Long-term depression-associated signaling is required for an in vitro model of NMDA receptor-dependent synapse pruning

Maile A Henson,Charles J Tucker,Meilan Zhao,Serena M Dudek

doi:10.1016/j.nlm.2016.10.013

Abstract

Activity-dependent pruning of synaptic contacts plays a critical role in shaping neuronal circuitry in response to the environment during postnatal brain development. Although there is compelling evidence that shrinkage of dendritic spines coincides with synaptic long-term depression (LTD), and that LTD is accompanied by synapse loss, whether NMDA receptor (NMDAR)-dependent LTD is a required step in the progression toward synapse pruning is still unknown. Using repeated applications of NMDA to induce LTD in dissociated rat neuronal cultures, we found that synapse density, as measured by colocalization of fluorescent markers for pre- and postsynaptic structures, was decreased irrespective of the presynaptic marker used, post-treatment recovery time, and the dendritic location of synapses. Consistent with previous studies, we found that synapse loss could occur without apparent net spine loss or cell death. Furthermore, synapse loss was unlikely to require direct contact with microglia, as the number of these cells was minimal in our culture preparations. Supporting a model by which NMDAR-LTD is required for synapse loss, the effect of NMDA on fluorescence colocalization was prevented by phosphatase and caspase inhibitors. In addition, gene transcription and protein translation also appeared to be required for loss of putative synapses. These data support the idea that NMDAR-dependent LTD is a required step in synapse pruning and contribute to our understanding of the basic mechanisms of this developmental process.

Highlights

In many brain regions, synapse number initially increases and subsequently decreases over the course of postnatal development[1]
Repeated periods of long-term depression (LTD)-inducing stimulation can be accompanied by synapse loss[23,50], and bath application of NMDA can induce synaptic depression in slices and in culture preparations[29,32,33,34,48,55,62,69], as well as a rapid loss of dendritic spines[70]
Two applications of NMDA were required for reliable detection of synapse loss, though, as one application induced a much smaller loss that was less consistent across individual dendrites (Figure 2B2). the reduction in synapse density was apparent at two hours post-NMDA treatment and did not reverse or progress further at four hours posttreatment (ANOVA; two hours: F(2,17) = 10.38; p = 0.0011; n = 20 biological samples; four hours: F(2,9) = 14.81; p = 0.0014; n = 12 biological samples); or normalized to same day mock-treatment branches in control coverslips (ANOVA; two hours: F(2,222) = 34.15; p < 0.0001; n = 225 dendritic segments; four hours: F(2,277) = 38.92; p < 0.0001; n = 280 dendritic segments)

Summary

Introduction

Synapse number initially increases and subsequently decreases over the course of postnatal development[1]. These changes in synapse number represent a period of time during which the rate of synapse formation exceeds that of pruning, followed by a net loss of synapses as the rate of pruning overtakes that of synapse formation[2,3,4,5,6]. During early postnatal and adolescent development, this experience- and activity-dependent process is required for the refinement and proper functioning of neuronal circuits; disruption of synapse pruning can lead to dysfunction underlying some neurodevelopmental and psychiatric disorders[9,10,11]. A gene recently identified as a risk factor for schizophrenia, C4, encoding complement protein C4, has been linked to synapse elimination in the lateral geniculate nucleus[19]

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Conclusion