Cortical and thalamic innervation of direct and indirect pathway medium-sized spiny neurons in mouse striatum

Abstract

The striatum receives major excitatory inputs from the cortex and thalamus that predominantly target the spines of medium-sized spiny neurons (MSNs). We aimed to determine whether there is any selectivity of these two excitatory afferents in their innervation of direct and indirect pathway MSNs. To address this, we used bacterial artificial chromosome transgenic mice, in which enhanced green fluorescent protein (EGFP) reports the presence of D(1) or D(2) dopamine receptor subtypes, markers of direct and indirect pathway MSNs, respectively. Excitatory afferents were identified by the selective expression of vesicular glutamate transporter type 1 (VGluT1) by corticostriatal afferents and vesicular glutamate transporter type 2 (VGluT2) by thalamostriatal afferents. A quantitative electron microscopic analysis was performed on striatal tissue from D(1) and D(2) mice that was double immunolabeled to reveal the EGFP and VGluT1 or VGluT2. We found that the proportion of synapses formed by terminals derived from the cortex and thalamus was similar for both direct and indirect pathway MSNs. Furthermore, qualitative analysis revealed that individual cortical or thalamic terminals form synapses with both direct and indirect pathway MSNs. Similarly, we observed a convergence of cortical and thalamic inputs onto individual MSNs of both direct and indirect pathway: individual EGFP-positive structures received input from both VGluT2-positive and VGluT2-negative terminals. These findings demonstrate that direct and indirect pathway MSNs are similarly innervated by cortical and thalamic afferents; both projections are thus likely to be critical in the control of MSNs and hence play fundamental roles in the expression of basal ganglia function.

Figure 1.

D₁- and D₂-expressing MSNs in the striatum, as revealed by immunoperoxidase labeling for EGFP. Light microscopic images showing neuronal cell bodies (some indicated by open arrowheads) and dendrites (some indicated by filled arrowheads) of MSNs labeled for EGFP (immunoperoxidase reaction product; TMB–DAB) in tissue from D₁ (left) and D₂ (right) BAC–EGFP mice. Note that the outlines of negative MSN cell bodies can be seen (some indicated by asterisks). Note also the dense meshwork of dendritic labeling in the neuropil. Scale bars, 50 μm.

Figure 2.

D₁ and D₂ MSNs receive synaptic input from VGluT1-positive terminals and VGluT2-positive terminals. A, A spine (sp) of a D₁ MSN, identified by peroxidase immunolabeling for EGFP, receiving asymmetric synaptic input (arrowhead) from a VGluT1-positive terminal (t), identified by immunogold labeling. Note the large crystal-like reaction product formed by TMB. B, A spine (sp) of a D₁ MSN, identified by immunoperoxidase labeling for EGFP, receiving asymmetric synaptic input (arrowhead) from a VGluT2-positive terminal (t), identified by immunogold labeling. Note the floccular electron dense peroxidase reaction product. C, A spine (sp) of a D₂ MSN identified by peroxidase labeling for EGFP receiving asymmetric synaptic input (arrowhead) from a VGluT1-positive terminal (t) identified by immunogold labeling. Note the large crystal-like reaction product formed by TMB. D, A spine (sp) of D₂ MSN, identified by peroxidase labeling for EGFP, receiving asymmetric synaptic input (arrowheads) from a VGluT2-positive terminal (t) identified by immunogold labeling. Note the large crystal-like reaction product formed by TMB. Scale bars, 250 nm.

Figure 3.

Quantitative analysis of the cortical and thalamic innervation of direct and indirect pathway MSNs in the striatum. A histogram showing the average percentage (±SEM) of VGluT-positive terminals forming asymmetric synapses with EGFP-positive (D₁ or D₂) structures out of a total of 150 synapses per set of tissue (D₁ VGluT1, D₁ VGluT2, D₂ VGluT1, or D₂ VGluT2; n = 3 animals per group). Of the 300 D₁-positive structures, 63 had presynaptic structures that were VGluT1 positive and 66 that were VGluT2 positive in their respectively labeled tissue. Similarly, for D₂-positive structures, 57 had presynaptic structures that were VGluT1 positive, and 50 that were VGluT2 positive. D₁-positive or D₂-positive structures did not form significantly different numbers of synapses with VGluT1 or VGluT2 terminals (p > 0.05, Fisher's exact test). Additionally, neither VGluT1- nor VGluT2-positive afferents formed significantly different numbers of synapses with D₁- or D₂-positive structures (p > 0.05, Fisher's exact test).

Figure 4.

Divergent cortical and thalamic input to direct and indirect pathway MSN spines. A, A VGluT1-positive terminal (t) makes asymmetric synaptic contact (arrowheads) with a D₁-positive spine (white asterisk) and a D₁-negative spine (black asterisk). B, A VGluT2-positive terminal (t) makes asymmetric synaptic contact (arrowheads) with a D₁-positive spine (white asterisk) and a D₁-negative spine (black asterisk). C, A VGluT1-positive terminal (t) makes asymmetric synaptic contact (arrowheads) with a D₂-positive (white asterisk) and a D₂-negative spine (black asterisk). D, A VGluT2-positive terminal (t) makes asymmetric synaptic contact (arrowheads) with a D₂-positive spine (white asterisk) and a D₂-negative spine (black asterisk). D₁- and D₂-positive spines are identified by the presence of peroxidase reaction product; clear synaptic specializations were seen over the seven serial sections. Scale bars, 250 nm.

Figure 5.

An individual direct pathway MSN may receive synaptic input from both thalamic (VGluT2-positive) and putative cortical (VGluT2-negative) terminals. A D₁-positive dendritic shaft (sh) receives asymmetric synaptic input (arrowhead) from a VGluT2-positive terminal (t). A spine (sp) emerging from this shaft receives asymmetrical synaptic input (arrowhead) from a VGluT2-negative terminal (n). Note that at the top of the image, another VGluT2-positive terminal (t) forms an asymmetric synapse (arrowhead) with a D₁-positive spine. Scale bar, 250 nm.