Elsevier

Neurobiology of Disease

Volume 86, February 2016, Pages 62-74
Neurobiology of Disease

Differential changes in thalamic and cortical excitatory synapses onto striatal spiny projection neurons in a Huntington disease mouse model

https://doi.org/10.1016/j.nbd.2015.11.020Get rights and content

Highlights

  • Thalamic input to striatal neurons undergoes early physiological alterations in HD.

  • Synaptic transmission, NMDAR distribution and release probability are altered.

  • Thalamostriatal coculture has been established and used for the first time.

  • Channelrhodopsin has been employed to confirm and extend the findings in slice.

Abstract

Huntington disease (HD), a neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin, predominantly affects the striatum, especially the spiny projection neurons (SPN). The striatum receives excitatory input from cortex and thalamus, and the role of the former has been well-studied in HD. Here, we report that mutated huntingtin alters function of thalamostriatal connections. We used a novel thalamostriatal (T-S) coculture and an established corticostriatal (C-S) coculture, generated from YAC128 HD and WT (FVB/NJ background strain) mice, to investigate excitatory neurotransmission onto striatal SPN. SPN in T-S coculture from WT mice showed similar mini-excitatory postsynaptic current (mEPSC) frequency and amplitude as in C-S coculture; however, both the frequency and amplitude were significantly reduced in YAC128 T-S coculture. Further investigation in T-S coculture showed similar excitatory synapse density in WT and YAC128 SPN dendrites by immunostaining, suggesting changes in total dendritic length or probability of release as possible explanations for mEPSC frequency changes. Synaptic N-methyl-d-aspartate receptor (NMDAR) current was similar, but extrasynaptic current, associated with cell death signaling, was enhanced in YAC128 SPN in T-S coculture. Employing optical stimulation of cortical versus thalamic afferents and recording from striatal SPN in brain slice, we found increased glutamate release probability and reduced AMPAR/NMDAR current ratios in thalamostriatal synapses, most prominently in YAC128. Enhanced extrasynaptic NMDAR current in YAC128 SPN was apparent with both cortical and thalamic stimulation. We conclude that thalamic afferents to the striatum are affected early, prior to an overt HD phenotype; however, changes in NMDAR localization in SPN are independent of the source of glutamatergic input.

Introduction

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the gene encoding huntingtin (Htt). The brain region most affected is the striatum, an area critical for cognition and motor control, where GABAergic spiny projection neurons (SPN) make up > 90% of all neurons and show severe loss in late-stage HD. The striatum receives excitatory inputs from the cortex and thalamus, and the role of the former has been extensively studied in HD models. In particular, increased cortical excitability is one of the early features of HD (Cepeda et al., 2003, Graham et al., 2009, Joshi et al., 2009), and coordinated firing in corticostriatal circuits is disrupted (Miller et al., 2011). Although the magnitude of thalamic volume loss has been shown to closely correlate with degree of cognitive impairment in HD patients (Kassubek et al., 2005), the role of excitatory thalamostriatal connections and their functional changes in HD remain under-explored.

Impaired synaptic transmission and cellular signaling, leading to cell damage and death, have been reported at the pre-manifest stages of disease, in HD patients and animal models (Raymond et al., 2011). SPN carrying the HD mutation are more sensitive to NMDA-induced toxicity and express a larger proportion of NMDAR at the cell surface (Fan et al., 2007). Specifically, NMDAR expression was shown to be increased at extrasynaptic sites, where their activation triggers cell death signaling (Milnerwood et al., 2010, Milnerwood et al., 2012). Blocking extrasynaptic NMDAR (exNMDAR) with the low-affinity, use-dependent antagonist memantine exerted a neuroprotective effect and restored levels of exNMDAR in YAC128 striatum to those of wild-type (WT) FVB/NJ mice (Okamoto et al., 2009, Milnerwood et al., 2010, Milnerwood et al., 2012, Dau et al., 2014). However, it is not known whether thalamic input contributes to progressive striatal synaptic changes, or to what extent any thalamostriatal synaptic alterations replicate those occurring at the corticostriatal synapse.

