription transduction pathway. Using the fluorescent dye FM1-43 to quantify endocytotic activity we show that both Epo and EV-3 increase the number of fluorescently labelled endocytotic vesicles. This reveals that binding of Epo to its neuroprotective receptor induces endocytosis, as it has been described for the mammalian homodimeric Epo-receptor expressed by erythroid progenitors. Reduction in Epo-stimulated endocytotic activity following pre-exposure to EV-3 indicated that both Epo and its splice variant bind to the same receptor on locust neurons. The shared neuroprotective potency of Epo and EV-3 in insect and mammalian neurons, in the absence of erythropoietic effects of EV-3 in mammals, suggests a greater similarity of the unidentified nervous Epo receptors (or receptor complexes) across phyla than between mammalian haematopoietic and neuroprotective receptors. Insects may serve as suitable models to evaluate the specific protective mechanisms mediated by Epo and its variants in non-erythropoietic mammalian tissues.

Saccadic movement strategy in a semiaquatic species - the harbour seal (Phoca vitulina).

Moving animals can estimate the distance of visual objects from image shift on their retina (optic flow) created during translational, but not rotational movements. To facilitate this distance estimation, many terrestrial and flying animals perform saccadic movements, thereby temporally separating translational and rotational movements, keeping rotation times short. In this study, we analysed whether a semiaquatic mammal, the harbour seal, also adopts a saccadic movement strategy. We recorded the seals' normal swimming pattern with video cameras and analysed head and body movements. The swimming seals indeed minimized rotation times by saccadic head and body turns, with top rotation speeds exceeding 350 deg s-1 which leads to an increase of translational movements. Saccades occurred during both types of locomotion of the seals' intermittent swimming mode: active propulsion and gliding. In conclusion, harbour seals share the saccadic movement strategy of terrestrial animals. Whether this movement strategy is adopted to facilitate distance estimation from optic flow or serves a different function will be a topic of future research.

Hearing regulates Drosophila aggression.

Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

Saccadic Movement Strategy in Common Cuttlefish (Sepia officinalis).

Most moving animals segregate their locomotion trajectories in short burst like rotations and prolonged translations, to enhance distance information from optic flow, as only translational, but not rotational optic flow holds distance information. Underwater, optic flow is a valuable source of information as it is in the terrestrial habitat, however, so far, it has gained only little attention. To extend the knowledge on underwater optic flow perception and use, we filmed the movement pattern of six common cuttlefish (Sepia officinalis) with a high speed camera in this study. In the subsequent analysis, the center of mass of the cuttlefish body was manually traced to gain thrust, slip, and yaw of the cuttlefish movements over time. Cuttlefish indeed performed short rotations, saccades, with rotational velocities up to 343°/s. They clearly separated rotations from translations in line with the saccadic movement strategy documented for animals inhabiting the terrestrial habitat as well as for the semiaquatic harbor seals before. However, this separation only occurred during fin motion. In contrast, during jet propelled swimming, the separation between rotational and translational movements and thus probably distance estimation on the basis of the optic flow field is abolished in favor of high movement velocities. In conclusion, this study provides first evidence that an aquatic invertebrate, the cuttlefish, adopts a saccadic movement strategy depending on the behavioral context that could enhance the information gained from optic flow.

Saccadic body turns in walking Drosophila.

Drosophila melanogaster structures its optic flow during flight by interspersing translational movements with abrupt body rotations. Whether these "body saccades" are accompanied by steering movements of the head is a matter of debate. By tracking single flies moving freely in an arena, we now discovered that walking Drosophila also perform saccades. Movement analysis revealed that the flies separate rotational from translational movements by quickly turning their bodies by 15 degrees within a tenth of a second. Although walking flies moved their heads by up to 20 degrees about their bodies, their heads moved with the bodies during saccadic turns. This saccadic strategy contrasts with the head saccades reported for e.g., blowflies and honeybees, presumably reflecting optical constraints: modeling revealed that head saccades as described for these latter insects would hardly affect the retinal input in Drosophila because of the lower acuity of its compound eye. The absence of head saccades in Drosophila was associated with the absence of haltere oscillations, which seem to guide head movements in other flies. In addition to adding new twists to Drosophila walking behavior, our analysis shows that Drosophila does not turn its head relative to its body when turning during walking.

Motor patterns during active electrosensory acquisition.

Motor patterns displayed during active electrosensory acquisition of information seem to be an essential part of a sensory strategy by which weakly electric fish actively generate and shape sensory flow. These active sensing strategies are expected to adaptively optimize ongoing behavior with respect to either motor efficiency or sensory information gained. The tight link between the motor domain and sensory perception in active electrolocation make weakly electric fish like Gnathonemus petersii an ideal system for studying sensory-motor interactions in the form of active sensing strategies. Analyzing the movements and electric signals of solitary fish during unrestrained exploration of objects in the dark, we here present the first formal quantification of motor patterns used by fish during electrolocation. Based on a cluster analysis of the kinematic values we categorized the basic units of motion. These were then analyzed for their associative grouping to identify and extract short coherent chains of behavior. This enabled the description of sensory behavior on different levels of complexity: from single movements, over short behaviors to more complex behavioral sequences during which the kinematics alter between different behaviors. We present detailed data for three classified patterns and provide evidence that these can be considered as motor components of active sensing strategies. In accordance with the idea of active sensing strategies, we found categorical motor patterns to be modified by the sensory context. In addition these motor patterns were linked with changes in the temporal sampling in form of differing electric organ discharge frequencies and differing spatial distributions. The ability to detect such strategies quantitatively will allow future research to investigate the impact of such behaviors on sensing.

A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons.

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.