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J Neurosci. 2008 Apr 30; 28(18): 4635–4639.
doi: 10.1523/JNEUROSCI.0908-08.2008
PMCID: PMC6670448
PMID: 18448640

GABAergic Depolarization of the Axon Initial Segment in Cortical Principal Neurons Is Caused by the Na–K–2Cl Cotransporter NKCC1

Stanislav Khirug, 1 ,* Junko Yamada, 1 ,* Ramil Afzalov, 1 Juha Voipio, 1 Leonard Khiroug, 2 and Kai Kailacorresponding author 1 , 2
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

GABAergic terminals of axo-axonic cells (AACs) are exclusively located on the axon initial segment (AIS) of cortical principal neurons, and they are generally thought to exert a powerful inhibitory action. However, recent work (Szabadics et al., 2006) indicates that this input from AACs can be depolarizing and even excitatory. Here, we used local photolysis of caged GABA to measure reversal potentials (E GABA) of GABAA receptor-mediated currents and to estimate the local chloride concentration in the AIS compared with other cellular compartments in dentate granule cells and neocortical pyramidal neurons. We found a robust axo-somato-dendritic gradient in which the E GABA values from the AIS to the soma and dendrites become progressively more negative. Data from NKCC1 −/− and bumetanide-exposed neurons indicated that the depolarizing E GABA at the AIS is set by chloride uptake mediated by the Na–K–2Cl cotransporter NKCC1. Our findings demonstrate that spatially distinct interneuronal inputs can induce postsynaptic voltage responses with different amplitudes and polarities as governed by the subcellular distributions of plasmalemmal chloride transporters.

Keywords: axon initial segment, NKCC1, KCC2, synapse, chloride, caged GABA

Introduction

The axon initial segment (AIS) of cortical principal neurons is selectively innervated by a subclass of interneurons, the chandelier or axo-axonic cells (AACs) (Freund and Buzsaki, 1996; Somogyi et al., 1998). This strategic location of GABAergic synapses on the AIS has generally been thought to endow the AACs with a powerful inhibitory action on the output of principal neurons. However, recent work on neocortical layer 2/3 pyramidal neurons in rats and humans has shown that AACs can evoke depolarizing and even excitatory instead of inhibitory responses, because the reversal potential of GABAA receptor-mediated currents (E GABA) in the AIS is at a much more depolarized level (by ∼20 mV) than the resting membrane potential (Szabadics et al., 2006). In contrast, somatic and dendritic GABAA receptor-mediated responses evoked by basket cells in these neurons are hyperpolarizing. This compartmentalized E GABA indicates a steady-state intracellular gradient of Cl− that was attributed to a differential subcellular distribution of the chloride-extruding K-Cl cotransporter, KCC2, with a high level of KCC2 in the somata and dendrites and a very low level in the AIS (Szabadics et al., 2006).

The central role of KCC2 in the generation of a transmembrane chloride gradient that results in hyperpolarizing GABAergic responses in cortical neurons is well established (Rivera et al., 1999; Payne et al., 2003). However, the mere absence of KCC2 cannot account for the strikingly positive E GABA values at the AIS, because in the complete absence of Cl− transport, E GABA would achieve a level close to and only slightly more depolarized than the resting membrane potential (Kaila et al., 1993; Farrant and Kaila, 2007). Thus, the depolarizing GABAA receptor-mediated response at the AIS requires the presence of a Cl− uptake mechanism. The present work focuses on the identification of this mechanism and on its role in the subcellular compartmentalization of E GABA.

Materials and Methods

Experimental animals.

The experiments were approved by the Animal Care and Use Committee, University of Helsinki. The animals used in this study included a mouse line with disruption of the Slc12a2 gene that codes for the Na–K–2Cl isoform NKCC1 (Pace et al., 2000), as well as a transgenic line expressing membrane-targeted enhanced green fluorescent protein (EGFP) in a small subset of dentate gyrus cells (DGCs) under the Thy1 promoter (Thy1–mGFPs, L21) (De Paola et al., 2003; Galimberti et al., 2006). These mice were generously provided by Dr. Beverly H. Koller (Department of Genetics, University of North Carolina, Chapel Hill, NC) and Dr. Pico Caroni (Friedrich Miescher Institut, Basel, Switzerland), respectively. In experiments on NKCC1 functionality, we used hippocampal slices from homozygous NKCC1−/− animals and their wild-type (WT) (NKCC1 +/+) littermates. Heterozygous Thy1-mGFPs L21 mice were used for subcellular real-time imaging of DGCs in gramicidin patch-clamp recordings (see below). Some experiments were performed on slices from Wistar rats.

Preparation and maintenance of slices.

