Abstract

In contrast to the prenatal topographic development of sensory cortices, striatal circuit organization is slow and requires the functional maturation of cortical and thalamic excitatory inputs throughout the first postnatal month. While mechanisms regulating synapse development and plasticity are quite well described at excitatory synapses of glutamatergic neurons in the neocortex, comparatively little is known of how this translates to glutamate synapses onto GABAergic neurons in the striatum. Here we investigate excitatory striatal synapse plasticity in an in vitro system, where glutamate can be studied in isolation from dopamine and other neuromodulators. We examined pre-and post-synaptic structural and functional plasticity in GABAergic striatal spiny projection neurons (SPNs), co-cultured with glutamatergic cortical neurons. After synapse formation, medium-term (24 h) TTX silencing increased the density of filopodia, and modestly decreased dendritic spine density, when assayed at 21 days in vitro (DIV). Spine reductions appeared to require residual spontaneous activation of ionotropic glutamate receptors. Conversely, chronic (14 days) TTX silencing markedly reduced spine density without any observed increase in filopodia density. Time-dependent, biphasic changes to the presynaptic marker Synapsin-1 were also observed, independent of residual spontaneous activity. Acute silencing (3 h) did not affect presynaptic markers or postsynaptic structures. To induce rapid, activity-dependent plasticity in striatal neurons, a chemical NMDA receptor-dependent “long-term potentiation (LTP)” paradigm was employed. Within 30 min, this increased spine and GluA1 cluster densities, and the percentage of spines containing GluA1 clusters, without altering the presynaptic signal. The results demonstrate that the growth and pruning of dendritic protrusions is an active process, requiring glutamate receptor activity in striatal projection neurons. Furthermore, NMDA receptor activation is sufficient to drive glutamatergic structural plasticity in SPNs, in the absence of dopamine or other neuromodulators.

Highlights

  • The striatum is a highly integrative structure

  • While striatal neurons develop poorly and have low viability in mono-culture (Segal et al, 2003; Kaufman et al, 2012; Burguière et al, 2013), those co-cultured with cortical neurons develop complex dendritic arbors and spines that stabilize around DIV20, and exhibit both morphological and electrophysiological properties resembling striatal projection neuron (SPN) in vivo (Segal et al, 2003; Tian et al, 2010; Randall et al, 2011; Kaufman et al, 2012; Milnerwood et al, 2012; Burguière et al, 2013; Lalchandani et al, 2013; Penrod et al, 2015)

  • To verify the nucleofection of isolated striatal cells before mixing in co-culture, and ensure correct visual identification of SPNs by the experimenter based on fluorescent fills, we quantified the co-expression of BFP plasmid-nucleofected striatal neurons in cultures prepared from germ-line SPN marker mice; for Drd1 dopamine receptor (D1R) SPNs we used Drd1a-tdTomato reporter mice, and for Drd2 dopamine receptor (D2R) SPNs we used Drd2-eGFP reporter mice

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Summary

Introduction

Each of the ∼2.5 million GABAergic medium-sized spiny projection neurons (SPNs) receives ∼25,000 glutamate afferents from most areas of the cortex and thalamus (Kincaid et al, 1998; Doig et al, 2010). These are modulated by nigrostriatal dopamine and form the only striatal output pathways (Tritsch and Sabatini, 2012). We developed assays to investigate excitatory synapse plasticity in a striatal in vitro system, where glutamate activity can be examined in isolation from dopamine and other neuromodulators

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