Biology subjects

Miles, G. B.

Publications and source records attributed to Miles, G. B..

13 recordsLinked to original sources

bioRxiv2026

Discovery of the Honeycomb Synapse in Spinal Motor Circuits

The structural and molecular diversity of synapses in the nervous system contributes to the specialisation of neural circuits underlying diverse behaviours. We have discovered a morphologically distinct postsynaptic specialisation in the mammalian spinal cord. We have named this the Honeycomb Synapse based on its elaborate postsynaptic nanostructure, comprising rings formed of ~6 scaffolding protein domains that create multiple perforations throughout the large postsynaptic domain. Analysis of the organisation of other synaptic proteins reveals that Honeycomb Synapses harbour mixed signalling properties structurally facilitated by a postsynaptic scaffold matrix supporting chemical transmission, with gap junction proteins occupying some of the perforations. We reveal anatomical diversity in the presence of Honeycomb Synapses on populations of -motoneurons across the lumbar spinal cord in mice from approximately 2 weeks of age through to adulthood. The Honeycomb Synapse was found to be a subtype of synapse within the Ia afferent monosynaptic stretch reflex circuit. Finally, we have identified that Honeycomb Synapses are highly vulnerable to degeneration in two different genetically engineered mouse models of Amyotrophic Lateral Sclerosis (ALS), contributing to monosynaptic stretch reflex circuit dysfunction. These findings suggest that synaptic diversity within circuits may confer selective vulnerability to distinct synaptic subclasses.

neuroscience↗

bioRxiv2026

HCN channels modulate the medium afterhyperpolarization and adjust the firing gain of fast alpha motoneurons in mice

Motoneuron subtypes exhibit distinct firing properties that are critical for the graded control of muscle force. A key determinant of these differences is the medium afterhyperpolarization (mAHP), which shapes discharge rate and firing gain. While subtype-specific variation in mAHP properties has traditionally been attributed to differences in small-conductance calcium-activated potassium (SK) channel expression, emerging evidence suggests that additional conductances may contribute. Here, we investigated the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in regulating the mAHP and excitability of mouse spinal motoneurons during postnatal development. Using whole-cell patch-clamp recordings, we show that, by the onset of the third postnatal week, an h current (Ih) is active at resting potential in fast motoneurons and is correlated with the amplitude of the mAHP. Pharmacological blockade of HCN channels with ZD7288 increased mAHP amplitude in fast but not slow motoneurons, without affecting mAHP duration, indicating a subtype-specific contribution to mAHP amplitude. In line with the mAHP regulating firing gain, ZD7288 also reduced firing gain in fast but not slow motoneurons. These findings support a contribution of HCN channel activity to the regulation of mAHP amplitude and firing gain in fast motoneurons, highlighting a potential interaction between Ih and SK channel-dependent mechanisms in shaping motoneuron excitability. Key PointsO_LIThe amplitude of the medium afterhyperpolarization (mAHP) is negatively correlated with h-current (Ih) amplitude measured near resting potential in mouse lumbar motoneurons. C_LIO_LIPharmacological blockade of HCN channels selectively increases mAHP amplitude in fast, delayed firing alpha motoneurons, with no effect observed in slow, immediate firing alpha motoneurons. C_LIO_LIInhibition of HCN channels reduces firing gain in fast motoneurons, while slow motoneurons remain unaffected. C_LIO_LIHCN channels regulate firing gain in fast motoneurons, at least in part, through modulation of mAHP amplitude. C_LI

neuroscience↗

bioRxiv2026

Developmental mechanisms contributing to non-linear firing dynamics in spinal motoneurons of the postnatal mouse

