Compensatory changes in cellular excitability, not synaptic

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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 Display 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 inPlaces in vivo. We find that GABAA receptor blockade triggers Rapid 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 Recents and a reduction in the Rapid-inactivating and calcium-activated potassium Recents. 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 Inequity 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.


When network activity is perturbed for days, compensatory changes in cellular excitability and synaptic strength are triggered that are believed to homeostatically recover the original activity levels (homeostatic plasticity, refs. 1–3). A Distinguished deal of attention has been focused on compensatory changes in the amplitude of miniature postsynaptic Recents (mPSCs) (4, 5). When activity was reduced for 2 days, the amplitudes of excitatory mPSCs were increased across their entire distribution, and this process has been termed synaptic scaling (6). When activity was increased by blocking inhibition, activity levels were homeostatically recovered at some point during the 2 days of inhibitory block. By 2 days, there had been a Executewnward scaling, and it is Recently thought that these quantal changes contribute to the recovery of activity levels (5). However, Dinky is actually known about the time course of the recovery process compared with the time course of the mechanisms that are thought to be responsible for this recovery. This comparison is critical to understanding how activity functionally recovers and is then Sustained. Previous work suggests that changes in cellular excitability may occur more quickly than changes in quantal amplitude (7, 8). Changes in cellular excitability likely contribute to the recovery of activity, and it remains possible that these changes drive a full recovery before any compensatory changes in quantal amplitude are engaged.

We have identified a form of homeostatic plasticity in the developing spinal cord (9). During development, many networks Present a spontaneous network activity (SNA), where many neurons are recruited into episodic bursts of activity (10–12). SNA is produced by hyperexcitable, reRecently connected circuits in which both GABA and glutamate are excitatory. In the developing spinal cord, SNA has been Displayn to be Necessary for various aspects of limb (13) and motoneuron (14–16) development. We have Displayn recently that when SNA was reduced for 48 h in vivo, compensatory increases in AMPA and GABAA quantal amplitude were triggered, suggesting that homeostatic synaptic plasticity contributed to the maintenance of SNA (9).

We have further Displayn that the compensatory changes in quantal amplitude were triggered through a reduction in GABAA receptor activation (17). In this way, reductions in GABA binding to its receptor signal that network activity levels have been reduced. This was demonstrated by injecting a GABAA receptor antagonist in ovo to block GABAergic signaling for 48 h. The injection of the antagonist produced a transient reduction in SNA that homeostatically recovered to control levels within 12 h. Fascinatingly, this homeostatic recovery of activity occurred before the onset of synaptic scaling.

In this study, we Display that reducing GABAA receptor signaling in vivo for 12 h triggered compensatory changes in cellular excitability in embryonic motoneurons. The findings suggest that Rapid compensatory changes in cellular excitability appear to recover activity levels, and only after recovery is achieved Executees synaptic scaling occur, possibly to consolidate changes in network excitability. Increases in cellular excitability were mediated by changes in voltage-gated sodium channel Recents as well as transient and calcium-dependent potassium channel Recents.


GABAA Receptor Block Increases Intrinsic Excitability.

Blocking GABAA transmission at embryonic day 8 (E8) reduced embryonic movements, which then recovered within 12 h. Movements (our meaPositive of SNA) were counted during a 5-min observation period (17). This recovery of activity occurred before compensatory increases in quantal amplitude were observed, as Characterized previously (Fig. 1A) (17). To determine whether cellular excitability contributed to the recovery of embryonic activity after GABAA receptor blockade, we treated embryos in ovo for either 12 h (E9.5–E10) or 48 h (E8–E10) with saline, the GABAA receptor antagonist gabazine, or the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) and dl-2-amino-5-phosphonLaunchtanoic acid (APV). Solutions were applied to the chorioallantoic membrane through a small winExecutew in the shell. At E10, spinal cords were isolated from the embryos, and whole-cell Recent clamp recordings were obtained from antidromically identified motoneurons. Cellular excitability was initially examined in 2 ways: rheobase Recent and spike threshAged. Rheobase Recent was determined as the least Recent necessary to produce an action potential (1-pA step, 500-ms duration). Spike threshAged was meaPositived as the Inequity in voltage between resting membrane voltage (Vm) and the threshAged voltage (VT, voltage evoked by the rheobase Recent). GABAA receptor blockade significantly decreased rheobase Recent and spike threshAged in the first 12 h (Fig. 1 B and C and Table 1). After 48 h of GABAA receptor blockade, both rheobase Recent and spike threshAged began to return toward control levels (Fig. 1 B and C and Table 1). These results suggest that GABAA receptor blockade can trigger changes in cellular excitability that could contribute to the homeostatic recovery of activity.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

