The Uniform and Nonuniform Nature of Slow and Rapid Scaling in Embryonic Motoneurons.

Neurons regulate the strength of their synapses in response to a perturbation to stabilize neuronal signaling through a form of homeostatic plasticity known as synaptic scaling. The process of scaling has the potential to alter all of a cell's miniature postsynaptic current (mPSC) amplitudes by a single multiplicative factor (uniform scaling), and in doing so could change action potential-dependent or evoked synaptic strength by that factor. However, recent studies suggest that individual synapses scale with different scaling factors (nonuniform). This could complicate the simple multiplicative transform from mPSC scaling to the evoked response. We have previously identified a slow AMPAergic and GABAergic synaptic scaling in chick embryo motoneurons following 2 d in vivo perturbations inhibiting neuronal activity or GABAAR function, and now show a rapid form of scaling following NMDAR blockade in vitro Slow GABAergic scaling appeared to be of a classical uniform pattern. Alternatively, other forms of rapid and slow scaling demonstrated a uniform and nonuniform component in their mPSC amplitude distributions. Slow and rapid AMPAergic scaling was mediated by insertion of GluA2-lacking AMPA receptors. The nonuniform pattern of scaling may contribute to the observed complexity of the changes in evoked responses. Scaling-induced changes in mPSC amplitudes were not associated with changes in probability of release (Pr). Together, our results demonstrate a new rapid form of scaling in embryonic motoneurons, that slow and rapid scaling is not purely uniform, and that upscaling does not translate to an increase in evoked responses in a simple manner.SIGNIFICANCE STATEMENT Different forms of homeostatic plasticity are thought to play a critical role in maintaining neural function. For example, altering the amplitudes of spontaneous currents through a form of homeostatic plasticity known as synaptic scaling could affect evoked transmission; however, this is rarely tested. Here we demonstrate two forms of scaling and show that in many cases synaptic strength scales differently for distinct synapses within an embryonic motoneuron. These results have functional consequences for evoked synaptic strength and suggest that, like Hebbian plasticity, scaling can change relative synaptic strengths within a cell. Furthermore, our results demonstrate how different forms of homeostatic plasticity influence neuronal communication as the nascent spinal network is first established in the embryonic period.

Divergent Synaptic Scaling of Miniature EPSCs following Activity Blockade in Dissociated Neuronal Cultures.

Neurons can respond to decreased network activity with a homeostatic increase in the amplitudes of miniature EPSCs (mEPSCs). The prevailing view is that mEPSC amplitudes are uniformly multiplied by a single factor, termed "synaptic scaling." Deviations from purely multiplicative scaling have been attributed to biological differences, or to a distortion imposed by a detection threshold limit. Here, we demonstrate in neurons dissociated from cortices of male and female mice that the shift in mEPSC amplitudes observed in the experimental data cannot be reproduced by simulation of uniform multiplicative scaling, with or without the distortion caused by applying a detection threshold. Furthermore, we demonstrate explicitly that the scaling factor is not uniform but is close to 1 for small mEPSCs, and increases with increasing mEPSC amplitude across a substantial portion of the data. This pattern was also observed for previously published data from dissociated mouse hippocampal neurons and dissociated rat cortical neurons. The finding of "divergent scaling" shifts the current view of homeostatic plasticity as a process that alters all synapses on a neuron equally to one that must accommodate the differential effect observed for small versus large mEPSCs. Divergent scaling still accomplishes the essential homeostatic task of modifying synaptic strengths in the opposite direction of the activity change, but the consequences are greatest for those synapses which individually are more likely to bring a neuron to threshold.SIGNIFICANCE STATEMENT In homeostatic plasticity, the responses to chronic increases or decreases in network activity act in the opposite direction to restore normal activity levels. Homeostatic plasticity is likely to play a role in diseases associated with long-term changes in brain function, such as epilepsy and neuropsychiatric illnesses. One homeostatic response is the increase in synaptic strength following a chronic block of activity. Research is focused on finding a globally expressed signaling pathway, because it has been proposed that the plasticity is uniformly expressed across all synapses. Here, we show that the plasticity is not uniform. Our work suggests that homeostatic signaling molecules are likely to be differentially expressed across synapses.

