The complex synaptic pathways onto a looming-detector neuron revealed using serial block-face scanning electron microscopy.

The ability of locusts to detect looming stimuli and avoid collisions or predators depends on a neuronal circuit in the locust's optic lobe. Although comprehensively studied for over three decades, there are still major questions about the computational steps of this circuit. We used fourth instar larvae of Locusta migratoria to describe the connection between the lobula giant movement detector 1 (LGMD1) neuron in the lobula complex and the upstream neuropil, the medulla. Serial block-face scanning electron microscopy (SBEM) was used to characterize the morphology of the connecting neurons termed trans-medullary afferent (TmA) neurons and their synaptic connectivity. This enabled us to trace neurons over several hundred micrometers between the medulla and the lobula complex while identifying their synapses. We traced two different TmA neurons, each from a different individual, from their synapses with the LGMD in the lobula complex up into the medulla and describe their synaptic relationships. There is not a simple downstream transmission of the signal from a lamina neuron onto these TmA neurons; there is also a feedback loop in place with TmA neurons making outputs as well as receiving inputs. More than one type of neuron shapes the signal of the TmA neurons in the medulla. We found both columnar and trans-columnar neurons connected with the traced TmA neurons in the medulla. These findings indicate that there are computational steps in the medulla that have not been included in models of the neuronal pathway for looming detection.

The soluble neurexin-1ß ectodomain causes calcium influx and augments dendritic outgrowth and synaptic transmission.

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1ß ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1ß ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1ß:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1ß ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.

Induced oscillatory signaling in the beta frequency of top-down pain modulation.

BACKGROUND: Induced synchronized brain activity, particularly in the beta-frequency range, has rarely been investigated in human electrophysiological studies of attentional modulation of the perception of nociceptive stimuli. METHODS: We measured time-resolved brain responses to nociceptive stimuli in healthy subjects (final data set: n = 17) using magnetoencephalography (MEG). In addition to investigating evoked responses as previous studies, we tested whether synchronized beta activity induced by nociceptive stimuli differs between 2 attentional conditions. Subjects were presented simultaneously with 2 stimulus modalities (pain-producing intraepidermal electrical stimuli and visual stimuli) in 2 different experimental conditions, ie, "attention to pain" and "attention to color." Pain ratings between conditions were compared using a 2-sided paired-sample t test; MEG data were analyzed with Brainstorm. RESULTS: Pain ratings were significantly higher in the "attention to pain" compared with the "attention to color" condition. Peak amplitudes of the evoked responses were significantly larger in the "attention to pain" condition bilaterally in the insula and secondary somatosensory cortex, and in the primary somatosensory cortex (SI) contralateral to stimulation. Induced responses to painful stimuli were significantly stronger in contralateral SI in the beta-frequency range in the "attention to pain" condition. CONCLUSIONS: This study replicates previous reports w.r.t. the attentional modulation of evoked responses and suggests a functional role of induced oscillatory activity in the beta frequency in top-down modulation of nociceptive stimuli.

Cell Morphology on Poly(methyl methacrylate) Microstructures as Function of Surface Energy.

Whilst the significance of substrate topography as a regulator of cell function is well established, a systematic analysis of the principles underlying this is still unavailable. Here we evaluate the hypothesis that surface energy plays a decisive role in substrate-mediated modulation of cell phenotype by evaluation of cell behaviour on synthetic microstructures exhibiting pronounced differences in surface energy. These microstructures, specifically cubes and walls, were fabricated from a biocompatible base polymer, poly(methyl methacrylate), by variotherm injection molding. The dimensions of the cubes were 1 µm x 1 µm x 1 µm (height x width x length) with a periodicity of 1:1 and 1:5 and the dimensions of the walls 1 µm x 1 µm x 15 mm (height x width x length) with a periodicity of 1:1 and 1:5. Mold inserts were made by lithography and electroplating. The surface energy of the resultant microstructures was determined by static contact angle measurements. Light scanning microscopy of the morphology of NT2/D1 and MC3T3-E1 preosteoblast cells cultured on structured PMMA samples in both cases revealed a profound surface energy dependence. "Walls" appeared to promote significant cell elongation, whilst a lack of cell adhesion was observed on "cubes" with the lowest periodicity. Contact angle measurements on walls revealed enhanced surface energy anisotropy (55 mN/m max., 10 mN/m min.) causing a lengthwise spreading of the test liquid droplet, similar to cell elongation. Surface energy measurements for cubes revealed increased isotropic hydrophobicity (87° max., H2O). A critical water contact angle of ≤ 80° appears to be necessary for adequate cell adhesion. A "switch" for cell adhesion and subsequently cell growth could therefore be applied by, for example, adjusting the periodicity of hydrophobic structures. In summary cell elongation on walls and a critical surface energy level for cell adhesion could be produced for NT2/D1 and MC3T3-E1 cells by symmetrical and asymmetrical energy barrier levels. We, furthermore, propose a water-drop model providing a common physicochemical cause regarding similar cell/droplet geometries and cell adhesion on the investigated microstructures.