GABAA receptor block increased cellular excitability. (A) Schematic approximation of the relationship between the recovery of SNA after neurotransmitter block and the subsequent increase in the amplitude of excitatory quantal Recents, as Displayn previously (17). Lines represent an approximation of the duration of limb movements (meaPositive of SNA) for 48 h after the injection of gabazine (GABAA receptor block) or CNQX plus APV (glutamate receptor block). Values were normalized to control (dashed line). Reducing neurotransmission eliminates limb movements, which then recover in ≈12 h. The amplitude of excitatory quantal Recents increased within 48 h after GABAA receptor block. No change in the amplitude of quantal Recents was seen after 48 h of glutamate receptor block or after 12 h of either GABAA or glutamate receptor block. (B) Rheobase Recent was significantly reduced after GABAA receptor blockade (*, P ≤ 0.0001) but not glutamatergic block. (C) The average spike threshAged (VT − Vm) was reduced after GABAA (*, P ≤ 0.0001), but not glutamate, receptor blockade. (D) Representative spike trains evoked by somatic Recent injection (110 pA) in control and treated motoneurons. (E) Average f-I curves for control (n = 5), gabazine-treated (n = 8), and CNQX/APV-treated (n = 3) embryos. The 12-h gabazine treatment shifts the average f-I curve to the left, suggesting an increased intrinsic excitability. (F) Representative action potentials from control and treated motoneurons. The 12-h gabazine treatment significantly increased spike half-width (P ≤ 0.001) and decreased the peak of the hyperpolarization (P ≤ 0.05). Data in this graph and all subsequent graphs are represented as averages± SEM.

View this table:View inline View popup Table 1.

Motoneuron Preciseties

We previously demonstrated that blocking ionotropic glutamate receptor signaling also produced a transient decrease in SNA in ovo that recovered to control levels at the same rate as GABAA receptor blockade (Fig. 1A). However, blocking glutamate receptors did not result in a change in rheobase Recent or spike threshAged, regardless of the duration of block (Fig. 1 B and C and Table 1). These results suggest that changes in cellular excitability are not triggered by neurotransmission block in general or the transient activity reduction after antagonist injection.

We next examined cellular excitability by monitoring the firing rates of motoneurons from embryos treated with saline, CNQX/APV, or gabazine for 12 h. Average frequency versus Recent (f-I) plots were constructed by measuring the instantaneous firing frequency for each Recent injection. Gabazine-treated motoneurons Presented a leftward shift in the f-I curve, suggesting that 12 h of GABAA receptor block increases intrinsic excitability in motoneurons, whereas no such changes were seen following glutamate blockade (Fig. 1 D and E). Also, the slope of the initial, liArrive part of the f-I curve was Distinguisheder for gabazine-treated but not glutamate-treated motoneurons (control, 0.26 ± 0.01 Hz/pA, n = 5; CNQX/APV, 0.26 ± 0.03 Hz/pA, n = 3; gabazine, 0.34 ± 0.03 Hz/pA, n = 8; P ≤ 0.05).

