Presynaptic glycine receptors on hippocampal mossy fibers

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Abstract

Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600 pS in young to ∼130 pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8–17 per young, and 1–3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber – CA3 synapse.

Introduction

Glycine is the major inhibitory neurotransmitter in spinal cord and brain stem [1], [2], where glycinergic interneurons control neuronal excitation through the activation of postsynaptic glycine receptors (GlyRs). GlyRs are pentameric ligand-gated chloride channels of the Cys-loop receptor family, which open in response to glycine binding [3]. They are also expressed presynaptically. Presynaptic GlyRs have been shown to contribute to the regulation of glutamatergic synaptic transmission in the brain stem (calyx of Held) [4]. There, GlyR activation induces weak depolarizations of the nerve terminal and thereby causes an enhancement of glutamate release [4]. In contrast, at axon terminals of rod bipolar cells in the retina presynaptic GlyRs inhibit transmission due to either hyperpolarization or shunting [5]. Thus, the direction in which transmitter release is regulated by presynaptic GlyRs varies within the nervous system.

GlyR expression is not restricted to brain regions with well established glycinergic projections. Evidence for GlyRs in the hippocampus has been reported [6], and a recent study has shown that activation of putative presynaptic GlyRs enhances spontaneous, action potential-dependent glutamate release onto isolated hilar neurons of the hippocampus [7]. However, in the same study evoked glutamate release in the hippocampal slice preparation was not altered by glycine application, whereas axonal excitability was slightly enhanced. Thus, discrepancies exist between results obtained under different experimental conditions, and the precise localization and function of GlyRs in the hippocampus have remained unclear. Pharmacological evidence suggests that transmitter release from large mossy fiber boutons (MFBs) of hippocampal granule cell axons in the dentate gyrus contributes substantially to the effects observed when using isolated cells [7].

Mossy fiber boutons allow for analysis of presynaptic ion channels by direct patch-clamp recordings [8]. We have previously used this approach to analyze presynaptic GABAA receptors [9], which had been suggested to be expressed at this synapse [10], [11]. Here, we examined the expression of GlyRs at MFBs. We found functional presynaptic GlyRs of low glycine affinity. Single-channel analysis provided evidence for presynaptic expression of both homo- and hetero-oligomeric GlyRs. Moreover, and in contrast to presynaptic GABAA receptors [9], the density of presynaptic GlyRs in hippocampal MFBs was found to decline substantially during postnatal development.

Section snippets

Methods

Hippocampal slice preparation. Animal care and use, as well as experiments were conducted according to the guidelines of the European Union. Wistar rats (P12 to 3 weeks, and older than 3 months) were decapitated under deep isoflurane anesthesia. The brain was removed, dissected, and sliced at a thickness of 300 μm using a custom-built vibratome [12]. The following solution was used for dissection and storage of slices (in mM): 75 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 25 glucose, 100 sucrose, 0.5

Strychnine-sensitive GlyRs on hippocampal MFBs

Glycine was focally puff-applied to whole-cell recorded, voltage-clamped MFBs (Fig. 1Aa). Application of 1 mM glycine induced transient inward currents in immature MFBs obtained from P12 animals when symmetrical chloride conditions were used (VH = −80 mV; mean peak amplitude 51 ± 4 pA; n = 10). These puff-evoked currents were completely abolished by bath application of 3 μM strychnine, a specific GlyR antagonist (Fig. 1Aa). A lower concentration of glycine (100 μM) evoked significantly smaller currents (VH

Discussion

In this study, we provide direct evidence for the expression of low-affinity strychnine-sensitive GlyRs on hippocampal MFBs. These presynaptic GlyRs are chloride-permeable and display single-channel conductances similar to those previously reported for postsynaptic GlyRs. Notably, GlyR expression on MFBs declines substantially during postnatal development. The highest GlyR expression levels found here in immature tissue (P12) produced a conductance density of ∼600 pS per MFB, which is

Acknowledgments

We thank I. Wüllenweber for technical assistance. This work was supported by the Hertie Foundation, Max-Planck Society, Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

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