The intrinsic properties of spinal motoneurons support flexible movement, including the maintenance of postural tone. Motoneurons can produce sustained action potential output that outlasts synaptic input, a phenomenon traditionally attributed to persistent inward currents (PICs) mediated by sodium and calcium channels. Using whole-cell patch clamp electrophysiology, we examined how specific ion channels contribute to PIC maturation and non-linear firing dynamics that allow motoneurons to sustain their output in fast and slow lumbar motoneurons across postnatal development in mice. PIC amplitude and non-linear firing dynamics increased after weight bearing in fast but not slow motoneurons. Blocking Nav1.6 channels reduced PIC amplitude at both pre- and post-weight-bearing stages, whereas L-type calcium channel blockade only reduced PICs after weight bearing emerged. However, reducing PIC amplitude--either individually or in combination--did not abolish sustained firing hysteresis. Unexpectedly, activation of muscarinic receptors increased PIC amplitude while promoting adaptive firing dynamics, suggesting that PICs alone do not drive this behavior. Instead, pharmacological manipulation of potassium currents mediated by KCNQ and Kv1.2 channels, which oppose PICs, produced substantial changes in firing dynamics. Strikingly, blocking HCN channels promoted sustained firing dynamics and led to the emergence of self-sustained firing in fast motoneurons. These results indicate that while PICs and non-linear firing dynamics mature together, sustained firing relies on mechanisms beyond PICs, with potassium and HCN channels playing key modulatory roles. Key PointsO_LIPersistent inward currents (PICs) and recruitment-derecruitment hysteresis increase in parallel in fast, but not slow, motoneurons following the onset of hindlimb weight bearing. C_LIO_LIIncreased expression or function of L-type calcium channels may contribute to enhanced PICs in fast motoneurons after weight bearing emerges. C_LIO_LINeither Nav1.6 nor L-type calcium channels are required for sustained firing hysteresis in fast motoneurons. C_LIO_LIKCNQ channels attenuate PICs and, together with Kv1.2 channels, shape recruitment-derecruitment asymmetry, thereby modulating firing hysteresis in fast motoneurons. C_LIO_LIHCN channels generate a resting H-current that delays recruitment, modulates firing hysteresis, and prevents the emergence of self-sustained firing in fast motoneurons. C_LI

neuroscience↗

bioRxiv2025

Intrinsic mechanisms contributing to the biophysical signature of mouse gamma motoneurons

Precise motor control relies on continuous sensory feedback from muscles, a process in which gamma motoneurons play a key role. These specialized spinal neurons innervate intrafusal muscle fibres, modulating their sensitivity to stretch and maintaining proprioceptive signalling during movement. Gamma motoneurons are characterized by a distinct biophysical profile, including low recruitment thresholds and high firing rates that enable rapid activation of intrafusal fibres at contraction onset. Despite their importance, the intrinsic mechanisms that underlie these properties remain poorly understood. In this study, we analysed published and unpublished data to identify a population of low-threshold, high-gain motoneurons with features consistent with gamma motoneurons, emerging during the third postnatal week in mice. Their low recruitment threshold was linked to lower membrane capacitance, higher input resistance, a more hyperpolarized activation of persistent inward currents (PICs), and a narrower axon initial segment. In contrast, higher firing rates were associated not with PIC amplitude but with shorter action potential durations and smaller medium afterhyperpolarizations. Notably, 92% of putative gamma motoneurons exhibited a sodium pump-mediated ultra-slow afterhyperpolarization (usAHP), which was absent in slow alpha motoneurons. This difference could not be attributed to h-current activity or expression of the alpha 3 subunit of the sodium-potassium ATPase. These findings reveal key intrinsic properties that support the unique excitability of gamma motoneurons, offering new insight into their contribution to motor control. This work provides a foundation for future studies into their development, regulation, and involvement in neuromuscular disorders. Key Points[bullet] A distinct cluster of motoneurons with low recruitment current and high firing gain, characteristic of gamma motoneurons, emerges in the third week of postnatal development. [bullet]Gamma motoneurons have a low recruitment current due to lower capacitance, higher input resistance, and a more hyperpolarized activation voltage for persistent inward currents. [bullet]Their high firing rates are not driven by differences in persistent inward current amplitude but are instead attributed to shorter duration action potentials and smaller amplitude medium afterhyperpolarizations. [bullet]A narrower axon initial segment in gamma motoneurons may contribute to their increased excitability compared to alpha motoneurons. [bullet]Gamma motoneurons present with a higher prevalence of ultra slow afterhyperpolarization than slow alpha motoneurons that cannot be accounted for by differences in h-current or expression of alpha 3 subunits of the sodium potassium ATPase pump.