An Unbiased Approach of Sampling TEM Sections in Neuroscience.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

Artemin and an Artemin-Derived Peptide, Artefin, Induce Neuronal Survival, and Differentiation Through Ret and NCAM.

Artemin (ARTN) is a neurotrophic factor from the GDNF family ligands (GFLs) that is involved in development of the nervous system and neuronal differentiation and survival. ARTN signals through a complex receptor system consisting of the RET receptor tyrosine kinase and a glycosylphosphatidylinositol-anchored co-receptor GFL receptor α, GFRα3. We found that ARTN binds directly to neural cell adhesion molecule (NCAM) and that ARTN-induced neuritogenesis requires NCAM expression and activation of NCAM-associated signaling partners, thus corroborating that NCAM is an alternative receptor for ARTN. We designed a small peptide, artefin, that could interact with GFRα3 and demonstrated that this peptide agonist induces RET phosphorylation and mimics the biological functions of ARTN - neuroprotection and neurite outgrowth. Moreover, artefin mimicked the binding of ARTN to NCAM and required NCAM expression and activation for its neurite elongation effect, thereby suggesting that artefin represents a binding site for NCAM within ARTN. We showed that biological effects of ARTN and artefin can be inhibited by abrogation of both NCAM and RET, suggesting a more complex signaling mechanism that previously thought. As NCAM plays a significant role in neurodevelopment, regeneration, and synaptic plasticity we suggest that ARTN and its mimetics are promising candidates for treatment of neurological disorders and warrant further investigations.

Histological processing of un-/cellularized thermosensitive electrospun scaffolds.

Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.

Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice.

Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for "enviromimetics", therapeutics which reproduce the beneficial effects of enhanced environmental stimulation.

Immunomodulator CD200 Promotes Neurotrophic Activity by Interacting with and Activating the Fibroblast Growth Factor Receptor.

The CD200 ligand is expressed by a variety of cell types, including vascular endothelia, kidney glomeruli, some subsets of T and B cells, and neurons in the brain and periphery. In contrast, the receptor of CD200, CD200R, has a limited expression pattern and is mainly expressed by cells of myeloid origin. A recently solved crystal structure of the CD200-CD200R ectodomain complex suggests involvement of the first immunoglobulin (Ig)-like modules in ligand-receptor binding, resulting in the inhibition of myeloid cell function. In the central nervous system, CD200 has been implicated in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR.

A novel unbiased counting method for the quantification of synapses in the mouse brain.

BACKGROUND: The numerical density of synapses and their ultrastructural features are best assessed with electron microscopy. Counting is done within counting frames placed on a pair of sections (disector technique). But this requires that the thin sections are taken from comparable brain regions and the disectors are placed in a uniform random fashion. Small brain areas like the polymorph layer of the mouse dentate gyrus are difficult to encounter, and manually moving the microscope stage for placing the micrographs seems arbitrary. NEW METHOD: Here the polymorph layer was approximated with 20µm thin, Nissl-stained vibratome sections. The subsequent vibratome section was processed for electron microscopy and serially thin sectioned. The microscope stage was moved using a random number generator, placing at least 20 disectors onto a pair of sections. The numerical synapse density, the numerical density of dense-core vesicles, and other ultrastructural features were compared between mice that had been kept in an enriched environment and mice kept under standard housing conditions. RESULTS: Environmental enrichment significantly decreased the numerical density of dense-core vesicles and synaptic cleft widths within the polymorph layer, associated with behavioral improvement in the Morris water maze, a hippocampus-dependent task of spatial learning and memory. COMPARISON WITH EXISTING METHODS: This procedure was easy to handle and enabled us to produce thin sections in small, defined brain areas. Furthermore, placing the disectors with random numbers excluded observer bias. CONCLUSIONS: Our procedure provides an uncomplicated way of assessing numerical densities in small brain areas in an unbiased manner.

Synaptic connections of first-stage visual neurons in the locust Schistocerca gregaria extend evolution of tetrad synapses back 200 million years.

The small size of some insects, and the crystalline regularity of their eyes, have made them ideal for large-scale reconstructions of visual circuits. In phylogenetically recent muscomorph flies, like Drosophila, precisely coordinated output to different motion-processing pathways is delivered by photoreceptors (R cells), targeting four different postsynaptic cells at each synapse (tetrad). Tetrads were linked to the evolution of aerial agility. To reconstruct circuits for vision in the larger brain of a locust, a phylogenetically old, flying insect, we adapted serial block-face scanning electron microscopy (SBEM). Locust lamina monopolar cells, L1 and L2, were the main targets of the R cell pathway, L1 and L2 each fed a different circuit, only L1 providing feedback onto R cells. Unexpectedly, 40% of all locust R cell synapses onto both L1 and L2 were tetrads, revealing the emergence of tetrads in an arthropod group present 200 million years before muscomorph flies appeared, coinciding with the early evolution of flight.

