This study demonstrated the following. (1) HA treatment through chronic depolarization only transiently increased the firing rate of hippocampal neurons. (2) HA-treated neurons had a muted response to acute depolarization compared with control neurons, thus displaying a homeostatic regulation of excitability. Furthermore, inhibitory synaptic strength increased after HA through coordinated modifications of presynaptic and postsynaptic elements, and this increase was necessary for neuronal firing rate stability. (3)HA decreased the internalization rate of GABAA receptors, resulting in their accumulation on the surface membrane, and (4) synaptic strength increased when these receptors were confined to synapses in coordination with presynaptic modifications. These findings support the theory of homeostatic plasticity such that as neuronal excitatory activity increased, inhibitory synapse strength was scaled up to balance the overall firing rate of the neuron.
It is known that scaling of excitatory synaptic strength occurs in response to chronic changes in neuronal activity according to the hypothesis that neurons maintain a stable firing rate through mechanisms of homeostatic plasticity (Turrigiano, 1999
). Furthermore, it has been shown that neurons homeostatically scale synaptic strength in response to their own firing rate (Ibata et al., 2008
). A stable firing rate is the result of a balance between the natural fluctuations of inhibition and excitation in neural circuits. As previous studies have reported changes in synaptic strength occurring at both inhibitory and excitatory synapses in response to long-term depolarization (Leslie et al., 2001
; Peng et al., 2010
), we examined whether increased inhibitory synaptic strength corresponded to the homeostatic regulation of the firing rate in hippocampal cultures.
In hippocampal neurons, the stabilization of firing rate after HA demonstrated reduced excitability in the face of acute depolarization. Similar homeostatic stabilization of firing rate after HA induced by chronic bicuculline application was initially demonstrated in cortical neurons (Turrigiano et al., 1998
). However, in addition to a return to baseline firing rates, the current study found that this is accompanied by a diminished response to depolarizing high potassium challenge. This altered sensitivity to changing excitability indicates compensatory homeostatic processes occur in hippocampal neurons. As previously hypothesized, these homeostatic mechanisms allow neurons to maintain stable firing rates in the face of perturbations in activity that accompany, for example, developmental changes or Hebbian plasticity (Davis and Bezprozvanny, 2001
; Turrigiano and Nelson, 2004
; Nelson and Turrigiano, 2008
Accompanying the observation of firing rate stabilization during HA was an increased accumulation of postsynaptic GABAA receptors at the surface membrane and the subsequent incorporation of those receptors into synapses. Three lines of evidence suggested that this initial increase in postsynaptic GABAA receptors during HA was in part due to a diminished internalization rate. First, the increase in GABAA receptor surface expression after HA treatment was not accompanied by an increase in the total expression of GABAA receptors, suggesting that the accumulation of receptors at the surface was due to changes in trafficking of GABAA receptors and not to an increase in receptor transcription or synthesis. Second, when forward trafficking of new receptors from the ER was blocked, the decrease in mIPSC amplitude was smaller in the HA-treated neurons compared with control neurons, suggesting that a larger fraction of receptors remained at synapses during the brefeldin A treatment period in HA-treated neurons. The explanation that a greater fraction of receptors were confined to synapses in HA-treated neurons is also consistent with immunocytochemistry observations. A smaller brefeldin A-induced reduction of γ2 subunit cluster size was found in HA-treated neurons than in control neurons. Finally, the surface dwell time of GABAA receptors was longer in HA-treated neurons than in control neurons.
Mechanisms of receptor accumulation during long term changes in activity have been described at both glutamatergic synapses (O’Brien et al., 1998
; Ehlers, 2003
) and GABAergic synapses (Saliba et al., 2007
). In addition to receptor internalization, there are other mechanisms of GABAA
receptor trafficking that could be altered during HA to result in receptor accumulation at synapses. It is possible that homeostatic plasticity results in receptor internalization rate reaching a new equilibrium after a period of chronic HA. It would be important to understand how other mechanisms of receptor trafficking scale in response to this altered condition. Synaptic activity has been found to regulate the diffusion of both AMPA receptors (Ehlers et al., 2007
) and GABAA
receptors (Bannai et al., 2009
) between synaptic and extrasynaptic domains. Recent work has described activity-dependent alterations in GABAA
receptor diffusion dynamics and the stabilization of GABAA
receptors at synapses by scaffolding molecules such as gephyrin (Dumoulin et al., 2009
; Tyagarajan and Fritschy, 2010
). However, further investigation is needed to better understand how these mechanisms might regulate homeostatic plasticity of synaptic strength after chronic changes in activity.
