Stefan Weigel

Effects of carbon nanotubes on primary neurons and glial cells

Carbon nanotubes (CNTs) are among the most promising novel nanomaterials and their unique chemical and physical properties suggest an enormous potential for many areas of research and applications. As a consequence, the production of CNT-based material and thus the occupational and public exposure to CNTs will increase steadily. Although there is evidence that nanoparticles (NPs) can enter the nervous system via the blood stream, olfactory nerves or sensory nerves in the skin, there is still only little knowledge about possible toxic effects of CNTs on cells of the nervous system. The goal of the present study was to analyse the influences of single-walled CNTs (SWCNTs) with different degrees of agglomeration on primary cultures derived from chicken embryonic spinal cord (SPC) or dorsal root ganglia (DRG). As measured by the Hoechst assay treatment of mixed neuro-glial cultures with up to 30mug/mL SWCNTs significantly decreased the overall DNA content. This effect was more pronounced if cells were exposed to highly agglomerated SWCNTs as compared to better dispersed SWCNT-bundles. Using a cell-based ELISA we found that SWCNTs reduce the amount of glial cells in both peripheral nervous system (PNS) and central nervous system (CNS) derived cultures. Neurons were only affected in DRG derived cultures, where SWCNT treatment resulted in a decreased number of sensory neurons, as measured by ELISA. Additionally, whole-cell patch recordings revealed a diminished inward conductivity and a more positive resting membrane potential of SWCNT treated DRG derived neurons compared to control samples. The SWCNT suspensions used in this study induced acute toxic effects in primary cultures from both, the central and peripheral nervous system of chicken embryos. The level of toxicity is at least partially dependent on the agglomeration state of the tubes. Thus if SWCNTs can enter the nervous system at sufficiently high concentrations, it is likely that adverse effects on glial cells and neurons might occur.

A simple fluorescent double staining method for distinguishing neuronal from non-neuronal cells in the insect central nervous system.

Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially developmental or comparative studies often require an estimate of neuron numbers. Since neuronal and glial cell bodies are in many cases indiscernible in situ, a method to distinguish neurons from non-neuronal cells that works in any given species is wanting. Another application is cell culturing. Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Here, we present a simple method that uses a commercially available antiserum directed against horseradish peroxidase, which specifically stains neurons but no other cell type in every insect species investigated. Counterstaining with DAPI, a fluorescent chromophore that binds to double-stranded DNA in the nuclei of all cells, yields the total number of cells in a given sample. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal.

A novel relay nucleus between the inferior colliculus and the optic tectum in the chicken (Gallus gallus).

Processing multimodal sensory information is vital for behaving animals in many contexts. The barn owl, an auditory specialist, is a classic model for studying multisensory integration. In the barn owl, spatial auditory information is conveyed to the optic tectum (TeO) by a direct projection from the external nucleus of the inferior colliculus (ICX). In contrast, evidence of an integration of visual and auditory information in auditory generalist avian species is completely lacking. In particular, it is not known whether in auditory generalist species the ICX projects to the TeO at all. Here we use various retrograde and anterograde tracing techniques both in vivo and in vitro, intracellular fillings of neurons in vitro, and whole‐cell patch recordings to characterize the connectivity between ICX and TeO in the chicken. We found that there is a direct projection from ICX to the TeO in the chicken, although this is small and only to the deeper layers (layers 13–15) of the TeO. However, we found a relay area interposed among the IC, the TeO, and the isthmic complex that receives strong synaptic input from the ICX and projects broadly upon the intermediate and deep layers of the TeO. This area is an external portion of the formatio reticularis lateralis (FRLx). In addition to the projection to the TeO, cells in FRLx send, via collaterals, descending projections through tectopontine‐tectoreticular pathways. This newly described connection from the inferior colliculus to the TeO provides a solid basis for visual‐auditory integration in an auditory generalist bird. J. Comp. Neurol. 525:513–534, 2017.

Spatiotemporal analysis of electrically evoked activity in the chicken optic tectum: a VSDI study.

The midbrain is an important processing area for sensory information in vertebrates. The optic tectum and its mammalian counterpart, the superior colliculus, receive multimodal, topographic information and contain a sensory map that plays a role in spatial attention and orientation movements. Many studies have investigated the tectal circuitry by cytochemistry and by characterization of particular cell types. However, only a few studies have investigated network activation throughout the depth of the tectum. Our study provides the first data on spatiotemporal activity profiles in the depth and width of the avian optic tectum. We used an optical imaging approach with voltage-sensitive dyes to investigate population responses at a high temporal and spatial resolution. With the necessary caution due to cell extension across several layers, we can thus link our findings tentatively with the general layout of the avian optic tectum. Single electrical stimuli in the retinorecipient layers 1-4 evoked a complex optical response pattern with two components: a short, strong transient response and a weaker persistent response that lasted several hundred milliseconds. The response started in layer 5 and spread within this layer before it propagated into deeper layers. This is in line with neuroanatomical and earlier physiological data. Analysis of temporal sequence and pharmacological manipulations revealed that these responses were mainly driven by postsynaptic activation. Thus tectal network responses to patterned input can be studied by voltage-sensitive dye imaging.