neuroscience↗

bioRxiv2025

Synaptopathy in the TDP43ΔNLS Mouse Model of Sporadic Amyotrophic Lateral Sclerosis

Sporadic cases of Amyotrophic Lateral Sclerosis (sALS) represent the most common form of motor neuron disease. sALS is characterised by pathological cytoplasmic inclusions of TDP-43, so-called reactive astrocyte pathology, and motor neuron degeneration. Early-stage alterations in certain subpopulations of synapses between neurons are thought to be a key driver of the early pathological mechanisms of ALS. However, we do not have a clear understanding of which types of synapses are impacted in ALS. Identifying vulnerable synapses affected in sALS models may provide insights into the key sites of disease pathogenesis. In this study we have performed quantitative high-resolution microscopy to survey different synapse subtypes, including excitatory (glutamatergic), inhibitory (glycinergic) and modulatory (cholinergic C-Bouton) synapses, in the spinal cord of a mouse model of sALS showing inducible TDP-43 pathology (TDP43{Delta}NLS) restricted to neurons. We have identified changes in cholinergic synapses and a subpopulation of excitatory synapses. Mice display robust neuronal TDP-43 pathology and evidence of TDP-43 changes at cholinergic C-boutons. We also observe no evidence of astrocytic pathology nor changes in the fraction of synapses that are contacted by astrocytes, demonstrating that synapse pathology is driven by cell-autonomous (neuronal) mechanisms. Overall, our findings highlight the selective vulnerability of distinct synapse populations in ALS.

neuroscience↗

bioRxiv2025

A cholinergic spinal pathway for the adaptive control of breathing

The ability to amplify motor neuron (MN) output is essential for generating high intensity motor actions. This is critical for breathing that must be rapidly adjusted to accommodate changing metabolic demands. While brainstem circuits generate the breathing rhythm, the pathways that directly augment respiratory MN output are not well understood. Here, we mapped first-order inputs to phrenic motor neurons (PMNs), a key respiratory MN population that initiates diaphragm contraction to drive breathing. We identified a predominant spinal input from a distinct subset of genetically-defined V0C cholinergic interneurons. We found that these interneurons receive phasic excitation from brainstem respiratory centers, augment phrenic output through M2 muscarinic receptors, and are highly activated under a hypercapnia challenge. Specifically silencing cholinergic interneuron neurotransmission impairs the breathing response to hypercapnia. Collectively, our findings identify a novel spinal pathway that amplifies breathing, presenting a potential target for promoting recovery of breathing following spinal cord injury.

neuroscience↗

bioRxiv2024

Episodic rhythmicity is generated by a distributed neural network in the developing mammalian spinal cord

Spinal circuits produce diverse motor outputs that coordinate the rhythm and pattern of locomotor movements. Despite the episodic nature of these behaviours, the neural mechanisms encoding these episodes are not well understood. This study investigated mechanisms producing episodic rhythms evoked by dopamine in isolated neonatal mouse spinal cords. Dopamine-induced rhythms were primarily synchronous and propagated rostro-caudally across spinal segments, with occasional asynchronous episodes. Electrical stimulation of the L5 dorsal root could entrain episodes across segments, indicating afferent control of the rhythm generator and a distributed rostro-caudal network. Episodic activity was observed in isolated thoracic or sacral segments after full spinal transection or bilateral ventrolateral funiculus (VLF) lesions, suggesting a distributed network coupled via VLF projections. Rhythmicity was recorded from axons projecting through the VLF and dorsal roots, but not from cholinergic recurrent excitation via motoneurons or isolated dorsal inhibitory circuits. The data suggest episodic rhythmicity is generated by a flexibly coupled network of spinal interneurons distributed throughout the spinal cord.

