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Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons. Supplementary Information The online version contains supplementary material available at 10.1007/s12640-021-00364-8.

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as beta-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2-4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

Over the past few years, molecular studies of phylogeny have challenged the traditional view of evolutionary relationships among protostomian animal phyla. Based on analysis of 18S ribosomal RNA gene sequences, it has been suggested that some traditional groups, like the articulata and the pseudocoelomata, should be completely abandoned and that instead the protostomians should be split into two major clades: the Ecdysozoa and the Lophotrochozoa. However, this new molecular phylogeny still awaits confirmation by independent methods. In this study, we present a cytological feature that supports the new classification. The carbohydrate epitope that is recognised by antisera against the plant glycoprotein horseradish peroxidase (HRP) is known to be selectively expressed by membrane proteins on the surface of neural tissue in insects. We found that the major ecdysozoan phyla show neural expression of HRP immunoreactivity, which is completely absent in the nervous tissue of lophotrochozoans, deuterostomians, and cnidarians. This suggests that the presence of anti-HRP-reactive glycoproteins in neural tissue is an ecdysozoan autapomorphy.

Primary cell cultures were prepared from specific regions of the pupal honeybee brain which are involved in proboscis extension learning. Defined areas could be dissociated purely by mechanical treatment. We show that cultured neurons regenerate new neurites and remain viable for up to three weeks in a serum-free, chemically-defined medium. Several labelling techniques were employed to identify subpopulations of cultured neurons. For example, acetylcholinesterase staining; fluorescent beads to distinguish identified cell populations of co-cultured brain areas; various markers for surface antigens such as a monoclonal antibody to olfactory projection neurons of the antennoglomerular tracts and monopolar cells of the optic lobes, as well as anti-HRP immunoreactivity and alpha-bungarotoxin binding; and various antisera for detecting transmitter phenotype. The appearance of transmitter-immunoreactive cells agreed closely with that expected from their known distribution in situ. Our results suggest that cultured cells retain surface properties and transmitter phenotype of their in vivo counterparts, despite differences in basic morphology. Thus our culture system provides the important initial step for future in vitro investigations of the cellular and electrophysiological properties of neurons mediating proboscis extension learning.

1. Sensory reception in the molluscPleurobranchaea californica was studied in whole animals and surgically reduced preparations by delivering chemical and mechanical stimuli while observing behavior or recording extracellularly the responses of the corresponding nerves. Sensory structures studied included the rhinophores, tentacles and oral veil. 2. Specimens reliably (50% or more) exhibited feeding responses to 10 of 18 amino acids tested (Table 1), including alanine and glycine. In 14 electrophysiological experiments on the rhinophore, medium to high centripetal responses to alanine and/or glycine were obtained in 7 preparations (Fig. 2), while little or no response was obtained in 7 preparations. Responses to different amino acids were sometimes mediated by the same centripetal unit (Fig. 2). 3. The rhinophore nerves showed vigorous excitatory responses to variations in the salt concentration (Figs. 3, 4), osmolarity (Figs. 5, 6), and pH (Figs. 7, 8) of sea water solutions directed onto the rhinophore sensory epithelium. The rhinophore and tentacle nerves showed strong excitatory responses to various salt solutions directed onto the corresponding sensory structure, including 1 osmolar NaCl, NaBr, NaI, Na2SO4 and KCl, but not LiCl (Table 2). Curves relating extracellular discharge to stimulus strength typically showed a minimum in the physiological range and increases to either side of this range (Figs. 4, 8). 4. All nerves studied showed excitatory responses to stimulation with mechanical stimuli (Figs. 9–11). Maps of receptive fields of different nerves (Fig. 10) delineated areas of functional innervation for each nerve and showed little overlap. The same centripetal unit(s) typically responded to mechanostimulation of a wide peripheral area (Fig. 11). 5. All nerves studied showed excitatory responses to application of liquefied food substances to the sensory structures (Figs. 12–15). Dose response curves for different food stimuli (Fig. 13) were similar except at higher stimulus strengths, where mean discharge rates were significantly different for different foods. These and other data furnish neurophysiological evidence for discrimination between different food stimuli, as suggested also by earlier behavioral studies (Davis et al. 1980). 6. For all stimuli, severing the afferent nerves leading from the peripheral sensory epithelium abolished electrophysiological responses. Therefore the responses observed were mediated by the sensory epithelium rather than by direct stimulation of peripheral ganglia or nerves. 7. It is concluded that the rhinophores, tentacles and oral veil participate not only in food detection but also have the sensory capacity to detect changes in several other environmental parameters. The data are consistent with the hypothesis that incoming afferent information is processed by peripheral ganglia before it is relayed centrally.

