ABSTRACT
Neuronal excitability contributes to rhythm generation in central pattern generating networks (CPGs). In spinal cord CPGs, such intrinsic excitability partly relies on persistent sodium currents (INaP), whereas respiratory CPGs additionally depend on calcium-activated cation currents (ICAN). Here, we investigated the contributions of INaP and ICAN to spontaneous rhythm generation in neuronal networks of the spinal cord and whether they mainly involve Hb9 neurons. We used cultures of ventral and transverse slices from the E13-14 embryonic rodent lumbar spinal cord on multielectrode arrays (MEAs). All cultures showed spontaneous bursts of network activity. Blocking synaptic excitation with the AMPA receptor antagonist CNQX suppressed spontaneous network bursts and left asynchronous intrinsic activity at about 30% of the electrodes. Such intrinsic activity was completely blocked at all electrodes by both the INaP blocker riluzole as well as by the ICAN blocker flufenamic acid (FFA) and the more specific TRPM4 channel antagonist 9-phenanthrol. All three antagonists also suppressed spontaneous bursting completely and strongly reduced stimulus-evoked bursts. Also, FFA reduced repetitive spiking that was induced in single neurons by injection of depolarizing current pulses to few spikes. Other antagonists of unspecific cation currents or calcium currents had no suppressing effects on either intrinsic activity (gadolinium chloride) or spontaneous bursting (the TRPC channel antagonists clemizole and ML204 and the T channel antagonist TTA-P2). Combined patch-clamp and MEA recordings showed that Hb9 interneurons were activated by network bursts but could not initiate them. Together these findings suggest that both INaP through Na+-channels and ICAN through putative TRPM4 channels contribute to spontaneous intrinsic and repetitive spiking in spinal cord neurons and thereby to the generation of network bursts.
ABSTRACT
Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD50-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish an in vitro assay as an alternative to the widely used in vivo mouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox® for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use.
ABSTRACT
Presently there exists no cure for spinal cord injury (SCI). However, transplantation of embryonic tissue into spinal cord (SC) lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated SC circuits. We tested the two hypotheses in an in vitro SC lesion model that is based on propagation of activity between two rat organotypic SC slices in culture. Transplantation of dissociated cells from E14 rat SC or forebrain (FB) re-established the relay of activity over the lesion site and thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays (MEAs) we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse FB cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated SC circuits. In contrast, transplantation of neurospheres (NS) induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated SC circuits.
ABSTRACT
Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.
Subject(s)
Coculture Techniques/methods , Nerve Regeneration/physiology , Spinal Cord/physiology , Tissue Culture Techniques/methods , Animals , Electrochemical Techniques/methods , Electrodes , Nerve Net/physiology , Neurons/physiology , Rats , Spinal Cord/cytologyABSTRACT
The accessory outer segment, a cytoplasmic structure running alongside the photoreceptor outer segment, has been described in teleost fishes, excluding the model organism zebrafish. So far, the function of the accessory outer segment is unknown. Here, we describe the ultrastructure of the zebrafish cone accessory outer segment by electron microscopy. Starting at the connecting cilium, the accessory outer segment runs parallel alongside the cone outer segment (COS). A thin plasma bridge connects the outer segment with the accessory outer segment, whose surface is enlarged by foldings and invaginations. Beside the morphological descriptions, we demonstrate that the Usher protein myosin VIIa (Myo7a) is a specific marker for the zebrafish cone accessory outer segment. Zebrafish cone photoreceptors possess a large and well-differentiated accessory outer segment, in which the unconventional motor protein Myo7a is highly enriched. The direct cytoplasmic contact with the COS as well as the surface enlargement of the accessory outer segment suggests an important role of this structure in transport and exchange of metabolites between the COS and the surrounding retinal pigment epithelium. In future studies of the outer retina, more attention should be paid to this often neglected structure.