Giorgia Fattorini

GABAergic signaling at mossy fiber synapses in neonatal rat hippocampus

In the adult rat hippocampus, granule cell mossy fibers (MFs) form excitatory glutamatergic synapses with CA3 principal cells and local inhibitory interneurons. However, evidence has been provided that, in young animals and after seizures, the same fibers can release in addition to glutamate GABA. Here we show that, during the first postnatal week, stimulation of granule cells in the dentate gyrus gave rise to monosynaptic GABAA-mediated responses in principal cells and in interneurons. These synapses were indeed made by MFs because they exhibited strong paired-pulse facilitation, high sensitivity to the metabotropic glutamate receptor agonist l-AP-4, and short-term frequency-dependent facilitation. MF responses were potentiated by blocking the plasma membrane GABA transporter GAT-1 with NO-711 or by allosterically modulating GABAA receptors with flurazepam. Chemical stimulation of granule cell dendrites with glutamate induced barrages of GABAA-mediated postsynaptic currents into target neurons. Furthermore, immunocytochemical experiments demonstrated colocalization of vesicular GABA transporter with vesicular glutamate transporter-1 and zinc transporter 3, suggesting that GABA can be taken up and stored in synaptic vesicles of MF terminals. Additional fibers releasing both glutamate and GABA into principal cells and interneurons were recruited by increasing the strength of stimulation. Both the GABAergic and the glutamatergic component of synaptic currents occurred with the same latency and were reversibly abolished by l-AP-4, indicating that they originated from the MFs. GABAergic signaling may play a crucial role in tuning hippocampal network during postnatal development. Low-threshold GABA-releasing fibers may undergo elimination, and this may occur when GABA shifts from the depolarizing to the hyperpolarizing direction.

A1R–A2AR heteromers coupled to Gs and Gi/0 proteins modulate GABA transport into astrocytes

Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R–A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse.

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.

PrPC Controls via Protein Kinase A the Direction of Synaptic Plasticity in the Immature Hippocampus

The cellular form of prion protein PrPC is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrPSc to cause neurodegenerative diseases. Its predominant synaptic localization suggests a crucial role in synaptic signaling. Interestingly, PrPC is developmentally regulated and its high expression in the immature brain could be instrumental in regulating neurogenesis and cell proliferation. Here, PrPC-deficient (Prnp0/0) mice were used to assess whether the prion protein is involved in synaptic plasticity processes in the neonatal hippocampus. To this aim, calcium transients associated with giant depolarizing potentials, a hallmark of developmental networks, were transiently paired with mossy fiber activation in such a way that the two events were coincident. While this procedure caused long-term potentiation (LTP) in wild-type (WT) animals, it caused long-term depression (LTD) in Prnp0/0 mice. Induction of LTP was postsynaptic and required the activation of cAMP-dependent protein kinase A (PKA) signaling. The induction of LTD was presynaptic and relied on G-protein-coupled GluK1 receptor and protein lipase C. In addition, at emerging CA3-CA1 synapses in WT mice, but not in Prnp0/0 mice, pairing Schaffer collateral stimulation with depolarization of CA1 principal cells induced LTP, known to be PKA dependent. Postsynaptic infusion of a constitutively active isoform of PKA catalytic subunit Cα into CA1 and CA3 principal cells in the hippocampus of Prnp0/0 mice caused a persistent synaptic facilitation that was occluded by subsequent pairing. These data suggest that PrPC plays a crucial role in regulating via PKA synaptic plasticity in the developing hippocampus.

GLT-1 expression and Glu uptake in rat cerebral cortex are increased by phencyclidine

Using western blottings, microdialysis, and functional assays we tested the hypothesis that phencyclidine (PCP) modifies the expression and function of glutamate (Glu) transporters in the rat frontal cortex. Western blotting studies revealed that administration of PCP (10 mg/kg/day; 7 days) increased significantly the expression of the astrocytic Glu transporter GLT‐1/EAAT2. Functional studies showed that PCP increased significantly Na+‐dependent Glu uptake in slices and in neuron/astrocyte co‐cultures, and microdialysis studies evidenced that PCP treatment reduced basal Glu output. In our experimental conditions, PCP did not induce toxicity. These studies show that PCP increases the expression of GLT‐1 in the cerebral cortex, thereby increasing Glu uptake and reducing extracellular [Glu].

