RESUMEN
Hyperexcitability of motor neurons and spinal cord motor circuitry has been widely reported in the early stages of Amyotrophic Lateral Sclerosis (ALS). Changes in the relative amount of excitatory to inhibitory inputs onto a neuron (E:I synaptic ratio), possibly through a developmental shift in synapse formation in favour of excitatory transmission, could underlie pathological hyperexcitability. Given that astrocytes play a major role in early synaptogenesis and are implicated in ALS pathogenesis, their potential contribution to disease mechanisms involving synaptic imbalances and subsequent hyperexcitability is also of great interest. In order to assess E:I ratios in ALS, we utilised a novel primary spinal neuron / astrocyte co-culture system, derived from neonatal mice, in which synapses are formed in vitro. Using multiple ALS mouse models we found that no combination of astrocyte or neuron genotype produced alterations in E:I synaptic ratios assessed using pre- and post-synaptic anatomical markers. Similarly, we observed that ephrin-B1, a major contact-dependent astrocytic synaptogenic protein, was not differentially expressed by ALS primary astrocytes. Further to this, analysis of E:I ratios across the entire grey matter of the lumbar spinal cord in young (post-natal day 16-19) ALS mice revealed no differences versus controls. Finally, analysis in co-cultures of human iPSC-derived motor neurons and astrocytes harbouring the pathogenic C9orf72 hexanucleotide repeat expansion showed no evidence of a bias toward excitatory versus inhibitory synapse formation. We therefore conclude, utilising multiple ALS models, that we do not observe significant changes in the relative abundance of excitatory versus inhibitory synapses as would be expected if imbalances in synaptic inputs contribute to early hyperexcitability.
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Esclerosis Amiotrófica Lateral , Astrocitos , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Neuronas Motoras , Médula Espinal , Sinapsis , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Ratones , Sinapsis/metabolismo , Sinapsis/fisiología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Médula Espinal/metabolismo , Médula Espinal/patología , Humanos , Potenciales Postsinápticos Excitadores , Ratones Transgénicos , Células Cultivadas , Transmisión SinápticaRESUMEN
The size principle is a key mechanism governing the orderly recruitment of motor units and is believed to be dependent on passive properties of the constituent motoneurons. However, motoneurons are endowed with voltage-sensitive ion channels that create non-linearities in their input-output functions. Here we describe a role for the M-type potassium current, conducted by KCNQ channels, in the control of motoneuron recruitment in mice. Motoneurons were studied with whole-cell patch clamp electrophysiology in transverse spinal slices and identified based on delayed (fast) and immediate (slow) onsets of repetitive firing. M-currents were larger in delayed compared to immediate firing motoneurons, which was not reflected by variations in the presence of Kv7.2 or Kv7.3 subunits. Instead, a more depolarized spike threshold in delayed-firing motoneurons afforded a greater proportion of the total M-current to become activated within the subthreshold voltage range, which translated to a greater influence on their recruitment with little influence on their firing rates. Pharmacological activation of M-currents also influenced motoneuron recruitment at the population level, producing a rightward shift in the recruitment curve of monosynaptic reflexes within isolated mouse spinal cords. These results demonstrate a prominent role for M-type potassium currents in regulating the function of motor units, which occurs primarily through the differential control of motoneuron subtype recruitment. More generally, these findings highlight the importance of active properties mediated by voltage-sensitive ion channels in the differential control of motoneuron recruitment, which is a key mechanism for the gradation of muscle force. KEY POINTS: M-currents exert an inhibitory influence on spinal motor output. This inhibitory influence is exerted by controlling the recruitment, but not the firing rate, of high-threshold fast-like motoneurons, with limited influence on low-threshold slow-like motoneurons. Preferential control of fast motoneurons may be linked to a larger M-current that is activated within the subthreshold voltage range compared to slow motoneurons. Larger M-currents in fast compared to slow motoneurons are not accounted for by differences in Kv7.2 or Kv7.3 channel composition. The orderly recruitment of motoneuron subtypes is shaped by differences in the contribution of voltage-gated ion channels, including KCNQ channels. KCNQ channels may provide a target to dynamically modulate the recruitment gain across the motor pool and readily adjust movement vigour.