Locust primary neuronal culture for the study of synaptic transmission

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.

Expression patterns of ion channels and structural proteins in a multimodal cell type of the avian optic tectum

The midbrain is an important subcortical area involved in distinct functions such as multimodal integration, movement initiation, bottom‐up, and top‐down attention. Our group is particularly interested in cellular computation of multisensory integration. We focus on the visual part of the avian midbrain, the optic tectum (TeO, counterpart to mammalian superior colliculus). This area has a layered structure with the great advantage of distinct input and output regions. In chicken, the TeO is organized in 15 layers where visual input targets the superficial layers while auditory input terminates in deeper layers. One specific cell type, the Shepherds crook neuron (SCN), extends dendrites in both input regions. The characteristic feature of these neurons is the axon origin at the apical dendrite. The molecular identity of this characteristic region and thus, the site of action potential generation are of particular importance to understand signal flow and cellular computation in this neuron. We present immunohistochemical data of structural proteins (NF200, Ankyrin G, and Myelin) and ion channels (Pan‐Nav, Nav1.6, and Kv3.1b). NF200 is strongly expressed in the axon. Ankyrin G is mainly expressed at the axon initial segment (AIS). Myelination starts after the AIS as well as the distribution of Nav channels on the axon. The subtype Nav1.6 has a high density in this region. Kv3.1b is restricted to the soma, the primary neurite and the axon branch. The distribution of functional molecules in SCNs provides insight into the information flow and the integration of sensory modalities in the TeO of the avian midbrain.

Local cholinergic interneurons modulate GABAergic inhibition in the chicken optic tectum

The chicken optic tectum (TeO) and its mammalian counterpart, the superior colliculus, are important sensory integration centers. Multimodal information is represented in a topographic map, which plays a role in spatial attention and orientation movements. The TeO is organised in 15 layers with clear input and output regions, and further interconnected with the isthmic nuclei (NI), which modulate the response in a winner‐takes‐all fashion. While many studies have analysed tectal cell types and their modulation from the isthmic system physiologically, little is known about local network activity and its modulation in the tectum. We have recently shown with voltage‐sensitive dye imaging that electrical stimulation of the retinorecipient layers results in a stereotypic response, which is under inhibitory control [S. Weigel & H. Luksch ( ) J. Neurophysiol., 107, 640–648]. Here, we analysed the contribution of acetylcholine (ACh) and the NI to evoked tectal responses using a pharmacological approach in a midbrain slice preparation. Application of the nicotinic ACh receptor (AChR) antagonist curarine increased the tectal response in amplitude, duration and lateral extent. This effect was similar but less pronounced when γ‐aminobutyric acidA receptors were blocked, indicating interaction of inhibitory and cholinergic neurons. The muscarinic AChR antagonist atropine did not change the response pattern. Removal of the NI, which are thought to be the major source of cholinergic input to the TeO, reduced the response only slightly and did not result in a disinhibition. Based on the data presented here and the neuroanatomical literature of the avian TeO, we propose a model of the underlying local circuitry.

Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.

BACKGROUND Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience. NEW METHOD We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer. RESULTS In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution. COMPARISON WITH EXISTING METHODS Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals. CONCLUSIONS With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.

Effects of early eye removal on the morphology of a multisensory neuron in the chicken optic tectum

The midbrain is a subcortical area involved in central functions such as integrating sensory modalities, movement initiation and bottom-up and top-down attention. In chicken, the midbrain roof is termed optic tectum (TeO) and consists of 15 layers with distinct in- and output regions. Visual input targets the superficial layers, while auditory input terminates in deeper layers. It has been shown that ablation of sensory epithelia leads to changes in the cellular patterning and structural organization of the sensory pathways. For the tectum, ablation of the eye anlagen was shown to affect retinorecipient neurons. While the gross morphology remained intact after enucleation, the shape of dendritic endings was changed presumably due to missing presynaptic input during synaptic pruning. We investigated the effect of deafferentation in a multisensory cell type, the Shepherds crook neuron (SCN) in the TeO. SCNs have distinct dendritic branches in retinorecipient layers (superficial layers 1 to 5 and 7) and in layers where auditory input terminates. To assess whether removal of a single sensory input only affects the dendrites recipient for that input, we removed the eye anlagen and retrogradely labeled SCNs later in embryogenesis to visualize the morphology in lesioned and non-lesioned embryos. We found no changes in the gross morphology or in the basal dendrites, but an altered growth of the fine structures at the apical dendrite of SCNs in the retinorecipient layers. Our data indicate that the neuronal morphology of SCNs is mostly predefined before retinal innervation affect the fine structure.