M-type potassium currents differentially affect activation of motoneuron subtypes and tune recruitment gain.

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.

Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy.

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.

Selective vulnerability of tripartite synapses in amyotrophic lateral sclerosis.

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.

A common role for astrocytes in rhythmic behaviours?

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.

Nanostructural Diversity of Synapses in the Mammalian Spinal Cord.

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.

Bi-Directional Communication Between Neurons and Astrocytes Modulates Spinal Motor Circuits.

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.

Modulation of spinal motor networks by astrocyte-derived adenosine is dependent on D1-like dopamine receptor signaling.

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.

Sodium Pumps Mediate Activity-Dependent Changes in Mammalian Motor Networks.

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. SIGNIFICANCE STATEMENT: The sodium pump is ubiquitously expressed and responsible for at least half of total brain energy consumption. The pumps maintain ionic gradients and the resting membrane potential of neurons, but increasing evidence suggests that activity- and state-dependent changes in pump activity also influence neuronal firing. Here we demonstrate that changes in sodium pump activity regulate locomotor output in the spinal cord of neonatal mice. We describe a sodium pump-mediated afterhyperpolarization in spinal neurons, mediated by spike-dependent increases in pump activity, which is affected by dopamine. Understanding how sodium pumps contribute to network regulation and are targeted by neuromodulators, including dopamine, has clinical relevance due to the role of the sodium pump in diseases, including amyotrophic lateral sclerosis, parkinsonism, epilepsy, and hemiplegic migraine.

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits.

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.

Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.

Enteric glia cells (EGCs) form a dense network around myenteric neurons in a ganglia and are likely to have not only a supportive role but may also regulate or be regulated by neural activity. Our aims were to determine if EGCs are activated during the colonic migrating motor complex (CMMC) in the isolated murine colon. Strips of longitudinal muscle were removed and Ca(2+) imaging (Fluo-4) used to study activity in EGCs within myenteric ganglia during CMMCs, followed by post hoc S100 staining to reveal EGCs. The cell bodies of EGCs and their processes formed caps and halos, respectively, around some neighbouring myenteric neurons. Some EGCs (36%), which were largely quiescent between CMMCs, exhibited prolonged tetrodotoxin (TTX; 1 µm)-sensitive Ca(2+) transients that peaked ∼39 s following a mucosal stimulus that generated the CMMC, and often outlasted the CMMC (duration ∼23 s). Ca(2+) transients in EGCs often varied in duration within a ganglion; however, the duration of these transients was closely matched by activity in closely apposed nerve varicosities, suggesting EGCs were not only innervated but the effective innervation was localized. Furthermore, all EGCs, even those that were quiescent, responded with robust Ca(2+) transients to KCl, caffeine, nicotine, substance P and GR 64349 (an NK2 agonist), suggesting they were adequately loaded with indicator and that some EGCs may be inhibited by substances released by neighbouring neurons. Intracellular Ca(2+) waves were visualised propagating between closely apposed glia and from glial cell processes to the soma (velocity 12 µm s(-1)) where they produced an accumulative rise in Ca(2+), suggesting that the soma acts as an integrator of Ca(2+) activity. In conclusion, Ca(2+) transients in EGCs occur secondary to nerve activity; their activation is driven by intrinsic excitatory nerve pathways that generate the CMMC.