Hippocampal slices and neocortical slices from postnatal day 16–20 mice and rats were used in the present study. The animals were anesthetized with pentobarbital, and 350 μm transverse brain slices were cut using a VT 1000S vibratome (Leica, Wetzlar, Germany). Slices were bathed in the standard physiological solution containing the following (in mm): 124 NaCl, 3 KCl, 2 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, 2 MgSO4, and 10 d-glucose (equilibrated with 95% O2 and 5% CO2, pH 7.4, at the experimental temperature of 32°C). Tetrodotoxin (1 μm; Sigma, St. Louis, MO) and CGP55845A [(2S)-3-[(15)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl)-(phenylmethyl)phosphinic acid] (3 μm; Sigma) were added to block Na+-dependent action potentials and GABAB receptors. The slices were allowed to recover at 36°C for 1 h before the experiments were started. Slices were positioned in a submerged-type recording chamber (volume, ∼1 ml). During the experiments, the chamber was continuously perfused at a rate of 1 ml/min. Bumetanide and bicuculline (Sigma) were applied at a concentration of 10 μm (Sipilä et al., 2006). The data on bumetanide are from slices that were exposed to the drug throughout the experiment.

Electrophysiology.

Gramicidin perforated patch-clamp recordings were done using an EPC 10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). The patch pipette solution contained 150 mm KCl, 10 mm HEPES (pH adjusted to 7.2 with KOH), and 50 μg/ml gramicidin D. Gramicidin was dissolved from a freshly thawed aliquot of a dimethylsulfoxide stock solution (50 mg/ml) just before each experiment. Patch pipettes were fabricated from borosilicate glass (Harvard Apparatus, Edenbridge, UK), and their resistance ranged from 4.5 to 6.5 MΩ. Within 30 min after gigaseal formation, the access resistance dropped to 50–100 MΩ, and all experiments with steady increases or sudden drops in this parameter were discarded. The voltage-clamp data were corrected off-line for the series-resistance effect. Under the present conditions, the recorded intracellular voltage is affected by a calculated −3.6 mV liquid-junction potential (Barry, 1994) and by a Nernst potential of approximately +4 mV across the patch, which is set by the K+ concentration that is higher in the pipette than in the neuron (Kim and Trussell, 2007). These errors practically cancel each other, and, hence, no liquid junction potential corrections were applied on the recorded voltages. In the whole-cell patch-clamp recordings, the composition of the patch pipette solution was the following (in mm): 6 KCl, 111 K-gluconate, 0.5 CaCl2, 2 NaOH, 10 glucose, 10 HEPES, 2 Mg-ATP, and 5 BAPTA, pH was adjusted to 7.3 with KOH. The resistance of the patch pipettes was 6.5–7.5 MΩ. The membrane potential values were corrected for a calculated liquid junction potential of −15.0 mV (Barry, 1994). To minimize the yield of neurons at an immature stage of development, the recordings were made in the outer granule cell layer in the upper blade of the dentate gyrus. The input resistance of the neurons was 412 ± 19 MΩ (cf. Liu et al., 1996). We did not find any correlation between input resistance and the E GABA gradients examined in the present work. It is worth noting that the axon of DGCs has a space constant of >400 μm (Alle and Geiger, 2006), and, with the 50 μm distance between the recording site and voltage-clamped soma, a loss of space clamp is not likely to have a significant effect on the present results. Here, one should also note that E GABA is fairly close to the resting potential, unlike, for example, reversal potentials for currents mediated by ionotropic glutamate receptors. Loss of space clamp is expected to be even smaller in the proximal parts of the dendrites (also recorded at a 50 μm distance from the soma) than at the AIS. In the present context, it is notable that, in the whole-cell clamp experiments in which the calculated somatic E GABA is −68 mV (corrected for intracellular bicarbonate; E Cl of −77 mV) (Kaila et al., 1993; Kaila, 1994; Farrant and Kaila, 2007), the DGCs were obviously able to cope with part of this minor imposed load (7 mm Cl−) even in the somatic compartment, and, hence, the measured somatic E GABA is more negative (−74.2 mV, corresponding to 4.9 mm intracellular Cl−). A similar deviation of the measured intracellular Cl− from the calculated passive distribution was observed in the neocortical layer 2/3 pyramidal neurons.

Local photolysis of caged GABA.