neuroscience↗

bioRxiv2023

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 expression 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 PointsO_LIM-currents exert an inhibitory influence on spinal motor output. C_LIO_LIThis 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. C_LIO_LIPreferential control of fast motoneurons may be linked to a larger M-current that is activated within the subthreshold voltage range compared to slow motoneurons. C_LIO_LILarger M-currents in fast compared to slow motoneurons are not accounted for by differences in Kv7.2 or Kv7.3 channel composition. C_LIO_LIThe orderly recruitment of motoneuron subtypes is shaped by differences in the contribution of voltage-gated ion channels, including KCNQ channels. C_LIO_LIKCNQ channels may provide a target to dynamically modulate the recruitment gain across the motor pool and readily adjust movement vigour. C_LI

neuroscience↗

bioRxiv2022

Peptidergic modulation of motor neuron output via CART signaling at C bouton synapses

The intensity of muscle contraction, and therefore movement vigour, 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 novel roles for a neuropeptide, Cocaine and Amphetamine Regulated Transcript (CART), in the control of movement vigour. 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 novel spinal modulatory mechanisms that control movement vigour to support movements that require a high degree of muscle force.

neuroscience↗

bioRxiv2022

Synaptic Expression of TAR-DNA-Binding Protein 43 in the Mouse Spinal Cord Determined Using Super-Resolution Microscopy

Cellular inclusions of hyperphosphorylated TAR-DNA-Binding Protein 43 (TDP-43) are a key hallmark of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). ALS is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synaptic function that may be associated with TDP-43 pathology. However, there has been little characterisation of the synaptic expression of TDP-43 in 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 phosphorylated TDP-43 (pTDP-43) in spinal cord synapses. We observe that TDP-43 is expressed in the majority of spinal cord synapses as nanoscale clusters as small as 60 nm in diameter. Synaptic TDP-43 expression is more frequently associated with presynaptic terminals than postsynaptic densities, and is more enriched in VGLUT1-associated synapses, 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. This research characterizes 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.

neuroscience↗

bioRxiv2021

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.

neuroscience↗

bioRxiv2021

Maturation of persistent and hyperpolarization-activated inward currents shape the recruitment of motoneuron subtypes during postnatal development

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 become 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. Impact StatementElectrophysiological recordings from mouse spinal motoneurons reveal key roles for ion channels in establishing the differential activation of motoneuron subtypes during postnatal development.

neuroscience↗

bioRxiv2019

Monitoring contractility in single cardiomyocytes and whole hearts with bio-integrated microlasers

Cardiac regeneration and stem cell therapies depend critically on the ability to locally resolve the contractile properties of heart tissue1,2. Current regeneration approaches explore the growth of cardiac tissue in vitro and the injection of stem cell-derived cardiomyocytes3-6 (CMs) but scientists struggle with low engraftment rates and marginal mechanical improvements, leaving the estimated 26 million patients suffering from heart failure worldwide without effective therapy7-9. One impediment to further progress is the limited ability to functionally monitor injected cells as currently available techniques and probes lack speed and sensitivity as well as single cell specificity. Here, we introduce microscopic whispering gallery mode (WGM) lasers into beating cardiomyocytes to realize all-optical recording of transient cardiac contraction profiles with cellular resolution. The brilliant emission and high spectral sensitivity of microlasers to local changes in refractive index enable long-term tracking of individual cardiac cells, monitoring of drug administration, and accurate measurements of organ scale contractility in live zebrafish. Our study reveals changes in sarcomeric protein density as underlying factor to cardiac contraction which is of fundamental importance for understanding the mechano-biology of cardiac muscle activation. The ability to non-invasively assess functional properties of transplanted cells and engineered cardiac tissue will stimulate the development of novel translational approaches and the in vivo monitoring of physiological parameters more broadly. Likewise, the use of implanted microlasers as cardiac sensors is poised to inspire the adaptation of the most advanced optical tools known to the microresonator community, like quantum-enhanced single-molecule biosensing or frequency comb spectroscopy10.

biophysics↗