Dominique Engel

Disruption of ClC-3, a Chloride Channel Expressed on Synaptic Vesicles, Leads to a Loss of the Hippocampus

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

Patch-clamp recording from mossy fiber terminals in hippocampal slices

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2–5 μm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

Presynaptic action potential amplification by voltage-gated Na+ channels in hippocampal mossy fiber boutons.

Action potentials in central neurons are initiated near the axon initial segment, propagate into the axon, and finally invade the presynaptic terminals, where they trigger transmitter release. Voltage-gated Na(+) channels are key determinants of excitability, but Na(+) channel density and properties in axons and presynaptic terminals of cortical neurons have not been examined yet. In hippocampal mossy fiber boutons, which emerge from parent axons en passant, Na(+) channels are very abundant, with an estimated number of approximately 2000 channels per bouton. Presynaptic Na(+) channels show faster inactivation kinetics than somatic channels, suggesting differences between subcellular compartments of the same cell. Computational analysis of action potential propagation in axon-multibouton structures reveals that Na(+) channels in boutons preferentially amplify the presynaptic action potential and enhance Ca(2+) inflow, whereas Na(+) channels in axons control the reliability and speed of propagation. Thus, presynaptic and axonal Na(+) channels contribute differentially to mossy fiber synaptic transmission.

Plasticity of rat central inhibitory synapses through GABA metabolism

1 The production of the central inhibitory transmitter GABA (γ‐aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity. 2 We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA‐transaminase (GABA‐T) was blocked by γ‐vinyl‐GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3‐mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole‐cell patch clamp technique. 3 Elevated intra‐synaptic GABA levels after block of GABA‐T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations. 4 The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs. 5 We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up‐regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity‐dependent regulation of GABA synthesis in mammals.

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na+ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca2+ channels, this generates a large and brief presynaptic Ca2+ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.

LAMINAR DIFFERENCE IN GABA UPTAKE AND GAT-1 EXPRESSION IN RAT CA1

1 The axonal plexus of most hippocampal interneurons is restricted to certain strata within the target region. This lamination suggests a possible functional heterogeneity of inhibitory synapses between different interneurons and CA1 pyramidal cells. 2 We therefore compared inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in CA1 pyramidal cells, which were evoked from two stimulation sites (stratum oriens and stratum radiatum). Stimulation in stratum oriens yielded faster decaying IPSPs and IPSCs than stimulation in stratum radiatum. 3 IPSP and IPSC kinetics were regulated by GABA uptake in both layers as indicated by the prolongation of the signals under tiagabine, a GAT‐1 (neuronal GABA plasma membrane transporter)‐specific GABA‐uptake blocker. However, the effect of tiagabine was significantly more pronounced following stimulation in stratum radiatum than in stratum oriens (prolongation of IPSC half‐decay time by 167 vs. 115 %, respectively). 4 In situ hybridization with antisense mRNA for the GABA‐synthesizing enzyme glutamate decarboxylase (GAD65/67) and the GABA transporter GAT‐1 showed that the proportion of interneurons expressing GAT‐1 was lower in stratum oriens than in stratum radiatum/lacunosum‐moleculare. 5 From these functional and molecular data we conclude that the regulation of IPSP and IPSC kinetics in CA1 pyramidal cells by neuronal GABA uptake differs between layers. Our findings suggest that this laminar difference is caused by a lower expression of GAT‐1 in interneurons in stratum oriens than in stratum radiatum/lacunosum‐moleculare.

Efficacy of background GABA uptake in rat hippocampal slices

GABA uptake is crucial for the termination of inhibitory synaptic events. In addition, GABA transporters may also control the level of diffusely distributed GABA in the extracellular space. We analysed this function by superfusing rat hippocampal slices with different concentrations of GABA. Whole-cell patch clamp recordings of CA1 pyramidal cells revealed small increases in chloride conductance at 5–10 μM GABA which increased dramatically upon addition of the GABA uptake blocker tiagabine. Tiagabine alone induced a significant chloride conductance indicating that spontaneous release of GABA in hippocampal slices is neutralized by GAT-1, the main hippocampal GABA transporter. Thus, GAT-1 clears the extracellular space in the hippocampus from diffusely distributed GABA with high efficacy.

Presence of γ-aminobutyric acid transporter mRNA in interneurons and principal cells of rat hippocampus

After release, neurotransmitters are removed from the extracellular space by high-affinity uptake. Specific sodium-dependent transporters serve this function for the inhibitory transmitter gamma-aminobutyric acid (GABA). However, it is largely unknown to which proportion GABA is taken up by GABAergic interneurons, glia cells or principal neurons. We analyzed the distribution of mRNA for the main GABA-transporter subtype in the hippocampus, GAT-1, in adult rats. Most interneurons were strongly stained for GAT-1 mRNA, indicating re-uptake by the GABA-releasing cells. Surprisingly, prominent signals for GAT-1 were also found throughout the principal cell layers (granule and pyramidal cells). These data indicate that GABA transporters may be present in non-GABAergic projection cells of the rat hippocampus which contribute to the clearance of GABA from the extracellular space.

Age-dependence of the anticonvulsant effects of the GABA uptake inhibitor tiagabine in vitro

Epileptic syndromes frequently start at childhood and therefore it is crucial to test new anticonvulsants at immature stages of the nervous system. We compared the effects of the gamma-aminobutyric acid (GABA) uptake inhibitor tiagabine [(R)-N-(4, 4-bis(3-methyl-2-thienyl)but)3-en-1-yl nipecotic acid] on low-Mg(2+)-induced epileptic discharges in brain slices from rat pups (p 5-8) and juvenile animals (p 15-20). In tissue from rat pups, tiagabine slightly reduced epileptiform activity in hippocampal area CA1 but had no effect in the entorhinal cortex. In juvenile rats, epileptiform discharges were unaffected in CA1 but suppressed by 60% in the entorhinal cortex. While tiagabine increases its efficacy with age, in-situ hybridisation and PCR analysis show that mRNA coding for the neuronal GABA-transporter GAT-1 is already present at p 5. We therefore conclude that the increasing efficacy of tiagabine during ontogenesis is due to functional maturation of GABAergic synapses rather than to up-regulation of GAT-1 expression.

Endogenous zinc depresses GABAergic transmission via T-type Ca2+ channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells

•  Zinc inhibits ionotropic receptors commonly found at central synapses, as well as a wide variety of voltage‐activated ion channels that modulate neuronal excitability and neurotransmitter release. •  We found that zinc chelation facilitated GABAergic signalling in dentate granule cells and that blocking T‐type Ca2+ channel activity abolished this effect. Zinc chelation reduced spike threshold, increased spike width and shifted the input–output relationship in dentate interneurones, which is consistent with increased excitability. •  In granule cells, zinc chelation narrowed the window for the integration of glutamatergic inputs originating from perforant path synapses. •  These results demonstrate that zinc modulates dentate interneurone function and regulates spike routing to local and hippocampal targets.