Bi-directional communication between neurons and astrocytes modulates spinal motor circuits

Funding was provided by the Biotechnology and Biological Science Research Grant (BB/M021793/1), the Motor Neurone Disease (MND) Association UK and the RS MacDonald Charitable Trust.Evidence suggests that astrocytes are not merely supportive cells in the nervous system but may actively participate in the control of neural circuits underlying cognition and behavior. In this study, we examined the role of astrocytes within the motor circuitry of the mammalian spinal cord. Pharmacogenetic manipulation of astrocytic activity in isolated spinal cord preparations obtained from neonatal mice revealed astrocyte-derived, adenosinergic modulation of the frequency of rhythmic output generated by the locomotor central pattern generator (CPG) network. Live Ca2+ imaging demonstrated increased activity in astrocytes during locomotor-related output and in response to the direct stimulation of spinal neurons. Finally, astrocytes were found to respond to neuronally-derived glutamate in a metabotropic glutamate receptor 5 (mGluR5) dependent manner, which in turn drives astrocytic modulation of the locomotor network. Our work identifies bi-directional signaling mechanisms between neurons and astrocytes underlying modulatory feedback control of motor circuits, which may act to constrain network output within optimal ranges for movement.Publisher PDFPeer reviewe

Gliotransmission and adenosinergic modulation : insights from mammalian spinal motor networks

D Acton was supported by funds from a Wellcome Trust Institutional Strategic Support Fund grant. GBM received support from Biotechnology and Biological Science Research Grant BB/M021793/1.Astrocytes are proposed to converse with neurons at tripartite synapses, detecting neurotransmitter release and responding with release of gliotransmitters, which in turn modulate synaptic strength and neuronal excitability. However, a paucity of evidence from behavioral studies calls into question the importance of gliotransmission for the operation of the nervous system in healthy animals. Central pattern generator (CPG) networks in the spinal cord and brainstem coordinate the activation of muscles during stereotyped activities such as locomotion, inspiration and mastication, and may therefore provide tractable models in which to assess the contribution of gliotransmission to behaviorally relevant neural activity. Here, we review evidence for gliotransmission within spinal locomotor networks, including studies indicating that adenosine derived from astrocytes regulates the speed of locomotor activity via metamodulation of dopamine signaling.PostprintPeer reviewe

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

This work is supported by the UK Engineering Physical Sciences Research Council (EPSRC).A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.Publisher PDFPeer reviewe

Long-term culture of SH-SY5Y neuroblastoma cells in the absence of neurotrophins : a novel model of neuronal ageing

LS is sponsored by a Wellcome Trust ISSF studentship and YC is a recipient of a China Scholarship Council award.Background Studying human ageing is of increasing importance due to the worldwide ageing population. However, it faces the challenge of lengthy experiments to produce an ageing phenotype. Often, to recreate the hallmarks of ageing requires complex empirical conditions that can confound data interpretation. Indeed, many studies use whole organisms with relatively short life spans, which may have little, or limited, relevance to human ageing. There has been extensive use of cell lines to study ageing in human somatic cells, but the modelling of human neuronal ageing is somewhat more complex in vitro. New Method We cultured the well-characterised SH-SY5Y human neural cell line to produce high purity cultures of cells differentiated to express a neuronal phenotype, and designed a protocol to maintain these cells in culture until they accumulated biomarkers of cellular ageing. Results Our data validate a novel and simple technique for the efficient differentiation and long-term maintenance of SH-SY5Y cells, expressing markers of neuronal differentiation and demonstrating electrical activity in culture. Over time in vitro, these cells progressively accumulate markers of ageing such as enhanced production of reactive oxygen species and accumulation of oxidative damage. Comparison to Existing Methods In comparison to existing techniques to model neuronal ageing our method is cost effective, requiring no specialist equipment or growth factors. Conclusions We demonstrate that SH-SY5Y cells, grown under these culture conditions, represent a simple model of neuronal ageing that is amenable to cell biological, biochemical and electrophysiological investigation.Publisher PDFPeer reviewe

Sodium pumps mediate activity-dependent changes in mammalian motor networks

Funding: Laurence Picton [grant number BB/JO1446X/1] and Matthew Broadhead [grant number BB/M021793/1] were supported by the Biotechnology and Biological Science Research Council (BBSRC). Filipe Nascimento was supported by The Alfred Dunhill Links Foundation.Ubiquitously expressed sodium pumps are best known for maintaining the ionic gradients and resting membrane potential required for generating action potentials. However, activity- and state-dependent changes in pump activity can also influence neuronal firing and regulate rhythmic network output. Here we demonstrate that changes in sodium pump activity regulate locomotor networks in the spinal cord of neonatal mice. The sodium pump inhibitor, ouabain, increased the frequency and decreased the amplitude of drug-induced locomotor bursting, effects that were dependent on the presence of the neuromodulator dopamine. Conversely, activating the pump with the sodium ionophore monensin decreased burst frequency. When more "natural" locomotor output was evoked using dorsal-root stimulation, ouabain increased burst frequency and extended locomotor episode duration, whereas monensin slowed and shortened episodes. Decreasing the time between dorsal-root stimulation, and therefore interepisode interval, also shortened and slowed activity, suggesting that pump activity encodes information about past network output and contributes to feedforward control of subsequent locomotor bouts. Using whole-cell patch-clamp recordings from spinal motoneurons and interneurons, we describe a long-duration (∼60 s), activity-dependent, TTX- and ouabain-sensitive, hyperpolarization (∼5 mV), which is mediated by spike-dependent increases in pump activity. The duration of this dynamic pump potential is enhanced by dopamine. Our results therefore reveal sodium pumps as dynamic regulators of mammalian spinal motor networks that can also be affected by neuromodulatory systems. Given the involvement of sodium pumps in movement disorders, such as amyotrophic lateral sclerosis and rapid-onset dystonia parkinsonism, knowledge of their contribution to motor network regulation also has considerable clinical importance.Publisher PDFPeer reviewe

An automated framework of inner segment/outer segment defect detection for retinal SD-OCT images

The integrity of inner segment/outer segment (IS/OS) has high correlation with lower visual acuity in patients suffering from blunt trauma. An automated 3D IS/OS defect detection method based on the SD-OCT images was proposed. First, 11 surfaces were automatically segmented using the multiscale 3D graph-search approach. Second, the sub-volumes between surface 7 and 8 containing IS/OS region around the fovea (diameter of mm) were extracted and flattened based on the segmented retinal pigment epithelium layer. Third, 5 kinds of texture based features were extracted for each voxel. A KNN classifier was trained and each voxel was classified as disrupted or nondisrupted and the responding defect volume was calculated. The proposed method was trained and tested on 9 eyes from 9 trauma subjects using the leave-one-out cross validation method. The preliminary results demonstrated the feasibility and efficiency of the proposed method

Improving Together: A National Framework for Quality and GP Clusters in Scotland

Improving together will complement the development of the Scottish national GP contract that sets out the role of GPs and their important contribution as clinical leaders and expert medical generalists working in a community setting. This framework will be reviewed by the Scottish Government and the Scottish General Practitioners Committee of the BMA on a periodic basis, attentive to feedback from those involved in delivering its intent. As such, it is a framework that will develop to its full potential over time, as elements of the transformation of primary care in Scotland create the capacity to do so

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead