Chloride regulatory mechanisms and their influence on neuronal excitability

The chloride concentration in neurons is in general established by the precise functional expression of the sodium-potassium-chloride cotransporter one (NKCC1) and the potassium-chloride cotransporter two (KCC2). NKCC1 raises the intracellular chloride concentration, while KCC2 extrudes chloride. The intracellular chloride concentration determines the strength and direction of γ-aminobutyric acid (GABA) receptor-mediated transmission. In general, the intracellular chloride concentration in neurons is low and causes GABA-mediated inhibition. However, the intracellular chloride concentration in immature neurons is high leading to GABAergic depolarization, which can cause excitation. The effects of excitatory GABA signaling in early development is still unclear. It has been speculated that excitatory GABA, causing general depolarization in neurons, has profound effects on neuronal activity and neuronal maturation. Therefore, I studied in collaboration with Carsten Pfeffer the development of the hippocampal network during the early phase of postnatal development under conditions when excitatory GABA action is abolished. Here, sodium-potassium-chloride cotransporter one (NKCC1) knockout mice (Nkcc1-/-) were used to reduce the intracellular chloride concentration in immature neurons. Young CA1 pyramidal neurons of Nkcc1-/- mice showed diminished GABAergic depolarization. I found that this reduction was sufficient to cause a delay in the maturation of glutamatergic and GABAergic synapses. This suggests that GABAergic excitation during early postnatal development, increasing the network activity, facilitates the maturation of synaptic networks. GABAergic depolarization in Nkcc1-/- mice was reduced but not completely abolished; suggesting that additional chloride loading mechanisms might exist. As the anionexchanger three (AE3) was proposed to contribute to chloride accumulation, AE3 knockout (Ae3-/-) mice were also studied. I could not detect any changes in intracellular chloride concentration after loss of AE3 at postnatal day one (P1). However at P5, the disruption of AE3 affected the early network activity pattern, indicating an effect of reduced intracellular chloride concentration. These data showed that NKCC1 establishes high intracellular chloride concentration in neurons providing the basis for GABAergic excitation. The role of AE3 is still not clear; it might contribute to the chloride accumulation in neurons. In addition to the function of chloride transporters, chloride conductive channels are likely to modulate the intracellular chloride concentration, and therefore could influence neuronal excitability. Especially ClC-2 has been suggested to contribute to chloride extrusion. I investigated the functional role of the voltage-gated chloride channel ClC-2. As specific blockers for ClC-2 are not available, I used ClC-2 knockout (Clcn2-/-) mice. It has been proposed that ClC-2 constitutes a pathway for chloride extrusion to maintain the inhibitory action of GABA in mature neurons. My data provide direct evidence that ClC-2 mediates fast chloride extrusion preventing chloride accumulation. Chloride extrusion by ClC-2 seemed to be important especially in adult hippocampal pyramidal neurons where GABAA receptor activation occurs in high frequency bursts. Interestingly, the chloride-conductance of ClC-2 occurs first in the second postnatal week of developing mice, suggesting that ClC-2 is important in fully developed neurons, but might not be important in immature neurons. Surprisingly, neurons in Clcn2-/- mice have a very high membrane resistance compared to WT animals, indicating that ClC-2 is active during the resting membrane potential. This might be a general feature of neurons, as I recorded the chloride conductance of ClC-2 in various neuron types. I showed that the resting conductance of ClC-2 affects resting membrane properties, which determine the neuronal excitability. As a consequence, the loss of ClC-2 increases the excitability of a neuron; however, it does not cause hyperexcitation of the hippocampal network. Even more, the network excitability is reduced in ClC-2 KO mice in comparison to the WT. This reduction is caused by an increased inhibition. I found that ClC-2 expressing interneurons increased their inhibitory action onto pyramidal cells after loss of ClC-2. Taken together, my data reveal that ClC-2 plays a dual role in adult neurons. First, ClC-2 contributes a fast mechanism to extrude chloride after chloride accumulation. Second, ClC-2 provides the chloride leak conductance under resting conditions. The loss of ClC-2 leads to a higher excitability of the neuron due to a strongly increased membrane resistance. Importantly, hyper-excitability of the neuronal network is circumvented by a parallel enhanced inhibition, which can explain the absence of an epileptic phenotype in mice