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation

In rat frontal cortex, extracellular levels of glutamate are raised by the anti‐psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT‐1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT‐1 down‐regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome‐associated protein of 25 kDa (SNAP‐25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high‐resolution confocal microscopy studies in vivo showed that GLT‐1 down‐regulation is closely associated with a significant increase in synaptophysin, but not SNAP‐25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP‐25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT‐1 expression is down‐regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.

Analysis of Synaptotagmin, SV2, and Rab3 Expression in Cortical Glutamatergic and GABAergic Axon Terminals

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, by performing a quantitative analysis of the degree of co-localization of synaptotagmin (SYT) 1 and 2, synaptic vesicle protein 2 (SV2) A and B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ terminals and synaptic vesicles (SVs) in rat cerebral cortex. Co-localization studies showed that VGLUT1 puncta had high levels of SV2A and B and of Rab3c, intermediate levels of SYT1, and low levels of SYT2 and Rab3c; VGLUT2 puncta exhibited intermediate levels of all presynaptic proteins studied; whereas vesicular GABA transporter (VGAT) puncta had high levels of SV2A and SYT2, intermediate levels of SYT1, Rab3a, and Rab3c, and low levels of SV2B. Since SV2B is reportedly expressed by glutamatergic neurons and we observed SV2B expression in VGAT puncta, we performed electron microscopic studies and found SV2B positive axon terminals forming symmetric synapses. Immunoisolation studies showed that the expression levels of the protein isoforms varied in the three populations of SVs. Expression of SYT1 was highest in VGLUT1–SVs, while SYT2 expression was similar in the three SV groups. Expression of SV2A was similarly high in all three SV populations, except for SV2B levels that were very low in VGAT SVs. Finally, Rab3a levels were similar in the three SV groups, while Rab3c levels were highest in VGLUT1–SVs. These quantitative results extend our previous studies on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of the respective release machineries can be generated by the differential complement of SV proteins involved in distinct stages of the release process.

A role for GAT-1 in Presynaptic GABA Homeostasis?

In monoamine-releasing terminals, neurotransmitter transporters – in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space – are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy.

Modulation of GABA Transport by Adenosine A1R–A2AR Heteromers, Which Are Coupled to Both Gs- and Gi/o-Proteins

Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A1R–A2AR that signal via two different G-proteins, Gs and Gi/o, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse.

Neuronal localization of the GABA transporter GAT-3 in human cerebral cortex: a procedural artifact?

Gamma-amino butyric acid (GABA) plasma membrane transporters (GATs) contribute to the modulation of GABAs actions and are implicated in neuropsychiatric diseases. In this study, the localization of GAT-3, the major glial GAT, was investigated in human cortex using immunocytochemical techniques. In prefrontal and temporal cortices, GAT-3 immunoreactivity (ir) was present throughout the depth of the cortex, both in puncta and in neurons. GAT-3-positive puncta were dispersed in the neuropil or closely related to cell bodies; neuronal staining was in perikarya, especially of pyramidal cells, and proximal dendrites. Electron microscopic studies showed that GAT-3 ir was in astrocytic processes as well as in neuronal elements. All GAT-3-positive neurons co-expressed heat shock protein 70. To test the possibility that the collection procedure of human samples induced the expression of GAT-3 in neurons which normally do not express it, we analyzed rat cortical tissue resected using the same procedure and found that numerous neurons are GAT-3-positive and that they co-express heat shock protein 70. Results show that in human cortex GAT-3 is expressed in astrocytic processes and in neurons and suggest that neuronal expression is related to the procedure used for collecting human samples.