Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans.

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Vesicular glutamate transporter-3 in the rodent brain: vesicular colocalization with vesicular γ-aminobutyric acid transporter.

Vesicular glutamate transporters (VGLUT1-3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ-aminobutyric acid (GABA)-ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT-positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light-microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals.

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals.

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.