Quantitative analysis of glutamatergic innervation of the mouse dorsal raphe nucleus using array tomography

Department of Anesthesiology, Perioperative, and Pain Medicine, Children's Hospital Boston, Boston, Massachusetts 02115, USA.
The Journal of Comparative Neurology (Impact Factor: 3.23). 12/2011; 519(18):3802-14. DOI: 10.1002/cne.22734
Source: PubMed


Serotonin (5-hydroxytryptamine, 5-HT) containing neurons located in the dorsal raphe nucleus (DR) comprise the main source of forebrain 5-HT and regulate emotional states in normal and pathological conditions including affective disorders. However, there are many features of the local circuit architecture within the DR that remain poorly understood. DR neurons receive glutamatergic innervation from different brain areas that selectively express three different types of the vesicular glutamate transporter (VGLUT). In this study we used a new high-resolution imaging technique, array tomography, to quantitatively analyze the glutamatergic innervation of the mouse DR. In the same volumetric images, we studied the distribution of five antigens: VGLUT1, VGLUT2, VGLUT3, the postsynaptic protein PSD-95, and a marker for 5-HT cells, the enzyme tryptophan hydroxylase (TPOH). We found that all three populations of glutamatergic boutons are present in the DR; however, the density of paired association between VGLUT2 boutons and PSD-95 was ≈2-fold higher than that of either VGLUT1- or VGLUT3-PSD-95 pairs. In addition, VGLUT2-PSD-95 pairs were more commonly found associated with 5-HT cells than the other VGLUT types. These data support a prominent contribution of glutamate axons expressing VGLUT2 to the excitatory drive of DR neurons. The current study also emphasizes the use of array tomography as a quantitative approach to understand the fine molecular architecture of microcircuits in a well-preserved neuroanatomical context.

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Available from: Mariano Soiza-Reilly
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    • "Subsequently, the images can be reconstructed and rendered in 3D, and the relationships between the immunolabeled antigens can be visualized and quantitatively analyzed (Micheva and Smith, 2007; Micheva et al., 2010). One of the key features of AT is that it allows multiple rounds of immunolabeling/elution with a highly reliable preservation of antigens (Soiza-Reilly and Commons, 2011b). This results in high-resolution visualization of multiple antigens (typically 6–12) at the same time in 3D space (Figure 2). "
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    • "For inhibitory post-synaptic specializations, we have used antibody against GABA-A receptor subunit α1, a post-synaptic receptor component of inhibitory GABA-A synapses (Vicini et al., 2001). These primary antibodies have been applied to the mouse central nervous system and validated extensively, VGLUT2 (Graziano et al., 2008; Jakovcevski et al., 2009), PSD-95 (Gazula et al., 2010; Soiza-Reilly and Commons, 2011; Spangler et al., 2011), VGAT (Dudanova et al., 2007; Panzanelli et al., 2007; Fortune and Lurie, 2009; Jakovcevski et al., 2009) and GABA-A receptor subunit α1 (Panzanelli et al., 2007; Belichenko et al., 2009; Patrizi et al., 2012). This subsequently allowed us to use commercially available software Imaris (BitPlane, South Windsor, CT, USA) to reconstruct the filled neuron and to determine the distribution and number of glutamatergic or GABAergic synapses contacting it. "
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