Regulation of synaptic scaling by action potential-independent miniature neurotransmission.

Synaptic scaling represents a homeostatic adjustment in synaptic strength that was first identified as a cell-wide mechanism to achieve firing rate homeostasis after perturbations to spiking activity levels. In this review, we consider a form of synaptic scaling that is triggered by changes in action potential-independent neurotransmitter release. This plasticity appears to be both triggered and expressed locally at the dendritic site of the synapse that experiences a perturbation. A discussion of different forms of scaling triggered by different perturbations is presented. We consider work from multiple groups supporting this form of scaling, which we call neurotransmission-based scaling. This class of homeostatic synaptic plasticity is compared in studies using hippocampal and cortical cultures, as well as in vivo work in the embryonic chick spinal cord. Despite differences in the tissues examined, there are clear similarities in neurotransmission-based scaling, which appear to be molecularly distinct from the originally described spike-based scaling.

Spontaneous Release Regulates Synaptic Scaling in the Embryonic Spinal Network In Vivo.

UNLABELLED: Homeostatic plasticity mechanisms maintain cellular or network spiking activity within a physiologically functional range through compensatory changes in synaptic strength or intrinsic cellular excitability. Synaptic scaling is one form of homeostatic plasticity that is triggered after blockade of spiking or neurotransmission in which the strengths of all synaptic inputs to a cell are multiplicatively scaled upward or downward in a compensatory fashion. We have shown previously that synaptic upscaling could be triggered in chick embryo spinal motoneurons by complete blockade of spiking or GABAA receptor (GABAAR) activation for 2 d in vivo Here, we alter GABAAR activation in a more physiologically relevant manner by chronically adjusting presynaptic GABA release in vivo using nicotinic modulators or an mGluR2 agonist. Manipulating GABAAR activation in this way triggered scaling in a mechanistically similar manner to scaling induced by complete blockade of GABAARs. Remarkably, we find that altering action-potential (AP)-independent spontaneous release was able to fully account for the observed bidirectional scaling, whereas dramatic changes in spiking activity associated with spontaneous network activity had little effect on quantal amplitude. The reliance of scaling on an AP-independent process challenges the plasticity's relatedness to spiking in the living embryonic spinal network. Our findings have implications for the trigger and function of synaptic scaling and suggest that spontaneous release functions to regulate synaptic strength homeostatically in vivo SIGNIFICANCE STATEMENT: Homeostatic synaptic scaling is thought to prevent inappropriate levels of spiking activity through compensatory adjustments in the strength of synaptic inputs. Therefore, it is thought that perturbations in spike rate trigger scaling. Here, we find that dramatic changes in spiking activity in the embryonic spinal cord have little effect on synaptic scaling; conversely, alterations in GABAA receptor activation due to action-potential-independent GABA vesicle release can trigger scaling. The findings suggest that scaling in the living embryonic spinal cord functions to maintain synaptic strength and challenge the view that scaling acts to regulate spiking activity homeostatically. Finally, the results indicate that fetal exposure to drugs that influence GABA spontaneous release, such as nicotine, could profoundly affect synaptic maturation.

Elevated intracellular Na+ concentrations in developing spinal neurons.