Excitatory thalamic input onto striatal SPN is less abundant than cortical input (Lacey et al., 2005, Lei et al., 2013, Zhang et al., 2013), with approximately 40% of excitatory terminals being formed by the thalamus and 60% by the cortex (Deng et al., 2013, Lei et al., 2013; but see also: Doig et al., 2010, Ellender et al., 2013). Additionally, axodendritic connections, as opposed to axospinous ones, are more commonly formed on striatal SPN by thalamic afferents (especially those from parafascicular nuclei: Raju et al., 2006, Lacey et al., 2007) than by cortical ones (Lacey et al., 2005, Moss and Bolam, 2008, Doig et al., 2010, Lei et al., 2013, Zhang et al., 2013). Together, these anatomical findings suggest distinct functional properties of thalamic- and cortical-SPN synapses; indeed, electrophysiological recordings in slices from rat brain have shown marked differences in NMDAR/AMPAR current ratio and NMDAR subunit composition (Ding et al., 2008, Smeal et al., 2008), as well as differences in release probability and short-term synaptic plasticity (Ding et al., 2008) between corticostriatal and thalamostriatal synapses. The two major studies, however, significantly differed in their final conclusions (Ding et al., 2008, Smeal et al., 2008). Notably, a recent study has shown thalamostriatal connections are preferentially lost in an HD mouse model before phenotype development, preceding the loss of corticostriatal connections (Deng et al., 2013, Deng et al., 2014); however, whether the functional properties of these connections are significantly altered was not known.

The aim of this study was to investigate the properties of thalamostriatal synaptic transmission and their possible changes in the early stages of Huntington disease, comparing them to corticostriatal synapses. We used a novel thalamostriatal coculture system to characterize basic electrophysiological features of thalamic inputs to striatal SPN, including miniature excitatory events, synapse formation and NMDAR currents. We also applied optical stimulation of thalamic afferents in slice, to measure changes in thalamostriatal synapses in a more physiological environment. We conclude that thalamostriatal synapses in HD undergo marked early changes and may therefore significantly contribute to the pathogenesis of the disease.

Section snippets

Transgenic mice

In all experiments, the YAC128 HD mouse model was used. YAC128 is a yeast artificial chromosome (YAC) transgenic mouse model of HD that expresses the full-length human HTT gene with 128 CAG repeats on the FVB/NJ background (Slow et al., 2003). For coculture preparation, WT (FVB/NJ strain) and YAC128 mice (line 55, which produces viable homozygotes) of both sexes were used; for viral injections and slice electrophysiology, only male WT (FVB/NJ strain) and YAC128 mice (line 53, which expresses

Results

We identified YFP-expressing SPN from both thalamostriatal and corticostriatal cocultures, as described previously (Gladding et al., 2012, Kaufman et al., 2012, Milnerwood et al., 2012). Briefly, YFP + striatal cells were selected based on small soma (longest diameter < 20 μm) and spiny dendrites. For electrophysiology we used additional criteria, including capacitance > 30 pF and membrane resistance < 500 MΩ; in a few selected cells, I–V recordings were made to ensure responses were typical for SPN

Discussion

In this study, we show for the first time that specific glutamatergic inputs into the striatum, from cortex or thalamus, differentially influence development and function of excitatory synapses on striatal SPN. Moreover, we report early changes in YAC128 compared to WT not only in the thalamostriatal coculture system, but also in a more physiological environment of acute brain slice, using optical stimulation of cortical or thalamic fibers. Ours is the first study to show that thalamic input to

Conclusion

Our results indicate that mHtt expression affects the function of not only striatum and cortex, but also thalamus, at a stage when the overt HD phenotype is not yet apparent. Moreover, some of the functional changes at thalamostriatal synapses precede those of corticostriatal synapses, consistent with previously reported anatomical changes in another HD mouse model (Deng et al., 2014). Others have shown that hypothalamus and hippocampus are also altered (Aziz et al., 2007, Petersén and Gabery,

Acknowledgements

This work was funded by the Huntington Society of Canada and Canadian Institutes for Health Research (CIHR MOP-12699). We would like to thank L.Zhang, L.Wang and R.Kang for their technical support with coculture preparations, M.D. Sepers and P.Wang for their assistance with viral injections, and A.M.Craig and K.She for the YFP and YFP-GluN2B plasmids. The authors declare no competing financial interests.

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