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Neuronas Motoras , Potasio , Humanos , Animales , Ratones , Potenciales de Acción/fisiología , Neuronas Motoras/fisiología , Fenómenos Electrofisiológicos , Canales IónicosRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterises the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.
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The intensity of muscle contraction, and therefore movement vigor, needs to be adaptable to enable complex motor behaviors. This can be achieved by adjusting the properties of motor neurons, which form the final common pathway for all motor output from the central nervous system. Here, we identify roles for a neuropeptide, cocaine- and amphetamine-regulated transcript (CART), in the control of movement vigor. We reveal distinct but parallel mechanisms by which CART and acetylcholine, both released at C bouton synapses on motor neurons, selectively amplify the output of subtypes of motor neurons that are recruited during intense movement. We find that mice with broad genetic deletion of CART or selective elimination of acetylcholine from C boutons exhibit deficits in behavioral tasks that require higher levels of motor output. Overall, these data uncover spinal modulatory mechanisms that control movement vigor to support movements that require a high degree of muscle force.
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Acetilcolina , Sinapsis , Animales , Ratones , Terminales Presinápticos , Neuronas Motoras , Sistema Nervioso CentralRESUMEN
Introduction: The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. Materials and methods: We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Results: Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Discussion: Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.
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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. Separate lines of evidence suggest that synapses and astrocytes play a role in the pathological mechanisms underlying ALS. Given that astrocytes make specialised contacts with some synapses, called tripartite synapses, we hypothesise that tripartite synapses could act as the fulcrum of disease in ALS. To test this hypothesis, we have performed an extensive microscopy-based investigation of synapses and tripartite synapses in the spinal cord of ALS model mice and post-mortem human tissue from ALS cases. We reveal widescale synaptic changes at the early symptomatic stages of the SOD1G93a mouse model. Super-resolution microscopy reveals that large complex postsynaptic structures are lost in ALS mice. Most surprisingly, tripartite synapses are selectively lost, while non-tripartite synapses remain in equal number to healthy controls. Finally, we also observe a similar selective loss of tripartite synapses in human post-mortem ALS spinal cords. From these data we conclude that tripartite synaptopathy is a key hallmark of ALS.
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Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Médula Espinal/patología , Superóxido Dismutasa , Superóxido Dismutasa-1/genética , Sinapsis/patologíaRESUMEN
The size principle underlies the orderly recruitment of motor units; however, motoneuron size is a poor predictor of recruitment amongst functionally defined motoneuron subtypes. Whilst intrinsic properties are key regulators of motoneuron recruitment, the underlying currents involved are not well defined. Whole-cell patch-clamp electrophysiology was deployed to study intrinsic properties, and the underlying currents, that contribute to the differential activation of delayed and immediate firing motoneuron subtypes. Motoneurons were studied during the first three postnatal weeks in mice to identify key properties that contribute to rheobase and may be important to establish orderly recruitment. We find that delayed and immediate firing motoneurons are functionally homogeneous during the first postnatal week and are activated based on size, irrespective of subtype. The rheobase of motoneuron subtypes becomes staggered during the second postnatal week, which coincides with the differential maturation of passive and active properties, particularly persistent inward currents. Rheobase of delayed firing motoneurons increases further in the third postnatal week due to the development of a prominent resting hyperpolarization-activated inward current. Our results suggest that motoneuron recruitment is multifactorial, with recruitment order established during postnatal development through the differential maturation of passive properties and sequential integration of persistent and hyperpolarization-activated inward currents.
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Fenómenos Electrofisiológicos , Neuronas Motoras/fisiología , Sistema Nervioso/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Tamaño de la Célula , Femenino , Masculino , Ratones Endogámicos C57BL , Técnicas de Placa-ClampRESUMEN
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.