Anti-inflammatory properties of a novel peptide interleukin 1 receptor antagonist.

BACKGROUND: Interleukin 1 (IL-1) is implicated in neuroinflammation, an essential component of neurodegeneration. We evaluated the potential anti-inflammatory effect of a novel peptide antagonist of IL-1 signaling, Ilantide. METHODS: We investigated the binding of Ilantide to IL-1 receptor type I (IL-1RI) using surface plasmon resonance, the inhibition of Il-1ß-induced activation of nuclear factor κB (NF-κB) in HEK-Blue cells that contained an IL-1ß-sensitive reporter, the secretion of TNF-α in macrophages, protection against IL-1-induced apoptosis in neonatal pancreatic islets, and the penetration of Ilantide through the blood-brain barrier using competitive enzyme-linked immunosorbent assay (ELISA). We studied the effects of the peptide on social behavior and memory in rat models of lipopolysaccharide (LPS)- and amyloid-induced neuroinflammation, respectively, and its effect in a rat model of experimental autoimmune enchephalomyelitis. RESULTS: Ilantide bound IL-1RI, inhibited the IL-1ß-induced activation of NF-κB, and inhibited the secretion of TNF-α in vitro. Ilantide protected pancreatic islets from apoptosis in vitro and reduced inflammation in an animal model of arthritis. The peptide penetrated the blood-brain barrier. It reduced the deficits in social activity and memory in LPS- and amyloid-treated animals and delayed the development of experimental autoimmune enchephalomyelitis. CONCLUSIONS: These findings indicate that Ilantide is a novel and potent IL-1RI antagonist that is able to reduce inflammatory damage in the central nervous system and pancreatic islets.

The CNTF-derived peptide mimetic Cintrofin attenuates spatial-learning deficits in a rat post-status epilepticus model.

Ciliary neurotrophic growth factor is considered a potential therapeutic agent for central nervous system diseases. We report first in vivo data of the ciliary neurotrophic growth factor peptide mimetic Cintrofin in a rat post-status epilepticus model. Cintrofin prevented long-term alterations in the number of doublecortin-positive neuronal progenitor cells and attenuated the persistence of basal dendrites. In contrast, Cintrofin did neither affect acute status epilepticus-associated alterations in hippocampal cell proliferation and neurogenesis nor reveal any relevant effect on seizure activity. Whereas status epilepticus caused a significant disturbance in spatial learning in reversed peptide-treated rats, the performance of Cintrofin-treated rats did not differ from controls. The study confirms that Cintrofin comprises an active sequence mimicking effects of its parent molecule. While the data argue against an antiepileptogenic effect, they indicate a putative disease-modifying impact of Cintrofin.

A peptide antagonist of ErbB receptors, Inherbin3, induces neurite outgrowth from rat cerebellar granule neurons through ErbB1 inhibition.

ErbB receptors not only function in cancer, but are also key developmental regulators in the nervous system. We previously identified an ErbB1 peptide antagonist, Inherbin3, that is capable of inhibiting tumor growth in vitro and in vivo. In this study, we found that inhibition of ErbB1 kinase activity and activation of ErbB4 by NRG-1ß induced neurite extension, suggesting that ErbB1 and ErbB4 act as negative and positive regulators, respectively, of the neuritogenic response. Inherbin3, inhibited activation not only of ErbB1 but also of ErbB4 in primary neurons, strongly induced neurite outgrowth in rat cerebellar granule neurons, indicating that this effect mainly was due to inhibition of ErbB1 activation.

Antiinflammatory properties of a peptide derived from interleukin-4.

