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

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.

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

“…Boutons of paraventricular nucleus fibers ending in the forebrain coexpress CR and VGluT2 . In a follow-up study (Wouterlood et al 2007b) we report on entorhinal innervation from the NRT. The projection of this nucleus is known in detail (Wouterlood et al 1990;Vertes et al 2006), its neurophysiology studied in some detail (Dolleman-van der Weel et al 1997;Bertram and Zhang 1999), and it contains CR immunoreactive neurons (Arai 1994) as well as neurons rich in VGluT2 mRNA (Hur and Zaborszky 2005;Barroso-Chinea et al 2007).…”

Pallidothalamic-projecting neurons in Macaca fascicularis co-express GABAergic and glutamatergic markers as seen in control, MPTP-treated and dyskinetic monkeys

“…Although a number of recent studies have demonstrated-both in developing systems as well in adult animals-that single neurons across different brain areas co-express GABA and glutamate (Gómez-Lira et al 2005;Danik et al 2005;Gritti et al 2006;Wouterlood et al 2008;Fattorini et al 2009;Zander et al 2010), these findings do not provide conclusive evidence for GABA and glutamate co-release. In this regard, recent evidence shows that dopaminergic neurons may also use glutamate as a neurotransmitter in the striatum (Descarries et al 2008).…”

“…Axon terminals containing vGlut1 are characterized by a lower probability of glutamate release than those using vGlut2 (Fremeau et al 2001). A number of recent studies have reported co-expression of GABAergic and glutamatergic markers in adult animals, such as in a subpopulation of neurons in the basal forebrain (Gritti et al 2006), calretinin-positive neurons in the entorhinal cortex (Wouterlood et al 2008), mossy fiber terminals of the hippocampus (Sandler and Smith 1991;Zander et al 2010), basket cell terminals of the cerebellum (Zander et al 2010), as well as in layer V cortical terminals (Fattorini et al 2009). …”

Confocal Laser Scanning: of Instrument, Computer Processing, and Men

“…Using the thresholds derived from that analysis we instructed software to search in all 3D pixel matrices for isodensity-bounded aggregates of voxels where the center of gravity in both channels coincided, with 100% overlap of the 3D objects identified in the 488 nm 3D dataset with those in the 543 nm dataset (assuming that clusters of VGluT2-expressing synaptic vesicles fit axon terminals, i.e., the diameters of VGluT2 3D objects are smaller than those of the encapsulating axon terminals). Indeed, we found aggregates of VGluT2 voxels completely internalized in swellings of fibers of thalamic origin (Figure 1.10; Wouterlood et al, 2008b). We even found triple colocalization, that is, VGluT2 and calretinin in endings of labeled thalamocortical fibers (Figure 1.10C).…”