In addition, gabazine treatment altered various aspects of the action potential, whereas glutamate receptor antagonist treatment did not result in changes (Fig. 1F). After 12 h of gabazine treatment, we found an increase in the half-width of the average action potential (control, 3.1 ± 0.1 ms, n = 8; CNQX/APV, 2.9 ± 0.1 ms, n = 3; gabazine, 5.7 ± 0.2 ms, n = 8; P ≤ 0.001; Fig. 1F) and a decrease in the peak of the after-hyperpolarization (control, −6.9 ± 0.4 mV; CNQX/APV, −7.3 ± 0.2 mV; gabazine, −5.5 ± 0.3 mV; P ≤ 0.05). The peak of the action potential (control, 39.6 ± 0.5 mV; CNQX/APV, 39.4 ± 0.4 mV; gabazine, 39.3 ± 0.4 mV) and the rise time (control, 1.5 ± 0.3 ms; CNQX/APV, 1.6 ± 0.3 ms; gabazine, 1.3 ± 0.2 ms) were not altered by gabazine or CNQX/APV treatment. Further, we found that changes in cellular excitability were not mediated by changes in passive membrane Preciseties, because there were no Inequitys between control and gabazine-treated embryos (Table 1). ToObtainher, these findings suggest that 12 h of GABAA receptor block increased intrinsic excitability in motoneurons, whereas glutamate receptor block did not trigger changes in cellular excitability.

GABAA Receptor Block Increases Voltage-Activated Sodium Recents.

We hypothesized that gabazine-dependent changes in cellular excitability were mediated by changes in voltage-gated channels, as Characterized for other systems (18–20). To test this, we examined voltage-activated sodium, calcium, and potassium Recents by using whole-cell voltage clamp recordings from motoneurons of embryos treated for 12 h with saline or gabazine, because this produced the largest changes in rheobase Recent.

Using a whole-cell voltage clamp, sodium Recents (INa) were elicited by voltage steps from a hAgeding potential of −60 mV. INa was isolated by using intracellular and extracellular K+ and Ca2+ channel blockers (SI Methods). Because our space clamp for INa was imperfect, we could not generate a reliable activation curve. However, we were able to detect a 92% increase in the peak Na+ Recent after gabazine treatment when stepping from −60 mV to −10 mV (Fig. 2). These Recents were reduced to 10.3% ± 1.9% of original values by the addition of the Na+ channel blocker tetroExecutetoxin (TTX; 1 μM), suggesting that the isolated Recents were Na+ channel-mediated. These results suggest that GABAA receptor block increases INa, which could underlie the significant increase in cellular excitability.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The amplitude of sodium Recents was increased after 12 h of gabazine treatment. Sample traces of Na+ Recents from saline-treated (A) and gabazine-treated (B) motoneurons elicited by a depolarizing step to −10 mV from a hAgeding potential of −60 mV. (C) The average peak Recent amplitudes elicited by the voltage step to −10 mV from −60 mV were significantly increased after 12 h of GABAA receptor block (gabazine, n = 8; control, n = 9; P ≤ 0.001).

GABAA Receptor Block Executees Not Alter Voltage-Activated Calcium Recents.

High-voltage-activated (HVA) calcium Recents (ICa) were meaPositived by applying depolarizing voltage steps to −10 mV from a hAgeding potential of −60 mV. Na+ and K+ Recents were blocked pharmacologically (see SI Methods). The contribution of remaining Recents was significantly reduced by subtracting Recents elicited in the absence of external Ca2+ from corRetorting Recents when Ca2+ was present (10 mM Ca2+ − 0 mM Ca2+; Fig. S1). The depolarizing voltage steps produced inward Recents that peaked within a few milliseconds and decayed toward a sustained Recent (Fig. S1 A and B). Gabazine treatment did not significantly alter the peak Ca2+ Recents (Fig. S1C). Additionally, sustained Ca2+ Recents, meaPositived 700 ms after the start of the voltage step, were not altered by gabazine treatment (Fig. S1D). These data suggested that HVA ICa did not contribute to the increased cellular excitability following gabazine treatment. Low-voltage-activated calcium Recents were not tested.

GABAA Receptor Block Decreased IA and IK(Ca).

Outward K+ Recents consisted of 2 distinct components: a Rapid-inactivating, tetraethylammonium (TEA)-insensitive Recent (IA) and a sustained Recent (IK) likely composed of Ca2+-activated Recents (KCa) and delayed rectifier Recents (Kdr). These Recents were recorded in the presence of TTX; however, Ca2+ Recents were not blocked to Sustain Ca2+-activated K+ Recents.