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Factores de Crecimiento Nervioso , Neuroblastoma , Diferenciación Celular , Línea Celular Tumoral , Humanos , Neuronas , TretinoinaRESUMEN
Astrocytes are a functionally diverse form of glial cell involved in various aspects of nervous system infrastructure, from the metabolic and structural support of neurons to direct neuromodulation of synaptic activity. Investigating how astrocytes behave in functionally related circuits may help us understand whether there is any conserved logic to the role of astrocytes within neuronal networks. Astrocytes are implicated as key neuromodulatory cells within neural circuits that control a number of rhythmic behaviours such as breathing, locomotion and circadian sleep-wake cycles. In this review, we examine the evidence that astrocytes are directly involved in the regulation of the neural circuits underlying six different rhythmic behaviours: locomotion, breathing, chewing, gastrointestinal motility, circadian sleep-wake cycles and oscillatory feeding behaviour. We discuss how astrocytes are integrated into the neuronal networks that regulate these behaviours, and identify the potential gliotransmission signalling mechanisms involved. From reviewing the evidence of astrocytic involvement in a range of rhythmic behaviours, we reveal a heterogenous array of gliotransmission mechanisms, which help to regulate neuronal networks. However, we also observe an intriguing thread of commonality, in the form of purinergic gliotransmission, which is frequently utilised to facilitate feedback inhibition within rhythmic networks to constrain a given behaviour within its operational range.
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Astrocitos , Neuroglía , Neuronas , Transducción de SeñalRESUMEN
Functionally distinct synapses exhibit diverse and complex organisation at molecular and nanoscale levels. Synaptic diversity may be dependent on developmental stage, anatomical locus and the neural circuit within which synapses reside. Furthermore, astrocytes, which align with pre and post-synaptic structures to form 'tripartite synapses', can modulate neural circuits and impact on synaptic organisation. In this study, we aimed to determine which factors impact the diversity of excitatory synapses throughout the lumbar spinal cord. We used PSD95-eGFP mice, to visualise excitatory postsynaptic densities (PSDs) using high-resolution and super-resolution microscopy. We reveal a detailed and quantitative map of the features of excitatory synapses in the lumbar spinal cord, detailing synaptic diversity that is dependent on developmental stage, anatomical region and whether associated with VGLUT1 or VGLUT2 terminals. We report that PSDs are nanostructurally distinct between spinal laminae and across age groups. PSDs receiving VGLUT1 inputs also show enhanced nanostructural complexity compared with those receiving VGLUT2 inputs, suggesting pathway-specific diversity. Finally, we show that PSDs exhibit greater nanostructural complexity when part of tripartite synapses, and we provide evidence that astrocytic activation enhances PSD95 expression. Taken together, these results provide novel insights into the regulation and diversification of synapses across functionally distinct spinal regions and advance our general understanding of the 'rules' governing synaptic nanostructural organisation.
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Médula Espinal/citología , Sinapsis/metabolismo , Animales , Astrocitos/citología , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía , Relación Señal-Ruido , Médula Espinal/diagnóstico por imagenRESUMEN
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.
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Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.
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Astrocitos/metabolismo , Proteína C9orf72/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Potenciales de Acción/fisiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Astrocitos/patología , Proteína C9orf72/metabolismo , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/patología , MutaciónRESUMEN
Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.
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Acetilcolina/metabolismo , Locomoción/fisiología , Neuronas Motoras/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Médula Espinal/metabolismo , Acetilcolina/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Diaminas/farmacología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/fisiología , Muscarina/farmacología , Piperidinas/farmacología , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/fisiología , Médula Espinal/fisiologíaRESUMEN
Cholinergic neuromodulation has been described throughout the brain and has been implicated in various functions including attention, food intake and response to stress. Cholinergic modulation is also thought to be important for regulating motor systems, as revealed by studies of large cholinergic synapses on spinal motor neurons, called C boutons, which seem to control motor neuron excitability in a task-dependent manner. C boutons on spinal motor neurons stem from spinal interneurons that express the transcription factor Pitx2. C boutons have also been identified on the motor neurons of specific cranial nuclei. However, the source and roles of cranial C boutons are less clear. Previous studies suggest that they originate from Pitx2+ and Pitx2- neurons, in contrast to spinal cord C boutons that originate solely from Pitx2 neurons. Here, we address this controversy using mouse genetics, and demonstrate that brainstem C boutons are Pitx2+ derived. We also identify new Pitx2 populations and map the cholinergic Pitx2 neurons of the mouse brain. Taken together, our data present important new information about the anatomical organization of cholinergic systems which impact motor systems of the brainstem. These findings will enable further analyses of the specific roles of cholinergic modulation in motor control.
