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, Oct 04, 2015
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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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    ABSTRACT: The dorsal raphe nucleus (DRN), representing the main source of brain's serotonin, is implicated in the pathophysiology and therapeutics of several mental disorders that can be debilitating and life-long including depression, anxiety and autism. The activity of DRN neurons is precisely regulated, both phasically and tonically, by excitatory glutamate and inhibitory GABAergic axons arising from extra-raphe areas as well as from local sources within the nucleus. Changes in serotonin neurotransmission associated with pathophysiology may be encoded by alterations within this network of regulatory afferents. However, the complex organization of the DRN circuitry remains still poorly understood. Using a recently developed high-resolution immunofluorescence technique called array tomography (AT) we quantitatively analyzed the relative contribution of different populations of glutamate axons originating from different brain regions to the excitatory drive of the DRN. Additionally, we examined the presence of GABA axons within the DRN and their possible association with glutamate axons. In this review, we summarize our findings on the architecture of the rodent DRN synaptic neuropil using high-resolution neuroanatomy, and discuss possible functional implications for the nucleus. Understanding of the synaptic architecture of neural circuits at high resolution will pave the way to understand how neural structure and function may be perturbed in pathological states.
    Frontiers in Neural Circuits 08/2014; 8:105. DOI:10.3389/fncir.2014.00105 · 3.60 Impact Factor
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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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    ABSTRACT: Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit α1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses.
    Frontiers in Neural Circuits 10/2013; 7:153. DOI:10.3389/fncir.2013.00153 · 3.60 Impact Factor
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    ABSTRACT: The laminar nerve endings are distributed in the laryngeal mucosa, and described as sensory receptors evoked by laryngeal pressure changes. The present study aimed to determine detailed morphological characteristics of the laryngeal laminar endings of the rat. Immunohistochemistry for Na(+)-K(+)-ATPase, α(3) subunit, showed that laminar endings were distributed in the entire laryngeal surface of the epiglottis. The parent axons of the endings were thick in diameter, and they were branched and continued to the endings. In some cases, several endings from different parent axons fused into a large complex structure of 500 μm in width. The laminar endings were also immunoreactive for vesicular glutamate transporter 1 (vGLUT1) and vGLUT2, but not for P2X(3) purinoceptor. Around the laminar endings, terminal Schwann cells with immunoreactivity for S-100 protein were closely associated with axon terminals. Use of scanning electron microscopy with alkaline maceration method showed that the terminal Schwann cells consisted of a rounded perinuclear region and lamellar cytoplasmic processes. Ultrastructurally, axon terminals with numerous mitochondria were partly covered with Schwann cell sheath, and some terminals intruded into the epithelial layer. Clear vesicles of 50 nm in diameter were also observed especially in small cytoplasmic processes of 400 nm to 1 μm in size. The results in the present study suggested that the laminar endings in epiglottic mucosa have morphological characteristics of slowly adapting mechanoreceptors and contribute to sensation of laryngeal pressure via mucosal tension.
    Histochemie 03/2012; 138(1):25-39. DOI:10.1007/s00418-012-0939-y · 3.05 Impact Factor
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