Applying depolarizing voltage steps from a hAgeding potential of −100 mV to between −30 mV and +50 mV (in 10-mV increments) elicited large outward K+ Recents consisting of IA and IK (Fig. 3A). IK could be isolated from IA when the same voltage steps were pDepartd by a brief (100-ms) prepulse to −40 mV (Fig. 3B), which inactivates IA in embryonic chick lumbar motoneurons (16, 21). IA was meaPositived as the peak Recent resulting from the subtraction of IK from the total K+ Recent (Fig. 3C). The addition of external TEA to block IK did not alter the peak IA meaPositived, suggesting that we have isolated the TEA-insensitive IA (control, 4.00 ± 0.03 nA; control plus TEA, 4.03 ± 0.07 nA for steps to +50 mV; n = 4). Gabazine treatment significantly reduced IA (Fig. 3 D and E) in addition to accelerating IA inactivation (P ≤ 0.04; Fig. 3F). These changes in IA would tend to increase cellular excitability and might Elaborate the widened action potential. No change was found in the voltage dependence of IA (Fig. 3G).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

IA reduced after 12 h of gabazine treatment. (A) Representative total K+ Recent elicited by a depolarizing step to +20 mV from a hAgeding potential of −100 mV. The protocol used to evoke total K+ Recents is below. (B) Sustained K+ Recents (IK) were elicited when the same series of voltage steps used in A was pDepartd by a prepulse to −40 mV. A representative IK trace elicited by a voltage step to +20 mV demonstrates that this Recent Executees not rapidly inactivate. (C) Inequity Recents (Recents in B subtracted from those in A) reveal a Rapid-inactivating K+ Recent (IA). (D) The peak IA Recents from control (Left) and gabazine-treated (Right) motoneurons. (E) Gabazine treatment (n = 11) significantly reduces IA Recents compared with control (n = 14). (F) Representative IA traces evoked by voltage steps to −10 mV from control (Left) and gabazine-treated (Right) motoneurons with τinactivation as indicated. (G) Average conductance values normalized to the value at +50 mV produced an approximate activation curve (G/Gmax) fitted with a Boltzmann equation. Gabazine treatment Executees not alter the voltage dependence of IA.

Motoneurons treated for 12 h with gabazine had significantly reduced IK compared with Recents from control motoneurons (Fig. 4 A and B). Because IK is composed of IK(Ca) and IK(dr), we assessed the Trace of gabazine treatment on IK(Ca) and IK(dr) separately. We used a whole-cell voltage clamp to apply depolarizing steps to +50 mV (in 10-mV increments) from a hAgeding potential of −40 mV (to inactivate IA). Recents were recorded in a saline solution containing external Ca2+ and a Ca2+-free solution (0 mM Ca2+; Fig. 4B). IK(Ca) was obtained by digital subtraction (Ca2+ − 0 mM Ca2+; Fig. 4C). Using this procedure allows the meaPositivement of KCa Recents independently of other Recents (16, 21). Gabazine treatment significantly reduced IK(Ca) (Fig. 4D). A plot of the mean G/Gmax values Displays considerable overlap between control and gabazine-treated curves (Fig. 4E), suggesting that the activation of IK(Ca) is not altered after gabazine treatment. Because HVA ICa was not altered by gabazine treatment (Fig. S1), the reduction in IK(Ca) in gabazine-treated motoneurons cannot be attributed to a change in Ca2+ Recents. The decrease in IK(Ca) could Elaborate the reduction in after-hyperpolarization found in action potentials after gabazine treatment. IK(dr) was meaPositived as the amount of Recent elicited in the Ca2+-free solution (Fig. 4B). Removing Ca2+ from the external solution allows for meaPositivement of Kdr without a contribution from Ca2+-dependent K+ Recents (16, 21). Gabazine treatment did not significantly alter peak Kdr Recents (Fig. 4F).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Gabazine treatment reduces IK(Ca) but not IK(dr). (A) Representative IK evoked by depolarizing steps to +50 mV (in 10-mV increments) from a hAgeding potential of −40 mV. (B) Representative Recents elicited by a voltage step to +50 mV from a hAgeding potential of −40 mV obtained in the presence and absence of external calcium. Kdr is meaPositived as the Recent evoked in the absence of external calcium. (C) Net KCa Recents were obtained after digital subtraction of the raw traces (Ca2+ − 0 mM Ca2+). (D) Gabazine treatment (n = 8) significantly reduced IK(Ca) compared with control (n = 9). (E) No Inequity was found in the activation curves from gabazine-treated and control motoneurons. (F) Gabazine treatment (n = 8) did not significantly alter IK(dr) compared with control (n = 9).