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Tronco Encefálico/citología , Neuronas Colinérgicas/citología , Interneuronas/citología , Neuronas Motoras/citología , Terminales Presinápticos/fisiología , Animales , Tronco Encefálico/fisiología , Neuronas Colinérgicas/metabolismo , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Médula Espinal/citología , Médula Espinal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Optogenetics, photostimulation of neural tissues rendered sensitive to light, is widely used in neuroscience to modulate the electrical excitability of neurons. For effective optical excitation of neurons, light wavelength and power density must fit with the expression levels and biophysical properties of the genetically encoded light-sensitive ion channels used to confer light sensitivity on cells-most commonly, channelrhodopsins (ChRs). As light sources, organic light-emitting diodes (OLEDs) offer attractive properties for miniaturized implantable devices for in vivo optical stimulation, but they do not yet operate routinely at the optical powers required for optogenetics. Here, OLEDs with doped charge transport layers are demonstrated that deliver blue light with good stability over millions of pulses, at powers sufficient to activate the ChR, CheRiff when expressed in cultured primary neurons, allowing live cell imaging of neural activity with the red genetically encoded calcium indicator, jRCaMP1a. Intracellular calcium responses scale with the radiant flux of OLED emission, when varied through changes in the current density, number of pulses, frequency, and pulse width delivered to the devices. The reported optimization and characterization of high-power OLEDs are foundational for the development of miniaturized OLEDs with thin-layer encapsulation on bioimplantable devices to allow single-cell activation in vivo.
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Neuronas , Optogenética/métodos , Estimulación Luminosa/métodos , Animales , Células Cultivadas , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Electrodos Implantados , Hipocampo/citología , Ratones , Neuronas/química , Neuronas/citología , Neuronas/metabolismo , Proteínas RecombinantesRESUMEN
Astrocytes modulate many neuronal networks, including spinal networks responsible for the generation of locomotor behavior. Astrocytic modulation of spinal motor circuits involves release of ATP from astrocytes, hydrolysis of ATP to adenosine, and subsequent activation of neuronal A1 adenosine receptors (A1Rs). The net effect of this pathway is a reduction in the frequency of locomotor-related activity. Recently, it was proposed that A1Rs modulate burst frequency by blocking the D1-like dopamine receptor (D1LR) signaling pathway; however, adenosine also modulates ventral horn circuits by dopamine-independent pathways. Here, we demonstrate that adenosine produced upon astrocytic stimulation modulates locomotor-related activity by counteracting the excitatory effects of D1LR signaling and does not act by previously described dopamine-independent pathways. In spinal cord preparations from postnatal mice, a D1LR agonist, SKF 38393, increased the frequency of locomotor-related bursting induced by 5-hydroxytryptamine and N-methyl-d-aspartate. Bath-applied adenosine reduced burst frequency only in the presence of SKF 38393, as did adenosine produced after activation of protease-activated receptor-1 to stimulate astrocytes. Furthermore, the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine enhanced burst frequency only in the presence of SKF 38393, indicating that endogenous adenosine produced by astrocytes during network activity also acts by modulating D1LR signaling. Finally, modulation of bursting by adenosine released upon stimulation of astrocytes was blocked by protein kinase inhibitor-(14-22) amide, a protein kinase A (PKA) inhibitor, consistent with A1R-mediated antagonism of the D1LR/adenylyl cyclase/PKA pathway. Together, these findings support a novel, astrocytic mechanism of metamodulation within the mammalian spinal cord, highlighting the complexity of the molecular interactions that specify motor output. NEW & NOTEWORTHY Astrocytes within the spinal cord produce adenosine during ongoing locomotor-related activity or when experimentally stimulated. Here, we show that adenosine derived from astrocytes acts at A1 receptors to inhibit a pathway by which D1-like receptors enhance the frequency of locomotor-related bursting. These data support a novel form of metamodulation within the mammalian spinal cord, enhancing our understanding of neuron-astrocyte interactions and their importance in shaping network activity.
