Mossy fibers are the sole excitatory projection from dentate gyrus granule cells to the hippocampus, where they release glutamate, dynorphin, and zinc. In addition, mossy fiber terminals show intense immunoreactivity for the inhibitory neurotransmitter GABA. Fast inhibitory transmission at mossy fiber synapses, however, has not previously been reported. Here, we show that electrical or chemical stimuli that recruit dentate granule cells elicit monosynaptic GABA(A) receptor-mediated synaptic signals in CA3 pyramidal neurons. These inhibitory signals satisfy the criteria that distinguish mossy fiber-CA3 synapses: high sensitivity to metabotropic glutamate receptor agonists, facilitation during repetitive stimulation, and NMDA receptor-independent long-term potentiation. GABAergic transmission from the dentate gyrus to CA3 has major implications not only for information flow into the hippocampus but also for developmental and pathological processes involving the hippocampus.

Heteromeric kainate receptors (KARs) containing both glutamate receptor 6 (GluR6) and KA2 subunits are involved in KAR-mediated EPSCs at mossy fiber synapses in CA3 pyramidal cells. We report that endogenous glutamate, by activating KARs, reversibly inhibits the slow Ca 2ϩ -activated K ϩ current I sAHP and increases neuronal excitability through a G-protein-coupled mechanism. Using KAR knockout mice, we show that KA2 is essential for the inhibition of I sAHP in CA3 pyramidal cells by low nanomolar concentrations of kainate, in addition to GluR6. In GluR6 Ϫ/Ϫ mice, both ionotropic synaptic transmission and inhibition of I sAHP by endogenous glutamate released from mossy fibers was lost. In contrast, inhibition of I sAHP was absent in KA2 Ϫ/Ϫ mice despite the preservation of KAR-mediated EPSCs. These data indicate that the metabotropic action of KARs did not rely on the activation of a KAR-mediated inward current. Biochemical analysis of knock-out mice revealed that KA2 was required for the interaction of KARs with G␣ q/11 -proteins known to be involved in I sAHP modulation. Finally, the ionotropic and metabotropic actions of KARs at mossy fiber synapses were differentially sensitive to the competitive glutamate receptor ligands kainate (5 nM) and kynurenate (1 mM). We propose a model in which KARs could operate in two modes at mossy fiber synapses: through a direct ionotropic action of GluR6, and through an indirect G-protein-coupled mechanism requiring the binding of glutamate to KA2.

Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl- is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the alpha2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus.

Although GABA A receptors are widely distributed at inhibitory synapses on dendrites and cell bodies of neurons, they also occur in other places, in particular at synapses made on axons and in extrasynaptic membranes. This review summarises some of the evidence that presynaptic receptors modulate transmission not only at primary afferents in the spinal cord, but also at a variety of sites in the brain, including hippocampal mossy fibres. These receptors modulate transmitter release via several different mechanisms. Another form of unconventional GABA A receptor-mediated signalling is the mediation of a tonic conductance, seen in granule cells of the cerebellum and dentate gyrus and also in hippocampal interneurons. Tonic signalling appears to be mediated by extrasynaptic receptors. The adaptive significance of this form of signalling remains poorly understood.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Presynaptic GABA(A) receptors (GABA(A)Rs) occur at hippocampal mossy fiber synapses. Whether and how they modulate orthodromic signaling to postsynaptic targets is poorly understood. We found that an endogenous neurosteroid that is selective for high-affinity delta subunit-containing GABA(A)Rs depolarized rat mossy fiber boutons, enhanced action potential-dependent Ca(2+) transients and facilitated glutamatergic transmission to pyramidal neurons. Conversely, blocking GABA(A)Rs hyperpolarized mossy fiber boutons, increased their input resistance, decreased spike width and attenuated action potential-dependent presynaptic Ca(2+) transients, indicating that a subset of presynaptic GABA receptors are tonically active. Blocking GABA(A)Rs also interfered with the induction of long-term potentiation at mossy fiber-CA3 synapses. Presynaptic GABA(A)Rs therefore facilitate information flow to the hippocampus both directly and by enhancing LTP.

Subthreshold somatic depolarization has been shown recently to modulate presynaptic neurotransmitter release in cortical neurons. To understand the mechanisms underlying this mode of signaling in the axons of dentate granule cells (hippocampal mossy fibers), we have combined two-photon Ca 2ϩ imaging with dual-patch recordings from somata and giant boutons forming synapses on CA3 pyramidal cells. In intact axons, subthreshold depolarization propagates both orthodromically and antidromically, with an estimated length constant of 200 -600 m depending on the signal waveform. Surprisingly, presynaptic depolarization sufficient to enhance glutamate release at mossy fiber-CA3 pyramidal cell synapses has no detectable effect on either basal Ca 2ϩ -dependent fluorescence or actionpotential-evoked fluorescence transients in giant boutons. We further estimate that neurotransmitter release varies with presynaptic Ca 2ϩ entry with a 2.5-power relationship and that depolarization-induced synaptic facilitation remains intact in the presence of highaffinity presynaptic Ca 2ϩ buffers or after blockade of local Ca 2ϩ stores. We conclude that depolarization-dependent modulation of transmission at these boutons does not rely on changes in presynaptic Ca 2ϩ .

Anatomical and electrophysiological evidence has raised the possibility that corelease of GABA and glutamate occurs at hippocampal mossy fibre synapses which, however, lack the vesicular GABA transporter VGAT. Here, we apply immunogold cytochemistry to show that GABA, like glutamate, has a close spatial relation to synaptic vesicles in rat mossy fibre terminals, implying that a mechanism exists to package GABA in synaptic vesicles. We also show that GABAA and AMPA receptors are colocalized at mossy fibre synapses. The expression of GABA and GABAA receptors is, however, weaker than in inhibitory synapses. Electrical stimuli that recruit mossy fibres evoke monosynaptic GABAA receptor-mediated signals in post-synaptic targets that show marked frequency-dependent facilitation and sensitivity to group II metabotropic receptors, two features that are characteristic of mossy fibre transmission. These results provide further evidence for GABA and glutamate cotransmission at mossy fibre synapses, although paired pre- and post-synaptic recordings will be required to determine the role of GABA at this unusual synapse.