Over 25 years ago it was first reported that intracellular chloride levels (Cl-in ) were higher in developing neurons than in maturity. This finding has had significant implications for understanding the excitability of developing networks and recognizing the underlying causes of hyperexcitability associated with disease and neural injury. While there is some evidence that intracellular sodium levels (Na+in ) change during the development of non-neural cells, it has largely been assumed that Na+in is the same in developing and mature neurons. Here, using the sodium indicator SBFI, we test this idea and find that Na+in is significantly higher in embryonic spinal motoneurons and interneurons than in maturity. We find that Na+in reaches ~ 60 mM in mid-embryonic development and is then reduced to ~ 30 mM in late embryonic development. By retrogradely labeling motoneurons with SBFI we can reliably follow Na+in levels in vitro for hours. Bursts of spiking activity, and blocking voltage-gated sodium channels did not influence observed motoneuron sodium levels. On the other hand, Na+in was reduced by blocking the Na+ -K+ -2Cl- cotransporter NKCC1, and was highly sensitive to changes in external Na+ and a blocker of the Na+ /K+ ATPase. Our findings suggest that the Na+ gradient is weaker in embryonic neuronal development and strengthens in maturity in a manner similar to that of Cl- .

In vivo synaptic scaling is mediated by GluA2-lacking AMPA receptors in the embryonic spinal cord.

When spiking activity within a network is perturbed for hours to days, compensatory changes in synaptic strength are triggered that are thought to be important for the homeostatic maintenance of network or cellular spiking activity. In one form of this homeostatic plasticity, called synaptic scaling, all of a cell's AMPAergic miniature postsynaptic currents (mEPSCs) are increased or decreased by some scaling factor. Although synaptic scaling has been observed in a variety of systems, the mechanisms that underlie AMPAergic scaling have been controversial. Certain studies find that synaptic scaling is mediated by GluA2-lacking calcium receptors (CP-AMPARs), whereas others have found that scaling is mediated by GluA2-containing calcium-impermeable receptors (CI-AMPARs). Spontaneous network activity is observed in most developing circuits, and in the spinal cord this activity drives embryonic movements. Blocking spontaneous network activity in the chick embryo by infusing lidocaine in vivo triggers synaptic scaling in spinal motoneurons; here we show that AMPAergic scaling occurs through increases in mEPSC conductance that appear to be mediated by the insertion of GluA2-lacking AMPA receptors at the expense of GluA2-containing receptors. We have previously reported that in vivo blockade of GABAA transmission, at a developmental stage when GABA is excitatory, also triggered AMPAergic synaptic scaling. Here, we show that this form of AMPAergic scaling is also mediated by CP-AMPARs. These findings suggest that AMPAergic scaling triggered by blocking spiking activity or GABAA receptor transmission represents similar phenomena, supporting the idea that activity blockade triggers scaling by reducing GABAA transmission.

GABAergic synaptic scaling is triggered by changes in spiking activity rather than AMPA receptor activation.

Homeostatic plasticity represents a set of mechanisms that are thought to recover some aspect of neural function. One such mechanism called AMPAergic scaling was thought to be a likely candidate to homeostatically control spiking activity. However, recent findings have forced us to reconsider this idea as several studies suggest AMPAergic scaling is not directly triggered by changes in spiking. Moreover, studies examining homeostatic perturbations in vivo have suggested that GABAergic synapses may be more critical in terms of spiking homeostasis. Here, we show results that GABAergic scaling can act to homeostatically control spiking levels. We found that perturbations which increased or decreased spiking in cortical cultures triggered multiplicative GABAergic upscaling and downscaling, respectively. In contrast, we found that changes in AMPA receptor (AMPAR) or GABAR transmission only influence GABAergic scaling through their indirect effect on spiking. We propose that GABAergic scaling represents a stronger candidate for spike rate homeostat than AMPAergic scaling.

Tonic and transient endocannabinoid regulation of AMPAergic miniature postsynaptic currents and homeostatic plasticity in embryonic motor networks.