Interleukin-4 (IL-4) is a potent antiinflammatory cytokine. However its use in the clinic is hampered by side effects. We here describe the identification of a novel synthetic peptide, termed Ph8, derived from α-helix C of IL-4, which interacts with IL-4 receptor α (IL-4Rα). Employing various cultured genetically engineered cell lines and primary lymphocytes, surface plasmon resonance, qPCR, ELISA and immunoblotting techniques we found that Ph8 bound IL-4Rα and mimicked the anti-inflammatory effects of IL-4 by inhibiting TNF-α production by macrophages in vitro. It induced phosphorylation of STAT6 65kD but inhibited phosphorylation of STAT6 110 kD induced by IL-4 in a B-cell line that expressed the type I receptor. It also inhibited the IL-4-stimulated expression of a STAT6-inducible reporter gene in cells that expressed the type II receptor. Ph8 inhibited the proliferation of Th1/2 cells and downregulated the production of IFN-γ in stimulated Th1 cells. Moreover, Ph8 did not induce any shift in Th1/Th2 profile. This is a favorable effect and it is indicating that Ph8 could block general T cell activation and inflammatory responses without further inducing the side effects generally associated with IL-4 signaling. These data collectively show that Ph8 is only a partial agonist of IL-4 mimicking its desirable properties. In agreement, Ph8 treatment of rats with collagen-induced arthritis, a Th1- and antibody- mediated disease of joint, delayed the manifestation of chronic inflammation and reduced acute inflammation in carrageenan-induced edema. Our findings indicate that Ph8 is a promising potential drug candidate for the treatment of inflammatory diseases.

Identification of a novel antagonist of the ErbB1 receptor capable of inhibiting migration of human glioblastoma cells.

BACKGROUND: Receptors of the ErbB family are involved in the development of various cancers, and the inhibition of these receptors represents an attractive therapeutic concept. Upon ligand binding, ErbB receptors become activated as homo- or heterodimers, leading to the activation of downstream signaling cascades that result in the facilitation of cell proliferation and migration. A region of the extracellular part of the receptor, termed the 'dimerization arm', is important for the formation of receptor dimers and represents an attractive target for the design of ErbB inhibitors. METHODS: An ErbB1 targeting peptide, termed Herfin-1, was designed based on a model of the tertiary structure of the EGF-EGFR ternary complex. The binding kinetics of this peptide were determined employing surface plasmon resonance analyses. ErbB1-4 expression and phosphorylation in human glioblastoma cell lines U87 and U118 were determined by Western blotting using specific antibodies. Cell proliferation was determined by MTS staining. Cell migration was examined using a Chemotaxis Migration Kit. Neurite outgrowth from primary cerebellar granule neurons was evaluated by fluorescence microscopy and image processing. RESULTS: The present study shows that Herfin-1 functions as an ErbB1 antagonist. It binds to the extracellular domain of ErbB1 with a KD value of 361 nM. In U87 and U118 cells, both expressing high levels of ErbB1, Herfin-1 inhibits EGF-induced ErbB1 phosphorylation and cell migration. Additionally, Herfin-1 was found to increase neurite outgrowth in cerebellar granule neurons, likely through the inhibition of a sustained weak ErbB1 activation. CONCLUSIONS: Targeting the ErbB1 receptor dimerization interface is a promising strategy to inhibit receptor activation in ErbB1-expressing glioma cells.

Peptide mimetic of the S100A4 protein modulates peripheral nerve regeneration and attenuates the progression of neuropathy in myelin protein P0 null mice.

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.

Prenatal prochloraz treatment significantly increases pregnancy length and reduces offspring weight but does not affect social-olfactory memory in rats.

Metabolites of the commonly used imidazole fungicide prochloraz are androgen receptor antagonists. They have been shown to block androgen-driven development and compromise reproductive function. We tested the effect of prochloraz on cognitive behavior following exposure to this fungicide during the perinatal period. Pregnant Wistar rats were administered a 200 mg/kg dose of prochloraz on gestational day (GD) 7, GD11, and GD15. The social recognition test (SRT) was performed on 7-week-old male rat offspring. We found an increase in pregnancy length and a significantly reduced pup weight on PND15 and PND40 but no effect of prenatal prochloraz exposure on social investigation or acquisition of social-olfactory memory.

Larval morphology of the antlion Myrmecaelurus trigrammus (Pallas, 1771) (Neuroptera, Myrmeleontidae), with notes on larval biology.

Morphology and behaviour of third instar larvae of the Holomediterranean antlion species Myrmecaelurus trigranunus (Pallas) are described. Larvae are facultative pit-builders, they either ambush their prey at the surface, or dig pitfall traps that prey fall in to. Dark brown spots on dorsal and ventral sides of the head and on dorsal side of the thorax are characteristic of the larvae. Eye tubercles are not prominent. Jaws are equipped with long bristles, campaniform sensilla, sensilla coeloconica, and digitiform sensilla. A unique feature is the shape of the tips of all three teeth that is screw-like with a polyhedral surface. The body surface is covered with longitudinally grooved bristles and plumose hairs. On the tip of the antennae and on terminal and subterminal parts of labial palps sensilla basiconica occur. On the 9th abdominal segment there are two bulges, each of them bearing four digging bristles. Non-prominent eye tubercles and numerous mandibular bristles are morphological traits of pit-builders. Most of the behavioural traits are related to pit builders, whereas forward movement, waiting for prey without a pit and frequent changing of ambush location are traits of non-pit builders.