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

Abstract

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 CRneurons: a glutamatergic excitatory and a GABAergic inhibitory.

Origin of calretinin‐containing, vesicular glutamate transporter 2‐coexpressing fiber terminals in the entorhinal cortex of the rat

“…In a separate paper, we reported the details of the distribution of vesicular glutamate transporters 1 and 2 in the entorhinal cortex and reported coexpression of VGluT2 and CR (Wouterlood et al, 2007). In agreement with the previous findings, VGluT2 and CR coexpression was observed in axon terminals in the entorhinal cortex.…”

“…In an earlier study, we observed CR-GABA coexpressing neuronal cell bodies in the entorhinal cortex intermingled with CR positive, GABA-negative cell bodies (Wouterlood et al, 2000). In a follow-up study on this cortical area, we noted the presence of axon terminals that coexpressed CR and VGluT2 (Wouterlood et al, 2007). Thus it is conceivable that CR-VGluT2-positive axon terminals belong to the intrinsic CR-positive and GABAnegative entorhinal neurons.…”

“…With antibodies against CR and GABA in sections studied under the confocal laser scanning microscope, we conjectured that a subpopulation of entorhinal CR-expressing interneurons is non-GABAergic (Wouterlood et al, 2000). A further investigation revealed the presence of vesicular glutamate transporter 2 (VGluT2) immunoreactivity in a considerable number of CR-positive axon terminals in the entorhinal cortex (Wouterlood et al, 2007). Because VGluT2 is associated with glutamatergic neurotransmission (Bellochio et al, 2000;Fremeau et al, 2001), we proposed that the entorhinal cortex hosts two subpopulations of CRimmunopositive interneurons, i.e., neurons that express CR and GABA (inhibitory interneurons) and neurons that express CR and VGluT2 (excitatory interneurons).…”

Complementary Modular Microcircuits of the Rat Medial Entorhinal Cortex

“…The parvalbumin cells in the superficial layers of this region are exclusively GABAergic (Wouterlood et al, 1995). The majority of calretinin neurons were initially thought to be GABAergic but several reports found also calretinin positive glutamatergic neurons (Miettinen et al, 1997; Wouterlood et al, 2000, 2007, 2008). Hence, based on the overall distribution of GABAergic neurons in the parahippocampal region, it is likely that the densely clustered calretinin positive neurons in the mMEC are glutamatergic (Miettinen et al, 1997), but their transmitter identity remains to be ascertained.…”

VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex

“…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.…”