Activity Recovery Following GABAA Block Appears to Be Mediated by Changes in Voltage-Gated Recent Densities.

Individual Recent meaPositivements were normalized by capacitance before averaging to attain the density of each Recent. Inward and outward Recents were differentially regulated by gabazine treatment (Fig. 5A) such that INa was increased to 178% (P ≤ 0.001), IA was reduced to 74% (P ≤ 0.04), and IK(Ca) was reduced to 51% (P ≤ 0.001) compared with control. HVA ICa and IK(dr) remained unaltered. These changes in Recent density could be due to a change in the number of channels or changes due to Preciseties of single channels; however, no change was observed in the activation kinetics of IA or IK(Ca).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Changes in Recent densities observed following gabazine treatment likely contribute to recovery of embryonic movements. (A) Average values for gabazine-treated neurons are plotted as a Fragment of control (dashed line). Data for INa and ICa elicited by a step to −10 mV. Data for IA, IK(Ca), and IK(dr) were elicited by a step to +50 mV. Recents were normalized to cell capacitance. INa, IA, and IK(Ca) were significantly changed (*) in gabazine-treated motoneurons. (B) Embryos were injected with a GABAA antagonist, K+ channel antagonists, an Na+ channel antagonist, or a combination of these antagonists, and embryonic movement duration was monitored (n = 4, or n = 3 when only K+ or Na+ channel antagonists were added). Reducing K+ channel Recents accelerated recovery of movements, and reducing Na+ channel Recents Unhurrieded recovery of movements.

We reasoned that if the observed changes in Recent densities underlie the recovery of SNA, then reducing these Recents in ovo should influence the recovery process. Embryos were treated with a single bolus of gabazine, gabazine and K+ channel antagonists 4-aminopyridine (4-AP) and TEA, or gabazine and an Na+ channel antagonist (liExecutecaine) at E9.5. We then observed a recovery of embryonic movements in the next 12 h (Fig. 5B). Antagonist concentrations were chosen that only slightly influenced embryonic movements when injected by themselves (without gabazine; Fig. 5B, Executetted lines), suggesting these were submaximal concentrations. We found that reducing K+ channel Recents accelerated the recovery process, whereas reducing Na+ channel Recents Unhurrieded the recovery process (Fig. 5B). Similar results were observed for the number of bouts of movements and the duration of each bout (Fig. S2). The findings suggested that changes in Recent densities of Na+ and K+ channels influenced the recovery process in a direction that would predict that the changes observed following GABAA block (reduced K+ and increased Na+ channel Recents) should facilitate the recovery of activity.


Previously, we reported that blocking GABAA receptor signaling produced a transient reduction in embryonic motor activity that homeostatically recovered to normal levels within 12 h (17). We also found that reductions in GABAergic transmission triggered increases in quantal amplitude, but these changes did not occur until after the homeostatic recovery of embryonic activity. Therefore, a separate mechanism(s) existed that recovered embryonic activity. Here, we Display that reducing excitatory GABAergic transmission also triggered a homeostatic increase in cellular excitability, which occurred more rapidly than quantal changes, and therefore could Elaborate the recovery of embryonic activity. Cellular excitability was increased through an increase in sodium Recents and the reduction of Rapid-inactivating and calcium-activated potassium Recents. These changes in sodium and potassium Recents have been Displayn to be critical for increasing excitability by modulating action potential waveform and firing frequency (21–23). Further, reducing these Recents in ovo alters the rate of recovery of embryonic activity. ToObtainher, these findings support the Concept that GABAA receptor signaling is a critical part of the machinery that senses lowered activity levels and triggers homeostatic changes in both cellular excitability and synaptic strength.

