The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

Certain neurons choose the neurotransmitter they use in an activity-dependent manner, and trophic factors are involved in this phenotypic differentiation during development. Developing hippocampal granule cells (GCs) constitutively express the markers of the glutamatergic and GABAergic phenotypes, but when development is completed, the GABAergic phenotype shuts off. With electrophysiological, single-cell reverse transcription-PCR and immunohistological techniques, we show here that short-term (24 h) cultures of fully differentiated adult glutamatergic GCs, which express glutamate, VGlut-1 (vesicular glutamate transporter) mRNA, calbindin, and dynorphin mRNA, can be induced to reexpress the GABAergic markers GABA, GAD₆₇(glutamate decarboxylase 67 kDa isoform), and VGAT (vesicular GABA transporter) mRNA, by sustained synaptic or direct activation of glutamate receptors and by activation of TrkB (tyrosine receptor kinase B) receptors, with brain-derived neurotrophic factor (BDNF) (30 min). The expression of the GABAergic markers was prevented by the blockade of glutamate receptors and sodium or calcium channels, and by inhibitors of protein kinases and protein synthesis. In hippocampal slices of epileptic rats and in BDNF-treated slices from naive rats, we confirmed the appearance of monosynaptic GABA_Areceptor-mediated responses to GC stimulation, in the presence of glutamate receptors blockers. Accordingly, GC cultures prepared from these slices showed the coexpression of the glutamatergic and GABAergic markers. Our results demonstrate that the neurotransmitter choice of the GCs, which are unique in terms of their continuing birth and death throughout life, depends on programmed and environmental factors, and this process is neither limited by a critical developmental period nor restricted by their insertion in their natural network.

Amyloid-beta oligomers (AβO) have been proposed as the most potent neurotoxic and inflammation inducers in Alzheimer’s disease (AD). AβO contribute to AD pathogenesis by impairing the production of several cytokines and inflammation-related signaling pathways, such as the Janus kinases/signal transducer of transcription factor-3 (JAK/STAT3) pathway. STAT3 modulates glial activation, indirectly regulates Aβ deposition, and induces cognitive decline in AD transgenic models. However, in vivo studies using an AβO microinjection rat model have not yet explored STAT3 role. The main purpose of this study was to elucidate if a single microinjection of AβO could promote an increased expression of STAT3 in glial cells favoring neuroinflammation and neurodegeneration. We designed a model of intrahippocampal microinjection and assessed glial activation, cytokines production, STAT3 expression, and neurodegeneration in time. Our results showed robust expression of STAT3 in glial cells (mainly in astrocytes) and neurons, correlating with neuronal death in response to AβO administration. A STAT3 inhibition assay conducted in rat primary hippocampal cultures, suggested that the induction of the transcription factor by AβO in astrocytes leads them to an activation state that may favor neuronal death. Notwithstanding, pharmacological inhibition of the JAK2/STAT3 pathway should be focused on astrocytes because it is also essential in neurons survival. Overall, these findings strongly suggest the participation of STAT3 in the development of neurodegeneration.