Endocannabinoid signaling has been shown to mediate synaptic plasticity by retrogradely inhibiting presynaptic transmitter release in several systems. We found that endocannabinoids act tonically to regulate AMPA miniature postsynaptic current (mPSC) frequency in embryonic motor circuits of the chick spinal cord. Further, strong postsynaptic depolarizations also induced a short-lived endocannabinoid-mediated suppression of mEPSC frequency. Unlike many previous studies, endocannabinoid signaling was not found to influence evoked transmitter release. The results suggest a special role for spontaneous glutamatergic mPSCs and their control by endocannabinoids in the developing spinal cord. We determined that blocking endocannabinoid signaling, which increases spontaneous glutamatergic release, increased spontaneous network activity in vitro and in vivo. Previous work in spinal motoneurons had shown that reducing spontaneous network activity (SNA) chronically in vivo led to homeostatic increases in AMPA and GABA mPSC amplitude (homeostatic synaptic plasticity). Blocking endocannabinoid signaling in vivo, and thus increasing SNA, triggered compensatory decreases of both AMPA and GABA mPSC amplitudes. These findings, combined with previous results, are consistent with the idea that this form of homeostatic synaptic plasticity is a bidirectional process in the living embryo. Together, our results suggest a role for tonic signaling of endocannabinoids as a potential mechanism to regulate the level of SNA, which is known to be critical for synaptic maturation in the embryonic spinal cord.

Plasticity in Preganglionic and Postganglionic Neurons of the Sympathetic Nervous System during Embryonic Development.

Sympathetic preganglionic neurons (SPNs) are the final output neurons from the central arm of the autonomic nervous system. Therefore, SPNs represent a crucial component of the sympathetic nervous system for integrating several inputs before driving the postganglionic neurons (PGNs) in the periphery to control end organ function. The mechanisms which establish and regulate baseline sympathetic tone and overall excitability of SPNs and PGNs are poorly understood. The SPNs are also known as the autonomic motoneurons (MNs) as they arise from the same progenitor line as somatic MNs that innervate skeletal muscles. Previously our group has identified a rich repertoire of homeostatic plasticity (HP) mechanisms in somatic MNs of the embryonic chick following in vivo synaptic blockade. Here, using the same model system, we examined whether SPNs exhibit similar homeostatic capabilities to that of somatic MNs. Indeed, we found that after 2-d reduction of excitatory synaptic input, SPNs showed a significant increase in intracellular chloride levels, the mechanism underlying GABAergic synaptic scaling in this system. This form of HP could therefore play a role in the early establishment of a setpoint of excitability in this part of the sympathetic nervous system. Next, we asked whether homeostatic mechanisms are expressed in the synaptic targets of SPNs, the PGNs. In this case we blocked synaptic input to PGNs in vivo (48-h treatment), or acutely ex vivo, however neither treatment induced homeostatic adjustments in PGN excitability. We discuss differences in the homeostatic capacity between the central and peripheral component of the sympathetic nervous system.

Compensatory changes in cellular excitability, not synaptic scaling, contribute to homeostatic recovery of embryonic network activity.

When neuronal activity is reduced over a period of days, compensatory changes in synaptic strength and/or cellular excitability are triggered, which are thought to act in a manner to homeostatically recover normal activity levels. The time course over which changes in homeostatic synaptic strength and cellular excitability occur are not clear. Although many studies show that 1-2 days of activity block are necessary to trigger increases in excitatory quantal strength, few studies have been able to examine whether these mechanisms actually underlie recovery of network activity. Here, we examine the mechanisms underlying recovery of embryonic motor activity following block of either excitatory GABAergic or glutamatergic inputs in vivo. We find that GABA(A) receptor blockade triggers fast changes in cellular excitability that occur during the recovery of activity but before changes in synaptic scaling. This increase in cellular excitability is mediated in part by an increase in sodium currents and a reduction in the fast-inactivating and calcium-activated potassium currents. These findings suggest that compensatory changes in cellular excitability, rather than synaptic scaling, contribute to activity recovery. Further, we find a special role for the GABA(A) receptor in triggering several homeostatic mechanisms after activity perturbations, including changes in cellular excitability and GABAergic and AMPAergic synaptic strength. The temporal difference in expression of homeostatic changes in cellular excitability and synaptic strength suggests that there are multiple mechanisms and pathways engaged to regulate network activity, and that each may have temporally distinct functions.

Homeostatic Regulation of Motoneuron Properties in Development.