Changes in postsynaptic GABAA receptor expression were accompanied by corresponding changes in the presynaptic marker GAD-65 as well (). Our results identify an initial increase in the number of postsynaptic receptors found at the synapse, followed by a slower increase in the size of presynaptic GAD-65 puncta. Presynaptic changes in transmitter release or uptake can affect the transmission of signaling across the synapse. Together, the data are consistent with the conclusion that a homeostatic response to chronic changes in neuronal activity occurs at inhibitory GABAergic synapses through coordinated presynaptic and postsynaptic changes that result in the scaling of synaptic strength.
Changes in mIPSC amplitude have been used previously as a measure of activity-dependent scaling of inhibitory synaptic strength (Hartman et al., 2006
; Swanwick et al., 2006a
), and presynaptic changes in neurotransmitter release have been suggested as a mechanism for decreasing mIPSC amplitude and frequency after chronic activity blockade (Hartman et al., 2006
). However, chronic activity blockade has been shown to reduce mIPSC amplitude but not frequency in cultures that have developed mature synaptic connections (Swanwick et al., 2006a
). The current study would suggest that reduced inhibitory synaptic strength likely results from accelerated internalization of receptors. Previous studies have shown that decreased activity by TTX-blockade and L-type voltage-gated calcium channels both lead to decreased surface expression of GABAA
receptor subunit β3 (Saliba et al., 2007
We found that mIPSC amplitude was larger after 12 h of HA treatment (). Furthermore, larger mIPSC amplitudes were observed only after the change in synaptic GABAA receptor clusters were seen at 6 h, suggesting surface expression of postsynaptic GABAA receptors increases before these receptors are incorporated into functional synapses. In addition, the size of GAD-65 puncta increases at 24 h of HA, earlier than the increase in mIPSC frequency found at 48 h of HA treatment. Altered GAD-65 labeling suggests that presynaptic changes have occurred at synapses, but these changes resulting in altered GAD-65 expression are not sufficient to drive increased probability of release. No change was found in the number of synaptic clusters, presynaptic puncta, or in the number of colocalized clusters. Therefore, the increase in probability of release that leads to an increase in the frequency of mIPSCs was not likely due to an increase in the number of synapses or an increase in the number of vesicle release sites. However, higher resolution imaging may be necessary to eliminate the possibility of an increase in the number of multiple receptor clusters colocalizing within larger presynaptic puncta.
Our data suggest that changes in synaptic strength arise from multiple homeostatic mechanisms occurring at different rates. The time scale of homeostatic change observed is similar to observations made at glutamatergic synapses under activity blockade (Turrigiano et al., 1998
). This similarity confirms that multiple mechanisms regulating homeostatic plasticity are globally scaling synaptic strength in coordination, thus preserving stable levels of excitability and the firing rate. We have shown here that the increase in GABAergic transmission at the synapse after HA is a necessary component to homeostatic plasticity. Application of a low concentration of bicuculline reduced GABAergic synaptic strength in HA-treated neurons to the level of control neurons. When this concentration was applied to spontaneously firing HA-treated neurons, the frequency of action potentials increased significantly, demonstrating that the increase in GABAergic synaptic strength was necessary to the maintenance of the homeostatically scaled firing rate.
