Co-expression of vesicular glutamate transporters 1and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

Graduate School of Neurosciences, Research Institute Neuroscience, Department of Anatomy & Neurosciences, Vrije Universiteit Medical Center, Rm MF-G-136, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands.
Brain Structure and Function (Impact Factor: 5.62). 01/2008; 212(3-4):303-19. DOI: 10.1007/s00429-007-0163-z
Source: PubMed


We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory.

Download full-text


Available from: Wolfgang Härtig, Feb 27, 2014
  • Source
    • "The following commercial antibodies were used: 1. anti-vGluT-1 polyclonal antibody, raised in guinea pig against a synthetic linear peptide from rat vGluT-1 (Millipore, Billerica, MA, USA) [34,64]; 2. anti-vGluT-2 polyclonal antibody, raised in rabbit against a strep-tag fusion protein of rat vGluT-2 (Synaptic System, Goettingen, Germany) [34,65]; 3. (a) anti-GAD monoclonal antibody, raised in mouse against human GAD-65-GST fusion protein (Stressgen, Victoria, BC, Canada), and (b) anti-GAD polyclonal antibody, raised in rabbit against a synthetic peptide corresponding to the rat GAD-65 C-terminus residues 572-585 (Chemicon), which react to both GAD-65 and GAD-67 [63]; 4. (a) anti-VAMP monoclonal antibody, raised in mouse against crude synaptic immunoprecipitate (human) (Millipore), and (b) anti-VAMP polyclonal antibody, raised in rabbit against a synthetic peptide based on rat VAMP-2 (Stressgen), which react to VAMP-2 [48]; 5. anti-SNAP-25 monoclonal antibody, raised in against crude synaptic immunoprecipitate (human), which reacts with both SNAP-25A and SNAP-25B (Chemicon) [15]; 6. anti-syntaxin monoclonal antibody, raised in mouse against clone HCP-1, which reacts to syntaxin-1 (ABCAM, Cambridge, UK) [57]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.
    Full-text · Article · Nov 2011 · BMC Neuroscience
  • [Show abstract] [Hide abstract]
    ABSTRACT: The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.
    No preview · Article · Jan 2008 · The Journal of Comparative Neurology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cortical and subcortical inputs to the striatum are functionally highly organized and they obey to some extent striatal patch-matrix topography. Whether this organization is reflected in the density of various glutamatergic endings is unknown. We therefore mapped boutons expressing the vesicular glutamate transporters VGluT1 and VGluT2, together with boutons immunoreactive for vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in patch and matrix throughout the striatum. We used triple-immunofluorescence staining followed by multichannel, high-magnification confocal laser scanning and 3D object recognition. Densities of VGluT1 and VGluT2 boutons were on average higher in matrix than in patches in all striatal sectors. The dorsal one-third of the striatum contained the highest densities of VGluT1 boutons. Subsequent 3D surface plotting revealed patterns of density "valleys" in the dorsomedial striatum coinciding with patch locations in the patch-matrix mapping. The density of VGluT1 boutons increased along three axes: ventrolateral-to-dorsomedial, ventral-to-dorsal, and lateral-to-medial. In contrast, VGluT2 showed a global increase in density from lateral to medial and a relatively high density in the ventral striatum. VGAT appeared more evenly distributed in the striatal patch-matrix than the VGluTs, with a tendency of bouton density to increase from medial to lateral. We noted a good correlation between the high VGluT1 bouton density dorsomedially with inputs from dorsal medial prefrontal cortex and related thalamic regions, and the enhanced VGluT2 input ventromedially with input from ventral medial prefrontal cortex and thalamic, amygdaloid, and hippocampal sources.
    No preview · Article · Jul 2012 · The Journal of Comparative Neurology
Show more