Homeostatic plasticity represents a set of compensatory mechanisms that are engaged following a perturbation to some feature of neuronal or network function. Homeostatic mechanisms are most robustly expressed during development, a period that is replete with various perturbations such as increased cell size and the addition/removal of synaptic connections. In this review we look at numerous studies that have advanced our understanding of homeostatic plasticity by taking advantage of the accessibility of developing motoneurons. We discuss the homeostatic regulation of embryonic movements in the living chick embryo and describe the spinal compensatory mechanisms that act to recover these movements (homeostatic intrinsic plasticity) or stabilize synaptic strength (synaptic scaling). We describe the expression and triggering mechanisms of these forms of homeostatic plasticity and thereby gain an understanding of their roles in the motor system. We then illustrate how these findings can be extended to studies of developing motoneurons in other systems including the rodents, zebrafish, and fly. Furthermore, studies in developing drosophila have been critical in identifying some of the molecular signaling cascades and expression mechanisms that underlie homeostatic intrinsic membrane excitability. This powerful model organism has also been used to study a presynaptic form of homeostatic plasticity where increases or decreases in synaptic transmission are associated with compensatory changes in probability of release at the neuromuscular junction. Further, we describe studies that demonstrate homeostatic adjustments of ion channel expression following perturbations to other kinds of ion channels. Finally, we discuss work in xenopus that shows a homeostatic regulation of neurotransmitter phenotype in developing motoneurons following activity perturbations. Together, this work illustrates the importance of developing motoneurons in elucidating the mechanisms and roles of homeostatic plasticity.

GABAergic synaptic scaling in embryonic motoneurons is mediated by a shift in the chloride reversal potential.

Homeostatic synaptic plasticity ensures that networks maintain specific levels of activity by regulating synaptic strength in a compensatory manner. When spontaneous network activity was blocked in vivo in the embryonic spinal cord, compensatory increases in excitatory GABAergic synaptic inputs were observed. This homeostatic synaptic strengthening was observed as an increase in the amplitude of GABAergic miniature postsynaptic currents. We find that this process is mediated by an increase in chloride accumulation, which produces a depolarizing shift in the GABAergic reversal potential (E(GABA)). The findings demonstrate a previously unrecognized mechanism underlying homeostatic synaptic scaling. Similar shifts in E(GABA) have been described following various forms of neuronal injury, introducing the possibility that these shifts in E(GABA) represent a homeostatic response.

GABAA transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude.

When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABA(A) transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABA(A) transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABA(A) receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABA(A) receptor. Therefore, GABA appears to be serving as a proxy for activity levels.

Mechanisms of GABAergic homeostatic plasticity.

Homeostatic plasticity ensures that appropriate levels of activity are maintained through compensatory adjustments in synaptic strength and cellular excitability. For instance, excitatory glutamatergic synapses are strengthened following activity blockade and weakened following increases in spiking activity. This form of plasticity has been described in a wide array of networks at several different stages of development, but most work and reviews have focussed on the excitatory inputs of excitatory neurons. Here we review homeostatic plasticity of GABAergic neurons and their synaptic connections. We propose a simplistic model for homeostatic plasticity of GABAergic components of the circuitry (GABAergic synapses onto excitatory neurons, excitatory connections onto GABAergic neurons, cellular excitability of GABAergic neurons): following chronic activity blockade there is a weakening of GABAergic inhibition, and following chronic increases in network activity there is a strengthening of GABAergic inhibition. Previous work on GABAergic homeostatic plasticity supports certain aspects of the model, but it is clear that the model cannot fully account for some results which do not appear to fit any simplistic rule. We consider potential reasons for these discrepancies.

Isolation and Electrophysiology of Murine Sympathetic Postganglionic Neurons in the Thoracic Paravertebral Ganglia.

