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.

Non-technical summary A ganglionated neural plexus, the myenteric plexus, within the colon generates a propagating contraction called the colonic migrating motor complex (CMMC) that underlies faecal pellet propulsion. Neurons in the myenteric plexus are surrounded by a network of enteric glia cells (EGCs), which were traditionally thought to be the glue that held the neurons together. Using imaging techniques, we demonstrate that 36% of EGCs respond with prolonged Ca 2+ transients following their activation by excitatory nerve fibres that generate the CMMC, suggesting that EGCs are innervated and are activated during the CMMC.Abstract 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.

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.

Spinal motor networks are formed by diverse populations of interneurons that set the strength and rhythmicity of behaviors such as locomotion. A small cluster of cholinergic interneurons, expressing the transcription factor Pitx2, modulates the intensity of muscle activation via ‘C-bouton’ inputs to motoneurons. However, the synaptic mechanisms underlying this neuromodulation remain unclear. Here, we confirm in mice that Pitx2+ interneurons are active during fictive locomotion and that their chemogenetic inhibition reduces the amplitude of motor output. Furthermore, after genetic ablation of cholinergic Pitx2+ interneurons, M2 receptor-dependent regulation of the intensity of locomotor output is lost. Conversely, chemogenetic stimulation of Pitx2+ interneurons leads to activation of M2 receptors on motoneurons, regulation of Kv2.1 channels and greater motoneuron output due to an increase in the inter-spike afterhyperpolarization and a reduction in spike half-width. Our findings elucidate synaptic mechanisms by which cholinergic spinal interneurons modulate the final common pathway for motor output.

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 astrocytederived, adenosinergic modulation of the frequency of rhythmic output generated by the locomotor central pattern generator (CPG) network. Live Ca 2+ 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. LAY SUMMARYWe have investigated how astrocytes, a type of supportive cell within the nervous system, may be directly involved in the control of movements, such as walking (locomotion). We found that astrocytes are active participants in the neural circuits of the mammalian spinal cord that control locomotion. The function of astrocytes within these networks appears to relate to the modulation of locomotor speed. We also identify the chemical signaling pathways involved in a modulatory feedback loop between astrocytes and neurons. Our findings suggest that this bi-directional communication between astrocytes and neurons may act to constrain spinal motor circuits within operational ranges for desired movements.

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 postsynaptic 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. Neuronal synapses show considerable structural, molecular and functional diversity throughout the nervous system. Their morphology, molecular composition and nanostructural organisation determines synaptic strength and function 1,2 , and must therefore be finely tuned to the circuits within which they operate 3. Synapse morphology may be determined by a number of factors, such as the type of neuron, the anatomical location, the activity state of the network, and development and ageing 4-9. In order to understand the basic logic of different neural circuits, it is pertinent to study the principles of synaptic morphology that underlie the function of neural circuits. Such information may help produce a set of basic 'rules' governing synaptic structure and therefore function. Key scaffolding proteins such as PSD95, one of the most abundantly expressed proteins from the Dlg family, form the core molecular architecture of the synapse 10-13. At a molecular level, PSD95 interacts with a host of different signalling enzymes and neurotransmitter receptors within the postsynaptic density (PSD) to form PSD95-dependent super-complexes (~1.5MDa in weight) 14-17. These super-complexes are heterogeneously distributed within the PSD, forming protein-enriched domains known as nanoclusters (NCs, ~140 nm diameter) 4,5,18,19 , which align with presynaptic release sites to form trans-synaptic nanocolumns 1. Visualising PSD95 enables us to determine the na...

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.

Activity in the submucous neurons appears to be secondary to that in the myenteric plexus and appears to be generated largely by activity in myenteric descending (serotonergic) interneurons. During the CMMC, there is likely to be an increase in secretion to lubricate and facilitate fecal pellet propulsion.