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Adenosina/metabolismo , Astrocitos/metabolismo , Neurotransmisores/metabolismo , Receptores de Dopamina D1/metabolismo , Médula Espinal/fisiología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Adenilil Ciclasas/metabolismo , Análisis de Varianza , Animales , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacología , Locomoción/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Receptor de Adenosina A1/metabolismo , Receptores de Dopamina D1/agonistas , Transducción de Señal/efectos de los fármacos , Médula Espinal/citología , Xantinas/farmacologíaRESUMEN
Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.
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Proteína C9orf72/genética , Neuronas Motoras/patología , Receptores AMPA/metabolismo , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/metabolismo , Sistemas CRISPR-Cas , Calcio/metabolismo , Expansión de las Repeticiones de ADN , Marcación de Gen , Humanos , Receptores AMPA/genética , Médula Espinal/metabolismo , Médula Espinal/fisiopatologíaRESUMEN
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 brain stem 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. 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.
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Adenosina/fisiología , Astrocitos/fisiología , Neuronas/fisiología , Médula Espinal/fisiología , Animales , Generadores de Patrones Centrales , Humanos , Locomoción , Vías Nerviosas/fisiologíaRESUMEN
Activation of N-methyl-d-aspartate receptors (NMDARs) requires the binding of a coagonist, either d-serine or glycine, in addition to glutamate. Changes in occupancy of the coagonist binding site are proposed to modulate neural networks including those controlling swimming in frog tadpoles. Here, we characterize regulation of the NMDAR coagonist binding site in mammalian spinal locomotor networks. Blockade of NMDARs by d(-)-2-amino-5-phosphonopentanoic acid (d-APV) or 5,7-dichlorokynurenic acid reduced the frequency and amplitude of pharmacologically induced locomotor-related activity recorded from the ventral roots of spinal-cord preparations from neonatal mice. Furthermore, d-APV abolished synchronous activity induced by blockade of inhibitory transmission. These results demonstrate an important role for NMDARs in murine locomotor networks. Bath-applied d-serine enhanced the frequency of locomotor-related but not disinhibited bursting, indicating that coagonist binding sites are saturated during the latter but not the former mode of activity. Depletion of endogenous d-serine by d-amino acid oxidase or the serine-racemase inhibitor erythro-ß-hydroxy-l-aspartic acid (HOAsp) increased the frequency of locomotor-related activity, whereas application of l-serine to enhance endogenous d-serine synthesis reduced burst frequency, suggesting a requirement for d-serine at a subset of synapses onto inhibitory interneurons. Consistent with this, HOAsp was ineffective during disinhibited activity. Bath-applied glycine (1-100 µM) failed to alter locomotor-related activity, whereas ALX 5407, a selective inhibitor of glycine transporter-1 (GlyT1), enhanced burst frequency, supporting a role for GlyT1 in NMDAR regulation. Together these findings indicate activity-dependent and synapse-specific regulation of the coagonist binding site within spinal locomotor networks, illustrating the importance of NMDAR regulation in shaping motor output.NEW & NOTEWORTHY We provide evidence that NMDARs within murine spinal locomotor networks determine the frequency and amplitude of ongoing locomotor-related activity in vitro and that NMDARs are regulated by d-serine and glycine in a synapse-specific and activity-dependent manner. In addition, glycine transporter-1 is shown to be an important regulator of NMDARs during locomotor-related activity. These results show how excitatory transmission can be tuned to diversify the output repertoire of spinal locomotor networks in mammals.