Vesicular glutamate transporters define two sets of glutamatergic afferents to the somatosensory thalamus and two thalamocortical projections in the mouse

Center for Neuroscience, University of California Davis, Davis, California 95616-8798, USA.
The Journal of Comparative Neurology (Impact Factor: 3.23). 03/2008; 507(2):1258-76. DOI: 10.1002/cne.21592
Source: PubMed


The ventral posterior nucleus of the thalamus (VP) receives two major sets of excitatory inputs, one from the ascending somatosensory pathways originating in the dorsal horn, dorsal column nuclei, and trigeminal nuclei, and the other originating from the cerebral cortex. Both systems use glutamate as neurotransmitter, as do the thalamocortical axons relaying somatosensory information from the VP to the primary somatosensory cortex (SI). The synapses formed by these projection systems differ anatomically, physiologically, and in their capacity for short-term synaptic plasticity. Glutamate uptake into synaptic vesicles and its release at central synapses depend on two isoforms of vesicular glutamate transporters, VGluT1 and VGluT2. Despite ample evidence of their complementary distribution, some instances exist of co-localization in the same brain areas or at the same synapses. In the thalamus, the two transcripts coexist in cells of the VP and other nuclei but not in the posterior or intralaminar nuclei. We show that the two isoforms are completely segregated at VP synapses, despite their widespread expression throughout the dorsal and ventral thalamus. We present immunocytochemical, ultrastructural, gene expression, and connectional evidence that VGluT1 in the VP is only found at corticothalamic synapses, whereas VGluT2 is only found at terminals made by axons originating in the spinal cord and brainstem. By contrast, the two VGluT isoforms are co-localized in thalamocortical axon terminals targeting layer IV, but not in those targeting layer I, suggesting the presence of two distinct projection systems related to the core/matrix pattern of organization of thalamocortical connectivity described in other mammals.

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    • "Cell subtypes that were filled with Lucifer yellow (LY) were identified either by retrograde labeling or by agglutinin labeling. Input sources were labeled by antibody staining with the vesicular glutamate transporter type 1 (VGluT1) or type 2 (VGluT2) to distinguish corticocortical and thalamocortical inputs, respectively (Fujiyama et al. 2001; Graziano et al. 2008). Putative synapses were identified by apposition of presynaptic label to postsynaptic cells observed with confocal laser scanning microscopy (CLSM) and validated by electron microscopic (EM) reconstructions. "
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    ABSTRACT: Most glutamatergic inputs in the neocortex originate from the thalamus or neocortical pyramidal cells. To test whether thalamocortical afferents selectively innervate specific cortical cell subtypes and surface domains, we investigated the distribution patterns of thalamocortical and corticocortical excitatory synaptic inputs in identified postsynaptic cortical cell subtypes using intracellular and immunohistochemical staining combined with confocal laser scanning and electron microscopic observations in 2 thalamorecipient sublayers, lower layer 2/3 (L2/3b) and lower layer 5 (L5b) of rat frontal cortex. The dendrites of GABAergic parvalbumin (PV) cells preferentially received corticocortical inputs in both sublayers. The somata of L2/3b PV cells received thalamic inputs in similar proportions to the basal dendritic spines of L2/3b pyramidal cells, whereas L5b PV somata were mostly innervated by cortical inputs. The basal dendrites of L2/3b pyramidal and L5b corticopontine pyramidal cells received cortical and thalamic glutamatergic inputs in proportion to their local abundance, whereas crossed-corticostriatal pyramidal cells in L5b exhibited a preference for thalamic inputs, particularly in their distal dendrites. Our data demonstrate an exquisite selectivity among thalamocortical afferents in which synaptic connectivity is dependent on the postsynaptic neuron subtype, cortical sublayer, and cell surface domain. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:
    Cerebral Cortex 06/2015; DOI:10.1093/cercor/bhv124 · 8.67 Impact Factor
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    • "Glutamate excites relay thalamocortical neurons through NMDA receptors, if the sensory stimulus is prolong and through non-NMDA receptors if the sensory stimulus is brief [33], [34]. Of the three isoforms of VGluT, we opted to study VGluT2 because it is expressed most densely in relay thalamic nuclei [35]–[39] and in ascending trigeminal sensory neurons that project to VPM and Po [40], [41]. Since VGluT1 axons originate in corticothalamic neurons, we interpreted the presence of VGluT2 on thalamic trigeminovascular neurons as constituting the main drive for activation of these neurons by glutamatergic input they receive from ascending trigeminothalamic (possibly dura-sensitive) neurons in SpV. "
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    ABSTRACT: Dynamic thalamic regulation of sensory signals allows the cortex to adjust better to rapidly changing behavioral, physiological and environmental demands. To fulfill this role, thalamic neurons must themselves be subjected to constantly changing modulatory inputs that originate in multiple neurochemical pathways involved in autonomic, affective and cognitive functions. Our overall goal is to define an anatomical framework for conceptualizing how a 'decision' is made on whether a trigeminovascular thalamic neuron fires, for how long, and at what frequency. To begin answering this question, we determine which neuropeptides/neurotransmitters are in a position to modulate thalamic trigeminovascular neurons. Using a combination of in-vivo single-unit recording, juxtacellular labeling with tetramethylrhodamine dextran (TMR) and in-vitro immunohistochemistry, we found that thalamic trigeminovascular neurons were surrounded by high density of axons containing biomarkers of glutamate, GABA, dopamine and serotonin; moderate density of axons containing noradrenaline and histamine; low density of axons containing orexin and melanin concentrating hormone (MCH); but not axons containing CGRP, serotonin 1D receptor, oxytocin or vasopressin. In the context of migraine, the findings suggest that the transmission of headache-related nociceptive signals from the thalamus to the cortex may be modulated by opposing forces (i.e., facilitatory, inhibitory) that are governed by continuous adjustments needed to keep physiological, behavioral, cognitive and emotional homeostasis.
    PLoS ONE 08/2014; 9(8):e103929. DOI:10.1371/journal.pone.0103929 · 3.23 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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