Patterns of spontaneous activity in unstructured and minimally structured spinal networks in culture

The rhythmic activity observed in locomotion is generated by local neuronal networks in the spinal cord. The alternating patterns are produced by reciprocal connections between these networks. Synchronous rhythmic activity, but not alternation, can be reproduced in disinhibited networks of dissociated spinal neurons of rats. This suggests that a specific network architecture is required for pattern generation but not for rhythm generation. Here we were interested in the recruitment of neurons to produce population bursts in unstructured and minimally structured cultures of rat spinal cord grown on multielectrode arrays. We tested whether two networks, connected by a small number of axons, could be functionally separated into two units and generate more complex patterns such as alternation. In the unstructured cultures, we found that the recruitment of the neurons into bursting populations is divided into two steps: the fast recruitment of a "trigger network”, consisting of intrinsically firing cells connected in networks with short delays, and slow recruitment of the rest of the network. One or several trigger networks were observed in a single culture and could account for variable patterns of propagation. In the minimally structured cultures, a functional separation between loosely connected networks was achieved. Such separation led either to an independent bursting between the networks or to synchronized bursting with long and variable delays. However, no qualitatively novel pattern such as alternation could be generated. In addition, we found that the strength of reciprocal inhibitory connections was modulated by spontaneous activit

Critical Components for Spontaneous Activity and Rhythm Generation in Spinal Cord Circuits in Culture

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

Functional regeneration of intraspinal connections in a new in vitro model

Abstract—Regeneration in the adult mammalian spinal cord is limited due to intrinsic properties of mature neurons and a hostile environment, mainly provided by central nervous system myelin and reactive astrocytes. Recent results indicate that propriospinal connections are a promising target for intervention to improve functional recovery. To study this functional regeneration in vitro we developed a model consisting of two organotypic spinal cord slices placed adjacently on multi-electrode arrays. The electrodes allow us to record the spontaneously occurring neuronal activity, which is often organized in network bursts. Within a few days in vitro (DIV), these bursts become synchronized between the two slices due to the formation of axonal connections. We cut them with a scalpel at different time points in vitro and record the neuronal activity 3 weeks later. The functional recovery ability was assessed by calculating the percentage of synchronized bursts between the two slices. We found that cultures lesioned at a young age (7–9 DIV) retained the high regeneration ability of embryonic tissue. However, cultures lesioned at older ages (>19 DIV) displayed a distinct reduction of synchronized activity. This reduction was not accompanied by an inability for axons to cross the lesion site. We show that functional regeneration in these old cultures can be improved by increasing the intracellular cAMP level with Rolipram or by placing a young slice next to an old one directly after the lesion. We conclude that co-cultures of two spinal cord slices are an appropriate model to study functional regeneration of intraspinal connections

Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation Is Essential for Functional Regeneration

Presently there exists no cure for spinal cord injury. However, transplantation of embryonic tissue into spinal cord 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 spinal cord circuits. We tested the two hypotheses in an in vitro spinal cord lesion model that is based on propagation of activity between two rat organotypic spinal cord slices in culture. Transplantation of dissociated cells from E14 rat spinal cord or forebrain 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 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 forebrain cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated spinal cord circuits. In contrast, transplantation of neurospheres 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 spinal cord circuits

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings

Apoptosis and proliferation in the trigeminal placode

The neurogenic trigeminal placode develops from the crescent-shaped panplacodal primordium which delineates the neural plate anteriorly. We show that, in Tupaia belangeri, the trigeminal placode is represented by a field of focal ectodermal thickenings which over time changes positions from as far rostral as the level of the forebrain to as far caudal as opposite rhombomere 3. Delamination proceeds rostrocaudally from the ectoderm adjacent to the rostral midbrain, and contributes neurons to the trigeminal ganglion as well as to the ciliary ganglion/oculomotor complex. Proliferative events are centered on the field prior to the peak of delamination. They are preceded, paralleled and, finally, outnumbered by apoptotic events which proceed rostrocaudally from non-delaminating to delaminating parts of the field. Apoptosis persists upon regression of the placode, thereby exhibiting a massive “wedge” of apoptotic cells which includes the postulated position of the “ventrolateral postoptic placode” (Lee et al. in Dev Biol 263:176–190, 2003), merges with groups of lens-associated apoptotic cells, and disappears upon lens detachment. In conjunction with earlier work (Washausen et al. in Dev Biol 278:86–102, 2005) our findings suggest that apoptosis contributes repeatedly to the disintegration of the panplacodal primordium, to the elimination of subsets of premigratory placodal neuroblasts, and to the regression of placodes

Cellular and network mechanisms of rhythm generation in spinal cord circuits

The generation of rhythmic electrical activity is a prominent feature of spinal cord circuits that is used for locomotion and also for circuit refinement during development. The mechanisms involved in rhythm generation in spinal cord networks are not fully understood. It is for example not known whether spinal cord rhythms are driven by pacemaker neurons and if yes, which neurons are involved in this function. We studied the mechanisms involved in rhythm generation in slice cultures from fetal rats that were grown on multielectrode arrays (MEAs). We combined multisite extracellular recordings from the MEA electrodes with intracellular patch clamp recordings from single neurons. We found that spatially restricted oscillations of activity appeared in most of the cultures spontaneously. Such activity was based on intrinsic activity in a percentage of the neurons that could activate the spinal networks through recurrent excitation. The local oscillator networks critically involved NMDA, AMPA and GABA / glycine receptors at subsequent phases of the oscillation cycle. Intrinsic spiking in individual neurons (in the absence of functional synaptic coupling) was based on persistent sodium currents. Intrinsic firing as well as persistent sodium currents were increased by 5-HT through 5-HT2 receptors. Comparing neuronal activity to muscle activity in co-cultures of spinal cord slices with muscle fibers we found that a percentage of the intrinsically spiking neurons were motoneurons. These motoneurons were electrically coupled among each other and they could drive the spinal networks through cholinergic recurrent excitation. These findings open the possibility that during development rhythmic activity in motoneurons is not only involved in circuit refinement downstream at the neuromuscular endplates but also upstream at the level of spinal cord circuits

Riluzole-induced oscillations in spinal networks

We previously showed in dissociated cultures of fetal rat spinal cord that disinhibition-induced bursting is based on intrinsic spiking, network recruitment, and a network refractory period after the bursts. A persistent sodium current (I(NaP)) underlies intrinsic spiking, which, by recurrent excitation, generates the bursting activity. Although full blockade of I(NaP) with riluzole disrupts such bursting, the present study shows that partial blockade of I(NaP) with low doses of riluzole maintains bursting activity with unchanged burst rate and burst duration. More important, low doses of riluzole turned bursts composed of persistent activity into bursts composed of oscillatory activity at around 5 Hz. In a search for the mechanisms underlying the generation of such intraburst oscillations, we found that activity-dependent synaptic depression was not changed with low doses of riluzole. On the other hand, low doses of riluzole strongly increased spike-frequency adaptation and led to early depolarization block when bursts were simulated by injecting long current pulses into single neurons in the absence of fast synaptic transmission. Phenytoin is another I(NaP) blocker. When applied in doses that reduced intrinsic activity by 80-90%, as did low doses of riluzole, it had no effect either on spike-frequency adaptation or on depolarization block. Nor did phenytoin induce intraburst oscillations after disinhibition. A theoretical model incorporating a depolarization block mechanism could reproduce the generation of intraburst oscillations at the network level. From these findings we conclude that riluzole-induced intraburst oscillations are a network-driven phenomenon whose major accommodation mechanism is depolarization block arising from strong sodium channel inactivation

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

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