At glutamatergic synapses, the expression of AMPA receptors have been found to homeostatically scale in response to changes in activity (O’Brien et al., 1998
; Wierenga et al., 2005
). Furthermore, homeostatic scaling of synaptic strength has been associated with similarly coordinated presynaptic and postsynaptic responses at excitatory synapses (Wierenga et al., 2006
). This coordinated process of synaptic modification could occur by retrograde signaling, which has been recently described for glutamatergic synapses through BDNF-TrkB signaling (Jakawich et al., 2010
). BDNF modulates activity-dependent scaling of GABAergic synaptic strength after chronic activity blockade as well (Swanwick et al., 2006a
), and has been identified as a potential messenger of retrograde signaling at GABAergic synapses after increases in activity (Peng et al., 2010
Studies investigating the time course of activity-dependent changes at excitatory synapses have also reported different temporal changes under different neuronal culture preparations (Turrigiano et al., 1998
; Ibata et al., 2008
). The role of growth factors from glial cells in culture is important to neuronal development in vitro
, and glial cytokine TNF-α has been shown to be important in synaptic scaling during both activity blockade in culture (Stellwagen and Malenka, 2006
) and during development in vivo
(Kaneko et al., 2008
). There is expanding knowledge of how factors like TNF-α and BDNF shape mechanisms of homeostatic plasticity in response to changes in activity. Differences in glial interaction during neuronal development could alter the processes that regulate homeostatic scaling at synapses. The recent report from Peng et al. describes changes in both amplitude and frequency of mIPSCs as early as 4 h into a HA treatment. Importantly, our finding that frequency of mIPSCs increased on a different time scale than mIPSC amplitude suggests that the overall increase in synaptic strength may not arise from a single mechanism or locus of change.
Furthermore, mIPSC decay was observed to change in HA-treated neurons, suggesting that the subunit composition of receptors at inhibitory synapses could also be altered with 48 h of HA treatment. Deactivation and desensitization properties of receptors on the postsynaptic membrane shape mIPSC kinetics (Jones and Westbrook, 1995
; Haas and Macdonald, 1999
; Bianchi et al., 2007
). The number of postsynaptic receptors clustering at a synapse can determine synaptic strength, and the subunit composition of these receptors can determine whether synapses are targeted to synapses (Essrich et al., 1998
receptor populations with varying subunit composition can have different trafficking characteristics, and differential expression of these receptor types during HA treatment may determine the changing properties of mIPSC kinetics seen as inhibitory synaptic strength increases. Subunit composition can also affect tonic inhibition through the distribution of extrasynaptic GABAA
receptors. There is evidence that an increase in extrasynaptic δ subunit-containing GABAA
receptors corresponds to decreased excitability during the estrous cycle in rats (Maguire et al., 2005
). In chronic epilepsy, a condition where action potential firing is not stably maintained by neuronal circuits, tonic inhibition is preserved while the expression of δ subunit-containing GABAA
receptors is decreased (Zhang et al., 2007
; Rajasekaran et al., 2010
). Future work will be needed to determine the relationship between the scaling of fast, synaptic GABAergic synaptic transmission during HA and the scaling of tonic inhibition in neurons. It also remains to be seen whether the changes in GABAergic synaptic strength after chronic high activity are similarly scaled over time in an in vivo
treatment. Kainate injection in rats was shown to induce increases in mIPSC amplitude and frequency as well BDNF protein level (Peng et al., 2010
). Similar to our findings, this suggests both postsynaptic and presynaptic mechanisms regulate in vivo
homeostatic scaling of GABAergic synapses in response to increased neuronal firing. A better understanding of how the trafficking of GABAA
receptors is altered with elevated activity in the intact brain is needed to understand the limits of homeostatic plasticity and its role in regulating neuronal activity in the healthy brain.
These data suggest that the temporal regulation of multiple mechanisms continues to occur at inhibitory synapses during HA, even after synaptic strength has ceased further increases and the firing rate has stabilized. Mechanisms beyond receptor accumulation are necessary for inhibitory synaptic scaling, as the initial increase in postsynaptic surface membrane receptors alone did not appear sufficient to increase inhibitory synaptic strength during HA. Our results predict that mechanisms of receptor trafficking lead to the accumulation of receptors targeted to synapses where corresponding presynaptic feedback is necessary for the homeostatic scaling of inhibitory synaptic strength and the regulation of the neuronal firing rate.