Evidence against GABA Release from Glutamatergic Mossy Fiber Terminals in the Developing Hippocampus

Abstract

Hippocampal mossy fibers of young rodents have been reported to corelease inhibitory neurotransmitter GABA in addition to excitatory transmitter glutamate. In this study, we aimed at re-evaluating this corelease hypothesis of both inhibitory and excitatory transmitters in the hippocampus. Electrophysiological examination revealed that, in juvenile mice and rats of the two to 3 weeks old, stimulation at the granule cell layer of the dentate gyrus elicited monosynaptic GABAergic IPSCs in CA3 neurons in the presence of ionotropic glutamate receptor (iGluR) blockers, only when rather strong stimuli were given. The group II mGluR agonist (2S,1ЈR,2ЈR,3ЈR)-2-(2,3-dicarboxycyclo-propyl)glycine (DCG-IV), which selectively suppresses transmission at the mossy fiber-CA3 synapse, abolished almost all postsynaptic responses elicited by the weak stimuli, whereas those by strong stimuli were inhibited only slightly. In addition, the minimal stimulation elicited GABAergic IPSCs in neonatal mice of the first postnatal week, whereas these responses are not sensitive to DCG-IV. Immunohistochemical examination revealed that mossy fiber terminals expressed GABA and the GABA-synthesizing enzyme GAD67, although the expression levels were much weaker than those in the inhibitory interneurons. Notably, the expression levels of the vesicular GABA transporter were much lower than those of GABA and GAD67, and almost below detection threshold. These results suggest that mossy fiber synapses are purely glutamatergic and apparent monosynaptic IPSCs so far reported are evoked by costimulation of inhibitory interneurons, at least in young mice and rats. Hippocampal mossy fiber terminals synthesize and store GABA, but have limited ability in vesicular release for GABA in the developing rodents.

Presynaptic But Not Postsynaptic GABA Signaling at Unitary Mossy Fiber Synapses

“…Dual glutamate/GABA transmission at MF synapses is supported by the following evidence: (1) GAD and VIAAT are expressed in MFTs (Sloviter et al, 1996;Gutiérrez et al, 2003;Zander et al, 2010), (2) GABA is released from isolated MF synaptosomes (Gó mez-Lira et al, 2002), (3) GABAA receptors are expressed in the postsynaptic density facing GAD-immunopositive MFTs (Bergersen et al, 2003), and (4) IPSCs are detected upon extracellular stimulation in st. granulosum (Walker et al, 2001;Gutiérrez et al, 2003;Safiulina et al, 2006; but see Mori et al, 2004;Uchigashima et al, 2007;Toni et al, 2008). Our results appear consistent with some of these observations but do not support the conclusion that postsynaptic GABAA receptors are activated by GABA release at MFTs upon phasic activity.…”

“…1), together with the presence of local interneurons in the granule cell layer and hilus (Lübke et al, 1998;Houser, 2007) complicates the interpretation of postsynaptic responses evoked by extracellular electrical stimulation (Walker et al, 2001;Safiulina et al, 2006;Uchigashima et al, 2007). We examined the postsynaptic responses evoked by photostimulation of individual, visually identified GCs using local photolysis of MNI-glutamate in hippocampal organotypic slices from G42 mice.…”

“…However, the complex dentate gyrus circuitry complicates the interpretation of these data, which might reflect direct stimulation of local interneurons (Uchigashima et al, 2007). Paired recordings from individual GCs and postsynaptic pyramidal neurons (Mori et al, 2004) are needed to demonstrate activation of postsynaptic GABAA receptors at MF synapses.…”

At Immature Mossy-Fiber–CA3 Synapses, Correlated Presynaptic and Postsynaptic Activity Persistently Enhances GABA Release and Network Excitability via BDNF and cAMP-Dependent PKA

“…During postnatal development, granule cells transiently express both mRNA coding for GAD 67 and GAD 67 protein (Dupuy and Houser, 1997;Frahm and Draguhn, 2001). This GABAergic phenotype has been attributed either to principal cells synthesizing GABA, which would be downregulated in adulthood (Frahm and Draguhn, 2001) or to GAD-positive interneurons which would transiently migrate to the upper and middle portions of the granule cell layer (Dupuy and Houser, 1997;Uchigashima et al, 2007). Whatever their origin, MF-mediated GPSCs exhibited all characteristics of MF responses such as strong paired pulse facilitation, short term frequency-dependent facilitation and sensitivity to the group III mGluR agonist L-AP4 (Salin et al, 1996;Safiulina et al, 2006).…”

Synaptic and Vesicular Coexistence of VGLUT and VGAT in Selected Excitatory and Inhibitory Synapses

“…For instance, electrophysiological experiments suggested that GABA is coreleased from glutamatergic hippocampal mossy fibers (Gutiérrez and Heinemann, 2001;Walker et al, 2001;Kasyanov et al, 2004) [see Uchigashima et al (2007) for an alternative view]. Furthermore, it has been suggested that coexpression of a vesicular glutamate transporter (VGLUT3) in a cholinergic neuron may improve vesicular acetylcholine transporter (VAChT)-mediated vesicular acetylcho-line uptake and storage (Gras et al, 2008).…”