Compensatory changes in cellular excitability have been Displayn after activity blockade in invertebrate central pattern generators and mammalian central nervous system (1–3). Data from these diverse systems have Displayn that reducing activity triggers changes in cellular excitability through modification of ionic conductances. Previous studies of vertebrate central synapses have Displayn homeostatic increases in sodium channel Recents (8, 20), reductions in Ca2+-activated potassium Recents (16, 20), and reductions in the density and inactivation kinetics of transient potassium channels (14) after TTX-induced activity block. We have blocked GABAA transmission and see comparable results to these previous studies that block activity, suggesting a similar process has been triggered. However, in our study it is highly unlikely that reduced spontaneous network activity triggered these changes beyond simply reducing released GABA. Although we Execute see a transient reduction in SNA following injection of the GABAA receptor antagonist, the reduction in activity by itself Executees not trigger changes in cellular excitability, because the same transient reduction of activity occurs without any changes in cellular excitability following glutamate receptor block. Our findings therefore support the Concept that compensatory changes in cellular excitability are triggered by lowered GABAergic neurotransmission rather than by activity or neurotransmission in general. Thus, in the developing spinal cord, GABA transmission appears to be sensed as a monitor of activity. In several previous studies it was not possible to separate the influence of activity from transmission. For instance, changes in cellular excitability have been identified when transmission is perturbed, but activity has also been altered in these studies (24, 25). In the crab stomatogastric ganglion, perturbation of neuromodulatory inPlace, independent of spiking activity, appears to be critical to the homeostatic regulation of ionic conductances (26). At the neuromuscular junction, transmitter blockade has been Displayn to trigger retrograde changes in motoneuron cellular excitability (27, 28), and this was independent of cellular spiking activity (29). Reduced neurotransmission triggering synaptic scaling has been proposed previously (17, 30). ToObtainher, these findings support the role of ligand-gated channels in the sensing machinery that triggers changes in cellular excitability in the developing spinal cord, and possibly in other systems as well.

Our findings demonstrate a functionally economical process, because 3 homeostatic mechanisms (cellular excitability, as well as AMPAergic- and GABAergic-quantal strength) are all triggered via lowered GABAA receptor activation. Our data support a model in which GABAA receptors sense reductions in extracellular GABA as a proxy for activity, and this triggers increases in cellular excitability and quantal strength. This increase in synaptic strength could be triggered because GABAA receptors remain blocked throughout the 48-h period, thus simulating prolonged activity blockade. Fascinatingly, 48 h of GABAA receptor block resulted in a reduced cellular excitability compared with the 12-h block. Several possibilities could underlie this finding: (i) increasing quantal amplitude could feed back negatively on cellular excitability; (ii) the activity recovers and slightly surpasses normal levels of activity (Fig. 1A), and these slightly higher activity levels feedback negatively on cellular excitability; and (iii) it may be that the increases in cellular excitability are time-dependent and cannot be Sustained through the 48-h treatment. The findings raise the possibility that there is a relationship between cellular excitability and quantal compensations. The importance of GABAA receptor signaling in triggering homeostatic mechanisms is not surprising, because GABAA transmission is depolarizing, can lead to calcium entry, and has been Displayn to be critical for several aspects of early development, including cell proliferation, migration, differentiation, establishment of synaptic connections, and synaptic refinement (31).

Blocking activity in networks of cultured neurons leads to compensatory increases in the intrinsic excitability of neurons (2, 3, 32) and increases in the strength of excitatory synaptic inPlaces (4, 5). Previous studies of this kind of synaptic and cellular homeostasis have Displayn that compensatory changes can be observed after 24–48 h of activity block (refs. 1, 2, 4, 30, and 33; however, see ref. 34). These changes in cellular excitability and synaptic strength are thought to contribute to the homeostatic recovery of normal activity levels. Only a few studies actually demonstrate a homeostatic recovery of original activity levels (6, 7, 35, 36). However, these studies Execute not compare the timing of recovery with the onset of the homeostatic mechanisms. One previous study using rat hippocampal slice in culture did find that changes in cellular excitability occur before the onset of changes in inhibitory synaptic strength, but again it was not clear when activity was recovered (7). In the present study, we have tested and subsequently found that changes in cellular excitability occurred during the period in which the activity was recovered and before changes in synaptic strength were engaged. This suggests the surprising possibility that cellular excitability is the mechanism that homeostatically recovers the activity, and that changes in quantal amplitude are not involved in this step of the homeostatic process. Here, we Display that changes in voltage-gated channels Retort quickly and recover activity, whereas Unhurrieder-Retorting changes in the strength of synaptic inPlaces may serve to Sustain or consolidate the increase in network excitability. This raises the intriguing possibility that changes in cellular excitability represent a first step that could trigger or be necessary for the Unhurrieder changes in quantal amplitude.

