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

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

Computer-assisted estimation in the CNS of 3D multimarker 'overlap' or 'touch' at the level of individual nerve endings: a confocal laser scanning microscope application

Presynaptic boutons and associated postsynaptic structures in the CNS express markers that are highly synapse type-specific. In multilabel immunofluorescence imaging, coexpression of such markers appears as overlap of signals in the same structures whereas closely related yet segregated markers, e.g., located pre-and postsynaptically, translate into signals that touch. 'Overlap' and 'touch' occur in three dimensions (3D). The instrument of choice to study overlap vs. touch of small objects in tissue volumes is the confocal laser scanning microscope (CSLM). To quantify overlap and touch we used two paradigms. Overlap was studied in rat brain sections triple-immunostained with antibodies against markers predominantly located presynaptically: glutamic acid decarboxylase, vesicular glutamate transporter 2, and calretinin. Touch was studied in rat temporal cortex where afferent, tracer-labeled entorhinohippocampal fibers in the subiculum were imaged together with possible postsynaptic target neurons immunostained with an antibody against the calcium binding protein, parvalbumin. Z-series of CLSM images were obtained in multiple channels. After post-acquisition deconvolution we further processed the images via software written in the C/C++ SCIL Image computer programming environment. The software receives parameters via scripts, after which it first identifies 3D objects through establishing isodensity envelopes around pixels representing small biologic structures (in our case: boutons) and then compares associated Z-series in which it determines whether there is overlap or touch between recognized 3D objects. Finally, graphic and numeric output is produced. With this script-commanded software we feel equipped to accurately and objectively quantify overlap and touch

Vesicular Glutamate and GABA Transporters Sort to Distinct Sets of Vesicles in a Population of Presynaptic Terminals

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal