Elsevier

Neuropharmacology

Volume 158, 1 November 2019, 107700
Neuropharmacology

Non-scaling regulation of AMPA receptors in homeostatic synaptic plasticity

https://doi.org/10.1016/j.neuropharm.2019.107700Get rights and content

Highlights

  • Synaptic AMPAR amounts are regulated at various proportions during HSP

  • Changes of AMPAR at individual synapses do not follow a scaling pattern during HSP

  • The basal synaptic AMPAR level is inversely correlated to the extent of HSP response


  • Neighboring synapses compete for AMPAR during HSP


Abstract

Homeostatic synaptic plasticity (HSP) as an activity-dependent negative feedback regulation of synaptic strength plays important roles in the maintenance of neuronal and neural circuitry stability. A primary cellular substrate for HSP expression is alterations in synaptic accumulation of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). It is widely believed that during HSP, AMPAR accumulation changes with the same proportion at each synapse of a neuron, a process known as synaptic scaling. However, direct evidence on AMPAR synaptic scaling remains largely lacking. Here we report a direct examination of inactivity-induced homeostatic scaling of AMPAR at individual synapse by live-imaging. Surprisingly, instead of uniform up-scaling, a scattered pattern of changes in synaptic AMPAR was observed in cultured rat hippocampal neurons. While the majority of synapses showed up-regulation after activity inhibition, a reduction of AMPAR could be detected in certain synapses. More importantly, among the up-regulated synapses, a wide range of AMPAR changes was observed in synapses of the same neuron. We also found that synapses with higher levels of pre-existing AMPAR tend to be up-regulated by lesser extents, whereas the locations of synapses relative to the soma seem not affecting AMPAR scaling strengths. In addition, we observed strong competition between neighboring synapses during HSP. These results reveal that synaptic AMPAR may not be scaled during HSP, suggesting novel molecular mechanisms for information processing and storage at synapses.

Introduction

In neurons, homeostatic plasticity serves to maintain a stable firing rate of action potentials. This can be achieved through adjustments in the strength of synaptic activity, neuronal excitability, neural cells connectivity, or the balance of excitation and inhibition (Turrigiano 2008, 2011; Pozo and Goda, 2010; Vitureira et al., 2012; Fernandes and Carvalho, 2016; Matsubara and Uehara, 2016). Among these possibilities, regulation of synaptic strength has been the most extensively studied and is believed to be the most crucial measure in homeostatic regulation, also known as homeostatic synaptic plasticity (HSP) (Turrigiano et al., 1998; Davis and Bezprozvanny, 2001; Davis, 2006; Shepherd et al., 2006; Stellwagen and Malenka, 2006; Turrigiano, 2008; Hou et al 2011, 2015). In addition to its roles in maintaining normal physiological brain functions, the contributions of HSP to neurological and psychiatric diseases, such as Alzheimer's disease and autism, have also been gradually revealed in recent years (Pratt et al., 2011; Blackman et al., 2012; Gilbert et al., 2016; Jewett et al., 2018; Lee et al., 2018; Styr and Slutsky, 2018).

At the cellular level, inactivity-dependent homeostatic plasticity causes a simultaneous up-regulation of AMPAR at all synapses (Stellwagen and Malenka, 2006; Sutton et al., 2006; Aoto et al., 2008; Hou et al., 2008; Ibata et al., 2008; Pozo and Goda, 2010; Vitureira et al., 2012; Wang et al., 2012; Soares et al., 2013; Diering and Huganir, 2018). It has been proposed that during the expression of homeostatic regulation, the amount of AMPAR added to each synapse is not by chance; rather, the response at individual synapse follows a common rule. In previous studies of HSP, the amplitudes of mEPSCs were often analyzed by cumulative plotting that ranks the miniature synaptic currents from the smallest to the largest (Turrigiano et al., 1998; Wierenga et al., 2005; Stellwagen and Malenka, 2006; Turrigiano, 2008). The multiplicative pattern of the cumulative curves is thought to be a result from proportional increases in transmission strength of all synapses, namely, synaptic scaling. Synaptic scaling is considered an important cellular mechanism by which the relative strength of individual synaptic input can be maintained following homeostatic plasticity. It has been hypothesized that synaptic scaling of mEPSCs is due to the proportional addition of AMPAR to the entire synapse population of a neuron (Ibata et al., 2008; Pozo and Goda, 2010; Turrigiano, 2008). However, this hypothesis, though of fundamental significance, has not been validated by direct experimental evidence. We therefore directly examined AMPAR synaptic scaling in cultured neurons by a series of live-imaging experiments to compare changes of synaptic AMPAR amounts during HSP. To our surprise, we found that while AMPAR amount was increased in most synapses after neural activity deprivation, AMPAR accumulation did not show the expected pattern of scaling. Instead, among the synapse population, the extents of synaptic AMPAR increases showed a wide range of variation during HSP.

Section snippets

Live-imaging shows homeostatic up-regulation of synaptic AMPAR induced by neural activity deprivation

In order to analyze AMPAR accumulation at single synapse during HSP, we performed live-imaging on cultured hippocampal neurons expressing GFP-tagged (GFP-GluA1, GFP-GluA2) or pHluorin-tagged (pH-GluA1) AMPAR subunits, respectively. Two-wk-old hippocampal neurons were transfected for 3 days to express the target protein before experiments (Fig. 1A). Immunostaining of a synaptic marker PSD-95 indicated that the GFP-GluA1/2 or pH-GluA1 was successfully expressed and localized at the synaptic sites

Discussion

By utilizing high-resolution live-imaging of GFP-tagged AMPAR subunits, we analyzed the homeostatic up-regulation of AMPARs at individual synapses. Most importantly, we found that during HSP, changes in AMPAR synaptic accumulation, while showing up-regulation in most synapses, did not scale up uniformly among the synapse population. Under activity deprivation by APV + TTX treatment, individual AMPAR puncta at the synapses of a neuron show a wide range of changes, including down-regulation,

Acknowledgements

We would like to thank Dr. Margaret Hastings for comments on the manuscript and the Man Lab members for helpful discussion. This work was supported by NIH grant R01 MH079407 (H.Y.M.) and International Fulbright Sci & Tech Outstanding Student Award (G.W.). The authors declare no conflicts of financial interests.

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