The thoracic paravertebral sympathetic chain postganglionic neurons (tSPNs) represent the predominant sympathetic control of vascular function in the trunk and upper extremities. tSPNs cluster to form ganglia linked by an interganglionic nerve and receive multisegmental convergent and divergent synaptic input from cholinergic sympathetic preganglionic neurons of the spinal cord (Blackman and Purves, 1969; Lichtman et al., 1980 ). Studies in the past have focused on cervical and lumbar chain ganglia in multiple species, but few have examined the thoracic chain ganglia, whose location and diminutive size make them less conducive to experimentation. Seminal studies on the integrative properties of preganglionic axonal projections onto tSPNs were performed in guinea pig (Blackman and Purves, 1969; Lichtman et al., 1980 ), but as mice have become the accepted mammalian genetic model organism, there is need to reproduce and expand on these studies in this smaller model. We describe an ex vivo approach that enables electrophysiological, calcium imaging, and optogenetic assessment of convergence, divergence, and studies on pre- to postganglionic synaptic transmission, as well as whole-cell recordings from individual tSPNs. Preganglionic axonal connections from intact ventral roots and interganglionic nerves across multiple segments can be stimulated to evoke compound action potential responses in individual thoracic ganglia as recorded with suction electrodes. Chemical block of synaptic transmission differentiates spiking of preganglionic axons from synaptically-recruited tSPNs. Further dissection, including removal of the sympathetic chain, enables whole-cell patch clamp recordings from individual tSPNs for characterization of cellular and synaptic properties.

Mitochondrial Structure and Polarity in Dendrites and the Axon Initial Segment Are Regulated by Homeostatic Plasticity and Dysregulated in Fragile X Syndrome.

Mitochondrial dysfunction has long been overlooked in neurodevelopmental disorders, but recent studies have provided new links to genetic forms of autism, including Rett syndrome and fragile X syndrome (FXS). Mitochondria show plasticity in morphology and function in response to neuronal activity, and previous research has reported impairments in mitochondrial morphology and function in disease. We and others have previously reported abnormalities in distinct types of homeostatic plasticity in FXS. It remains unknown if or how activity deprivation triggering homeostatic plasticity affects mitochondria in axons and/or dendrites and whether impairments occur in neurodevelopmental disorders. Here, we test the hypothesis that mitochondria are structurally and functionally modified in a compartment-specific manner during homeostatic plasticity using a model of activity deprivation in cortical neurons from wild-type mice and that this plasticity-induced regulation is altered in Fmr1-knockout (KO) neurons. We uncovered dendrite-specific regulation of the mitochondrial surface area, whereas axon initial segment (AIS) mitochondria show changes in polarity; both responses are lost in the Fmr1 KO. Taken together, our results demonstrate impairments in mitochondrial plasticity in FXS, which has not previously been reported. These results suggest that mitochondrial dysregulation in FXS could contribute to abnormal neuronal plasticity, with broader implications to other neurodevelopmental disorders and therapeutic strategies.

FMRP attenuates activity dependent modifications in the mitochondrial proteome.

Homeostatic plasticity is necessary for the construction and maintenance of functional neuronal networks, but principal molecular mechanisms required for or modified by homeostatic plasticity are not well understood. We recently reported that homeostatic plasticity induced by activity deprivation is dysregulated in cortical neurons from Fragile X Mental Retardation protein (FMRP) knockout mice (Bulow et al. in Cell Rep 26: 1378-1388 e1373, 2019). These findings led us to hypothesize that identifying proteins sensitive to activity deprivation and/or FMRP expression could reveal pathways required for or modified by homeostatic plasticity. Here, we report an unbiased quantitative mass spectrometry used to quantify steady-state proteome changes following chronic activity deprivation in wild type and Fmr1-/y cortical neurons. Proteome hits responsive to both activity deprivation and the Fmr1-/y genotype were significantly annotated to mitochondria. We found an increased number of mitochondria annotated proteins whose expression was sensitive to activity deprivation in Fmr1-/y cortical neurons as compared to wild type neurons. These findings support a novel role of FMRP in attenuating mitochondrial proteome modifications induced by activity deprivation.