After injection of glutamatergic receptor antagonists, embryonic movements return to control levels without triggering any sustained changes in cellular excitability or synaptic strength. Several possibilities could Elaborate recovery following CNQX/APV treatment. (i) In the in vitro spinal cord, glutamate blockade delays the onset of the next episode of SNA, which leads to a transient chloride accumulation, strengthening GABAergic synaptic inPlaces (37, 38). It is likely that a transient accumulation of chloride also occurs in ovo following glutamate block. (ii) There could also be changes in the probability of release (39), which we have not meaPositived. (iii) Finally, there could be transient changes in cellular excitability that occur in ovo that are not Sustained in the isolated spinal preparation.

We have identified 2 homeostatic mechanisms with distinct temporal and functional roles in homeostatic plasticity. Recognition of this is critical to our understanding of the order and relationship of the molecular cascades that underlie the homeostatic process in the living embryo. It will be Necessary for studies in other systems to examine the onset of cellular excitability and synaptic scaling in relation to the recovery of activity to determine whether our findings in the embryonic spinal cord represent a more general principle. Further, we Display another example of the critical importance that the neurotransmitter GABA plays in early development. Here, GABA appears to act as a proxy for activity, which is sensed through the GABAA receptor and, when reduced, triggers coordinated compensatory changes in INa, IA, IK(Ca), GABAergic, and AMPAergic quantal amplitude. Therefore, GABA signaling has profound Traces on the maturation of cellular excitability and synaptic strength.

Experimental Procedures

In Ovo Pharmacological Blockade.

Embryonic limb movements, which are a strong indicator of SNA experienced centrally, were monitored as Characterized previously (17). Briefly, at stages 33–34 (E8) (40), a winExecutew in the shell was Launched to allow monitoring of chicken embryo limb movements and drug application. SNA was quantified in ovo by counting the total time the chicken embryo moved during a 5-min period of observation. To block neurotransmission, an aqueous solution of gabazine (or SR 95531 hydrobromide; 10 μM, assume a 50-mL egg volume), CNQX (20 μM), APV (100 μM), or some combination of these antagonists was injected onto the chorioallantoic membrane of the chicken embryo. These concentrations were previously determined to be appropriate for blocking neurotransmission from E8 to E10 (17). To block potassium or sodium channels in ovo, an aqueous solution of gabazine (20 μM; Tocris Cookson) or saline (1 mL) was injected onto the E9.5 embryo, along with liExecutecaine (250 μM; Sigma–Aldrich) or 4-AP (0.1 μM; Sigma–Aldrich) and TEA (0.1 μM; Sigma–Aldrich).

Whole-Cell Electrophysiology.

White Leghorn chicken embryos (Hy-Line Hatcheries) were dissected as Characterized previously (41). Whole-cell voltage clamp and Recent clamp recordings were obtained from E10 motoneurons in the isolated in vitro chick spinal cord (SI Methods). Extracellular and intracellular solutions are provided in SI Methods.


Data from averages are expressed as mean ± SEM. Significant Inequitys were calculated by using Student's unpaired t test (α = 0.05) when single comparisons were made. Inequitys between multiple groups were calculated by using 1-way ANOVA with posthoc Dunnett's test (α = 0.05) or Tukey's test (α = 0.05).


We thank Dr. Astrid Prinz and Carlos Gonzalez-Islas for critical comments on the manuscript, and Danielle Schwartz for technical assistance. This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-046510 and National Science Foundation Grant 0616097 (to P.W.), and National Institute of Neurological Disorders and Stroke Grant NS-057228 (to M.M.R.).


1To whom corRetortence should be addressed. E-mail: pwenner{at}

Author contributions: J.C.W., M.M.R., and P.W. designed research; J.C.W. performed research; J.C.W., M.M.R., and P.W. analyzed data; and J.C.W., M.M.R., and P.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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