Molecular and Electrophysiological Characterization of GFP-Expressing CA1 Interneurons in GAD65-GFP Mice

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials

Patch them all - fully HTS-compatible automated patch clamping of 384 cells at once for massively parallel ion channel screening

<p>Automated patch clamping is well established within academic research and drug discovery efforts. Still, there is a longstanding desire to have gold standard electrophysiology compatible with primary ion channel drug screening requirements. We here present a chip-based, modular approach for massively parallel patch clamp recordings. Using microstructured glass bottom microtitre plates, recordings from 384 cells are performed in an automated fashion. The recording system contains 384 patch clamp amplifiers and is integrated into existing, state-of-the-art 384 head pipetting robots, so all experiments are done in parallel, resulting in a throughput of several thousand data point per hour. Such a robust and modular approach is themselves easily integrable into HTS environment. Finally patch clamp has gone HTS! Success rates achieved are routinely over 85 %. A complete run takes less than 15 minutes. From single point application to cumulative dose responses, there are no limitations with the number of additions. Solution onset is fast, <50 ms, current responses are highly reproducible and brief compound exposures, <1s, can be obtained.</p

GFP-positive GABAergic interneurons.

<p>A: Maximal projection image illustrating the distribution of GFP-labeled profiles in the CA1 layers. Calbindin (red), labeling a fraction of pyramidal cells, is only shown to facilitate recognition of the layers. B: Distribution of GFP-labeled interneurons in the hippocampal CA1 area in GAD65-GFP mice. C: Percentage of GFP-labeled cells that expressed GABA (blue), GAD67 (red) or both (purple). Data from 268 GFP cells; 10 sections. D: Example of triple immunostaining for GFP (green), GABA (blue) and GAD67 (red). Scale bars are 30 µm. Abbreviations of CA1 layers: Or - oriens; Pyr – pyramidale; Rad – radiatum; LM – lacunosum moleculare.</p

Firing properties of GFP-positive interneurons.

<p>A: Representation of all recorded GFP-labeled interneurons, arranged according to characteristics of their firing patterns and their classification in 5 groups. Each segment represents a single interneuron. Inner ring: adapting (Ad; dark blue) and strongly adapting (S-Ad; dark red) cells. Second ring: cells showing delayed onset (DO; blue) and immediate onset (IO; red) firing. Third ring: cells displaying regular (reg; light blue) and irregular (irr; light red) firing. Interneurons were divided in five groups (1–5) as indicated with yellow and green colors. The letters correspond to example cells (a–g) in B and C. B: Examples of firing patterns of example cells a–g (as indicated in A). Upper traces show firing around threshold, middle traces show responses to hyperpolarizing steps (100 pA step size) and intermediate firing and lower traces show maximal firing. C: Morphology of example cells a–g (same as in B). Dendrites are shown in black, axons in red.</p

The majority of GFP-positive interneurons contain reelin or VIP.

<p>A: Immunohistochemistry for reelin (red) and vasoactive intestinal peptide (VIP; blue). Most GFP-positive interneurons (green) contained either reelin (yellow arrowheads) or VIP (blue arrowheads). Very few GFP-positive cells were lacking both (green arrowheads). B: Immunohistochemistry for parvalbumin (PV; red) and somatostatin (SOM; blue), showing minimal overlap with GFP-positive interneurons (green arrowheads). Red and blue arrowheads point to GFP-negative parvalbumin- and somatostatin-positive interneurons. C: Summary for all GFP-positive interneurons. D: Distribution of reelin- and VIP-containing GFP-labeled interneurons in the layers of the CA1 area. Overlap between both markers are indicated with purple. Scale bars are 30 µm.</p

<b>Table 2.</b> Morphological properties of GFP-labeled interneurons.

<p>*Tangential cells have an orientation close to 90 degrees; 0 degrees reflect radially oriented cells (similar to pyramidal cells).</p><p>No significant differences in morphological parameters between groups were detected (ANOVA). Values are given as mean ± standard error.</p

NKCC1-dependent GABAergic excitation drives synaptic network maturation during early hippocampal development.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl-/HCO3- exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl- accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses