Peter Wenner

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.

Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent “Hypothesis and Theory Article,” the excitatory action of GABA in early brain development is suggested to be “an experimental artefact” (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

Sensing and expressing homeostatic synaptic plasticity

Chronic changes in the level of neuronal activity (over a period of days) trigger compensatory changes in synaptic function that seem to contribute to the homeostatic restoration of neuronal activity. Changes in both quantal amplitude and vesicle release contribute to homeostatic synaptic plasticity, but they are often considered as the same phenomenon. In this review, we propose a new approach to studying how neuronal activity is sensed and changes in synaptic function are expressed during synaptic compensation. Changes in quantal amplitude and vesicle release should be considered separately in an attempt to identify the sensors that trigger homeostatic synaptic plasticity. Although data are limited, current evidence suggests that the sensors triggering changes in the quantal amplitude and vesicle release exist at different locations. Furthermore, it is important to recognize that at least two different mechanisms underlie changes in quantal amplitude during homeostatic synaptic plasticity: changes in both the number of postsynaptic receptors and loading of synaptic vesicles with neurotransmitter. Finally, modulation of the probability of neurotransmitter release contributes to the changes in vesicle release associated with homeostatic synaptic plasticity. An improved understanding of where and how neuronal activity is sensed, in addition to the types of changes in synaptic function that are induced, will be needed both to design future experiments and to understand the consequences of synaptic compensation following injury to the nervous system.

Mechanisms of Spontaneous Activity in the Developing Spinal Cord and Their Relevance to Locomotion

Abstract: The isolated lumbosacral cord of the chick embryo generates spontaneous episodes of rhythmic activity. Muscle nerve recordings show that the discharge of sartorius (flexor) and femorotibialis (extensor) motoneurons alternates even though the motoneurons are depolarized simultaneously during each cycle. The alternation occurs because sartorius motoneuron firing is shunted or voltage‐clamped by its synaptic drive at the time of peak femorotibialis discharge. Ablation experiments have identified a region dorsomedial to the lateral motor column that may be required for the alternation of sartorius and femorotibialis motoneurons. This region overlaps the location of interneurons activated by ventral root stimulation. Whole‐cell recordings from interneurons receiving short latency ventral root input indicate that they fire at an appropriate time to contribute to the cyclical pause in firing of sartorius motoneurons. Spontaneous activity was modeled by the interaction of three variables: network activity and two activity‐dependent forms of network depression. A “slow” depression which regulates the occurrence of episodes and a “fast” depression that controls cycling during an episode. The model successfully predicts several aspects of spinal network behavior including spontaneous rhythmic activity and the recovery of network activity following blockade of excitatory synaptic transmission.

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

During early development, gamma-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (EGABA) for GABAA miniature postsynaptic currents. When whole cell patch solutions contained 17-52 mM chloride, we found that synaptic EGABA was around -30 mV. Because of the low HCO3- permeability of the GABAA receptor, this value of EGABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na+-K+-2Cl- cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized EGABA and reduced nerve potentials evoked by local application of a GABAA agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO3- and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

Dye screening and signal-to-noise ratio for retrogradely transported voltage-sensitive dyes

Using a novel method for retrogradely labeling specific neuronal populations, we tested different styryl dyes in attempt to find dyes whose staining would be specific, rapid, and lead to large activity dependent signals. The dyes were injected into the ventral roots of the isolated chick spinal cord from embryos at days E9-E12. The voltage-sensitive dye signals were recorded from synaptically activated motoneurons using a 464 element photodiode array. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. Over the 24 h period we examined, these dyes bound specifically to the cells with axons in the ventral roots. The dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic depolarization of the motoneurons. The signal-to-noise ratio obtained in a single trial from a detector that received light from a 14 x 14 microns2 area of the motoneuron population was about 10:1. Nonetheless, signals on neighboring diodes were similar, suggesting that we were not detecting the activity of individual neurons. Retrograde labeling and optical recording with voltage-sensitive dyes provides a means for monitoring the activity of identified neurons in situations where microelectrode recordings are not feasible.

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 GABAA 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 GABAA 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.

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.

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 GABAA transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABAA 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 GABAA 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 GABAA receptor. Therefore, GABA appears to be serving as a proxy for activity levels.

Imaging the spatiotemporal organization of neural activity in the developing spinal cord

In this review, we discuss the use of imaging to visualize the spatiotemporal organization of network activity in the developing spinal cord of the chick embryo and the neonatal mouse. We describe several different methods for loading ion‐ and voltage‐sensitive dyes into spinal neurons and consider the advantages and limitations of each one. We review work in the chick embryo, suggesting that motoneurons play a critical role in the initiation of each cycle of spontaneous network activity and describe how imaging has been used to identify a class of spinal interneuron that appears to be the avian homolog of mammalian Renshaw cells or 1a‐inhibitory interneurons. Imaging of locomotor‐like activity in the neonatal mouse revealed a wave‐like activation of motoneurons during each cycle of discharge. We discuss the significance of this finding and its implications for understanding how locomotor‐like activity is coordinated across different segments of the cord. In the last part of the review, we discuss some of the exciting new prospects for the future.