We have used immunohistochemical methods to investigate the morphology of identified, presumptive serotonergic neurons in the antennal lobes and suboesophageal ganglion of the worker honeybee. A large interneuron (deutocerebral giant, DCG) is described that interconnects the deutocerebral antennal and dorsal lobes with the suboesophageal ganglion and descends into the ventral nerve chord. This neuron is accompanied by a second serotonin-immunoreactive interneuron with projections into the protocerebrum. Two pairs of bilateral immunoreactive serial homologues were identified in each of the three suboesophageal neuromeres and were also found in the thoracic ganglia. With the exception of the frontal commissure, no immunoreactive processes could be found in the peripheral nerves of the brain and the suboesophageal ganglion. The morphological studies on the serial homologues were extended by intracellular injections of Lucifer Yellow combined with immunofluorescence.

Aspects of brightness perception in two visual mutants ofDrosophila melanogaster were investigated in avoidance conditioning experiments: 1. The eye colour mutantwhite (w) and the phototransduction mutanttransient receptor potential with eye colour pigments missing (w; trp) could be conditioned to discriminate intensity differences of 2 log units over a light intensity range of approximately 6 log units (Fig. 2). 2. w; trp mutants could be conditioned to discriminate intensity differences even under high light intensity conditions which largely eliminate the sustained component of the photoreceptor potential. Under these illumination conditions no significant differences betweenw andw; trp mutants in visual memory or in the discrimination of small intensity differences (Fig. 3) could be found. 3. In thew mutant functional inactivation of photoreceptors R1–6 by PDA induction increased the threshold for intensity discrimination of monochromatic 406 nm light stimuli in the conditioning experiment by 2–3 log units (Fig. 5). 4. Brightness perception as determined by conditioning experiments was compared to phototactic choice behaviour forw andw; trp flies (Figs. 4 and 6). Implications for the interpretation of phototaxis experiments are discussed.

Populations ofDrosophila melanogaster wildtype (strain CS) and of the photoreceptor mutantrdgB with functionally inactivated retinula cells R1–6 could be conditioned to avoid different intensities of a 406 nm light, which had been paired with aversive stimulation (shaking). Thus general visual learning ability is not impaired by therdgB mutation implying that conditionable intensity discrimination can be mediated by retinal receptors R7 and R8. Although therdgB mutant shows no conditionable intensity discrimination below an intensity threshold of 0.015 W·m−2, both the wildtype and the mutant could be conditioned to discriminate intensity differences as small as 0.5 log units at higher intensities (Fig. 4 and 5). Experiments in which two spectrally different stimuli (399 nm and 512 nm) are employed, show that conditioning effects for therdgB mutant and also for the wildtype are dependent on the given combinations of intensitiy values (Fig. 6). These experiments are in accordance with the expectation that the wildtype discriminates both wavelengths and intensities. ForrdgB flies no positive evidence for the existence of colour discrimination could be found (Fig. 7 and 8, Table 2).

Neuromodulators and hormones elicit and modify well-defined behaviours. Their mode of action can be studied to advantage in arthropods, where the natural releasing cells and neuronal target circuits are concisely identified. The coordinated actions of biogenic amines and peptides on both central and peripheral neural activity and metabolic processes bias the whole organism to perform a coherent behavioural routine.