Bi-directional control of motor neuron dendrite remodeling by the calcium permeability of AMPA receptors

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Abstract

Motor neurons express particularly high levels of the AMPA receptor subunit GluR1(Q)flip (GluR1(Q)i) during the period in early postnatal life when their dendritic tree grows and becomes more branched. To investigate how GluR1-containing AMPA receptors contribute to dendrite morphogenesis, we characterized a mutant form of GluR1 (containing a histidine in the Q/R editing site) with unique electrophysiological properties. Most notably, AMPA receptors assembled from GluR1(H)i display less calcium permeability than AMPA receptors assembled from GluR1(Q)i. Expression of GluR1(Q)i in vivo or in vitro led to an increase in dendrite branching with no net change in the overall tree size while GluR1(H)i led to a loss of branches and a net reduction in overall tree size. GluR1(H)i-dependent dendrite atrophy is mediated by protein phosphatase 2B. The results suggest that the electrophysiological properties of cell surface AMPA receptors, specifically their permeability to calcium, can be a central determinant of whether the dendrites undergo activity-dependent branching or atrophy.

Introduction

Neurons receive a broad spatio-temporal array of electrical and chemical signals that are processed by dendrites and transmitted to other neurons via patterned action potential output (Hausser et al., 2000). The factors that impact upon the computational work of dendrites include the size and geometrical arrangement of branches, the molecular composition of particular domains of the tree (Helmchen, 1999) and the quantitative and qualitative nature of afferent inputs (Hume and Purves, 1981, Purves and Lichtman, 1985, Spruston et al., 1999). The mechanisms by which dendrites acquire their mature features occur over an extended period in pre- and early postnatal life (Scott and Luo, 2001). The early events in tree elaboration are likely to be guided by biochemical processes that are not affected by synaptic activity, and subsequently activity-dependent mechanisms sculpt dendrites into their final form (Goodman and Shatz, 1993). Why these events are largely confined to the young nervous system is not well understood but it is likely to be, in part, a function of the unique molecular composition of immature neurons (Nedivi et al., 1996).

Glutamate receptors mediate the majority of excitatory neurotransmission within the vertebrate central nervous system (CNS) so it might be thought, a priori, that they are key molecular elements in activity-dependent plasticity. Consistent with this idea, developing spinal motor neurons express very high levels of the GluR1i subunit of the AMPA-type glutamate receptor (Jakowec et al., 1995a, Jakowec et al., 1995b), coinciding with their period of activity-dependent dendrite growth (Kalb, 1994). Large-scale changes in the motor neuron dendritic tree do not occur after the third postnatal week of life, a time when the abundance of GluR1 in motor neurons is low or absent. Re-introduction of GluR1 into motor neurons using a recombinant HSV from postnatal day 23 (P23) to P28 leads to a remodeling of dendritic architecture (Inglis et al., 2002) with increased branching but no alteration in the overall tree size. These findings support the notion that the glutamate receptor phenotype of neurons is a determinant of activity-dependent refinement of dendritic architecture.

The prominent effects of GluR1 on the architecture of motor neuron dendrites depend on the amino acid occupying the “Q/R” editing site (Inglis et al., 2002). Expression of GluR1(Q)i, but not GluR1(R)i, by motor neurons leads to an enhancement of dendrite branching and shortening of segment lengths. GluR1(Q)i is the physiological version of the protein expressed during development, and while arginine normally occupies the Q/R editing site in GluR2, GluR1(R)i is not present in the normal spinal cord. Upon activation, AMPA receptors composed of GluR1(Q)i are calcium-permeable and display an inwardly rectifying current–voltage (IV) relationship. AMPA receptors composed of GluR1(R)i are essentially calcium impermeable and display a linear IV relationship (Inglis et al., 2002). The experiments described herein aim to gain further insight into how the electrophysiological signature of glutamate receptors regulates dendrite remodeling.

Due to hetero- and homo-oligomerization of AMPA receptor subunits, individual neurons (and in all likelihood individual synapses) express AMPA receptors with a range of electrophysiological properties. We focused on the calcium permeability of AMPA receptors and in particular, the effects on dendrites of AMPA receptors with calcium permeability intermediate between those assembled from GluR1(Q)i versus GluR1(R)i. Would AMPA receptors with intermediate calcium permeability lead to a graded effect on dendrites (a “quantitative effect”-less branches than GluR1(Q)i-expressing motor neurons but more than GluR1(R)i-expressing motor neurons)? Or would an AMPA receptor with intermediate calcium permeability lead to a phenotypically distinct effect on dendritic architecture? To simply address this issue, we needed a GluR1 subunit that upon assembly into a functional AMPA receptor would yield receptor-channels with a calcium permeability less than those assembled from GluR1(Q)i and more than GluR1(R)i. While this would not precisely match the natural situation (wherein calcium permeability of AMPA receptors is controlled by the relative proportion of edited GluR2 in hetero-oligomeric assemblies Geiger et al., 1995, Jonas et al., 1994a), it would enable us to approach the question: what are the effects on dendrite architecture of expression of AMPA receptors with differing calcium permeabilities?

A report in 1992 by Curutchet et al. suggested that substituting a histidine into the Q/R editing site might yield a subunit which, when assembled into AMPA receptors, would be calcium permeable and potentially less so than AMPA receptors assembled from GluR1(Q)i (Curutchet et al., 1992). These investigators used an indirect measure of calcium permeability (the ability to evoke a chloride current via Ca2+-activated Cl channels) and the observations were qualitative. In the present report, we directly measure and quantify the calcium permeability of GluR1(H)i-containing AMPA receptors and establish that it is intermediate between those assembled from GluR1(R)i and GluR1(Q)i. When GluR1(H)i is expressed in motor neurons, in vivo or in vitro, it leads to a reduction in the overall size of the dendritic arbor and a loss of branches, the opposite effect seen with GluR1(Q)i. These effects are likely to be mediated by the calcium-activated phosphatase PP2B. These novel and unexpected effects on dendrite morphology might be linked to calcium-dependent synaptic plasticity.

Section snippets

Characterization of GluR1 isoforms in oocytes

The Q/R site has been shown in vivo and in vitro to control the rectification properties as well as the Ca2+ permeabilities of the GluR1–GluR4 and GluR6 ion pores in physiological extracellular solutions (Egebjerg and Heinemann, 1993, Hume et al., 1991, Verdoorn et al., 1991). The electrophysiological properties of the GluR1(H)i variant, however, which had been described as uncoupling Ca2+ permeability from rectification properties (Curutchet et al., 1992), have not been characterized in

Discussion

The present study begins to explore the link between specific electrophysiological properties of GluR1-containing AMPA receptors and their morphogenic effects on motor neuron dendrites. Previously, we found that transgenic expression of wild type GluR1 (containing a Q in the Q/R editing site) in mature motor neurons leads to increases in branching while not affecting the overall size of the dendritic tree (Inglis et al., 2002). Here we studied a mutant version of GluR1 (containing a histidine

Study design

The aim of these investigations was to explore the relationship between the electrophysiological properties of GluR1-containing AMPA receptors and dendrite remodeling. To accomplish this, we expressed variants of GluR1 after the naturally occurring critical period of activity-dependent development (beyond P21), when the dendritic tree is morphologically stable and there is little or no endogenous GluR1 expression (Inglis et al., 2002). In these in vivo studies, motor neurons expressed the

Acknowledgments

This work was supported by grants from the National Institutes of Health (NS29837) and NASA (NAG2-951) to R.G.K. The authors thank Dr. Fiona Inglis for critical commentary early in this project.

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