The Type 2 Diabetes Factor Methylglyoxal Mediates Axon Initial Segment Shortening and Alters Neuronal Function at the Cellular and Network Levels.

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or location along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment. To determine the key type 2 diabetes-related factor that produces AIS shortening we modified levels of insulin, glucose, or the reactive glucose metabolite methylglyoxal in cultures of dissociated cortices from male and female mice and quantified AIS geometry using immunofluorescent imaging of the AIS proteins AnkyrinG and ßIV spectrin. Neither insulin nor glucose modification altered AIS length. Exposure to 100 but not 1 or 10 µm methylglyoxal for 24 h resulted in accumulation of the methylglyoxal-derived advanced glycation end-product hydroimidazolone and produced reversible AIS shortening without cell death. Methylglyoxal-evoked AIS shortening occurred in both excitatory and putative inhibitory neuron populations and in the presence of tetrodotoxin (TTX). In single-cell recordings resting membrane potential was depolarized at 0.5-3 h and returned to normal at 24 h. In multielectrode array (MEA) recordings methylglyoxal produced an immediate ∼300% increase in spiking and bursting rates that returned to normal within 2 min, followed by a ∼20% reduction of network activity at 0.5-3 h and restoration of activity to baseline levels at 24 h. AIS length was unchanged at 0.5-3 h despite the presence of depolarization and network activity reduction. Nevertheless, these results suggest that methylglyoxal could be a key mediator of AIS shortening and disruptor of neuronal function during type 2 diabetes.

Spontaneous network activity in the embryonic spinal cord regulates AMPAergic and GABAergic synaptic strength.

Spontaneous network activity (SNA) has been described in most developing circuits, including the spinal cord, retina, and hippocampus. Despite the widespread nature of this developmental phenomenon, its role in network maturation is poorly understood. We reduced SNA in the intact embryo and found compensatory increases in synaptic strength of spinal motoneuron inputs. AMPAergic miniature postsynaptic current (mPSC) amplitude and frequency increased following the reduction of activity. Interestingly, excitatory GABAergic mPSCs also increase in amplitude through a process of synaptic scaling. Finally, the normal modulation of GABAergic mPSC amplitude was accelerated. Together, these compensatory responses appear to increase the excitability of the cord and could act to maintain appropriate SNA levels, thus demonstrating a distinct functional role for synaptic homeostasis. Because spontaneous network activity can regulate AMPAergic and GABAergic synaptic strength during development, SNA is likely to play an important role in a coordinated maturation of excitatory and inhibitory synaptic strength.

Homeostatic Recovery of Embryonic Spinal Activity Initiated by Compensatory Changes in Resting Membrane Potential.

When baseline activity in a neuronal network is modified by external challenges, a set of mechanisms is prompted to homeostatically restore activity levels. These homeostatic mechanisms are thought to be profoundly important in the maturation of the network. It has been shown that blockade of either excitatory GABAergic or glutamatergic transmission in the living chick embryo transiently blocks the movements generated by spontaneous network activity (SNA) in the spinal cord. However, the embryonic movements then begin to recover by 2 h and are completely restored by 12 h of persistent receptor blockade. It remains unclear what mechanisms mediate this early recovery (first hours) after neurotransmitter blockade, or even if the same mechanisms are triggered following GABAergic and glutamatergic antagonists. Here we find two distinct mechanisms that could underlie this homeostatic recovery. First, we see a highly robust compensatory mechanism observed shortly after neurotransmitter receptor blockade. In the first 2 h of GABAergic or glutamatergic blockade in vitro, there was a clear depolarization of resting membrane potential (RMP) in both motoneurons and interneurons. These changes reduced threshold current and were observed in the continued presence of the antagonist. Therefore, it appears that fast changes in RMP represent a key fast homeostatic mechanism for the maintenance of network activity. Second, we see a less consistent compensatory change in the absolute threshold voltage in the first several hours of in vitro and in vivo neurotransmitter blockade. These mechanisms likely contribute to the homeostatic recovery of embryonic movements following neurotransmitter blockade.