GABA was photolyzed from the α-carboxy-2-nitrobenzyl (CNB)-caged GABA compound (Invitrogen, Carlsbad, CA) using local uncaging as described previously (Khirug et al., 2005). Caged GABA (2.5 mm) was dissolved in the physiological solution and delivered at a flow rate of 1 μl/min to the vicinity of the patch-clamped cell using an UltraMicroPump II syringe pump (WPI, Sarasota, FL) and a syringe with an inner tip diameter of 100 μm. For local photolysis of caged GABA, the 351–364 nm output of a continuous emission argon–ion laser (Coherent Enterprise 653; Coherent, Santa Clara, CA) was delivered to the slice via a multimode optical fiber through an Olympus (Tokyo, Japan) LUMPlanFl 60× water-immersion objective. The UV beam yielded an uncaging spot of ∼10 μm in diameter (Khirug et al., 2005) that was focused either at the soma or at the AIS or dendrite at a distance of 50 μm from the soma. As shown in the above study, the spatial profile of GABAA receptor activation under these conditions precludes any “mixed” responses in which, for instance, the somatic response would be influenced by GABAergic currents generated by the AIS or dendrites. This conclusion was verified in control experiments with flashes of variable positions, durations, and intensities within the range used presently. UV laser power (1–10 mW at the objective output) and flash duration (10–50 ms) were set at a level that provided a good signal-to-noise ratio for uncaging-evoked currents. In general, less power and shorter flashes were required to induce somatic responses, perhaps mainly because larger membrane surface areas were covered by the uncaging spot at the soma compared with dendrites and the AIS. At each location, current–voltage relationship was determined by varying the holding potential from −100 to −40 mV (10 mV steps). Voltage was stepped 200 ms before each photolysis event and held for 600 ms. Control experiments showed that changing the voltage-step protocol from hyperpolarizing–depolarizing to depolarizing–hyperpolarizing had no effect on the estimated E GABA. Peak current amplitudes were measured and plotted against the holding potential to obtain an estimate of E GABA. For each cell, the somatic, dendritic, and axonal sites were tested in randomized manner. The gradient (ΔE GABA) at the AIS or dendrite was defined as the difference of the local value of E GABA and the E GABA at the soma. Control experiments showed that the laser flash evoked no responses in the absence of the CNB-caged GABA. Two types of control experiments were made to ensure that the present technique does not lead to activity-dependent ionic shifts (intracellular anion shifts and/or generation of extracellular K+ transients) that might have an effect on E GABA (Kaila et al., 1997; Farrant and Kaila, 2007). First, we found that changing the intensity of the laser pulse within the range used presently had no effect on E GABA. Second, detailed data obtained from the three subcellular compartments examined in five neurons showed that the value of E GABA was identical when derived from data points taken at different delays from the onset of the uncaging pulse (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Finally, we have never observed a time-dependent depolarizing shift in E GABA in response to the uncaging, which would be expected in response to an ion shift. Hence, the E GABA values recorded in this study are reliable estimates of the steady-state value of this parameter under the present experimental conditions.

Visualization of the neurons.

The overall appearance of the neurons in the slices was visualized using Dodt infrared optics (Luigs and Neumann, Ratingen, Germany). Gramicidin patch recordings were made from EGFP-expressing DGCs in slices from the Thy1–mGFPs L21 mice (see above). In addition, gramicidin patch recordings were made from DGCs from NKCC1−/− mice and their littermates. The DGCs were visualized by single-cell electroporation according to Haas et al. (2001). Briefly, a glass electrode similar to patch pipette (6–8 mΩ) was filled with intracellular solution containing 50–75 μm Alexa Fluor 488 (Invitrogen). The electrode was positioned in the immediate vicinity of a targeted cell, which was then electroporated using single hyperpolarizing square pulses (5–10 ms, 16–18 V) from Axoporator 800A (Molecular Devices, Sunnyvale, CA). In the whole-cell patch-clamp experiments, visualization of the neurons and their axons and dendrites was achieved by adding Alexa Fluor 488 (50 μm) directly to the patch pipette solution. Both EGFP and Alexa Fluor 488 were excited at 488 nm (Dual Calypso laser; Cobolt, Stockholm, Sweden), and their fluorescence was recorded using a 520 nm long-pass filter. A confocal microscope (Radiance 2100; Bio-Rad, Hemel Hempstead, UK) was used for real-time imaging and three-dimensional reconstruction of confocal image stacks. In the DGCs, identification of the axons is straightforward because these cells are devoid of basal dendrites in their axonal field. In the layer 2/3 neocortical pyramidal neurons, axons were readily distinguished from dendrites by lack of branching and absence of dendritic spines. Care was taken to select pyramidal neurons that had a morphology and orientation that permitted directing the uncaging beam to the AIS in a manner that did not affect any dendritic processes.

Statistical analysis.

Averaged data are presented as mean ± SEM. Statistical significance was assessed using Wilcoxon's signed-rank test or Student's t test as indicated in the figure legends.

Results

First, we examined whether an E GABA gradient can be observed under nonperturbed conditions in DGCs that are known to be heavily innervated by AACs (Freund and Buzsaki, 1996; Somogyi et al., 1998). Currents evoked by uncaging of GABA were recorded using the gramicidin perforated patch-clamp technique in hippocampal slices in which EGFP was expressed in a subset of DGCs (De Paola et al., 2003; Galimberti et al., 2006) (see Materials and Methods). Local uncaging of GABA (Khirug et al., 2005) on AISs, somata, and dendrites produced currents with E GABA values of −59.4 ± 1.5 mV (n = 14), −65.8 ± 1.2 mV (n = 14), and −70.9 ± 1.5 mV (n = 10), respectively (Fig. 1). These data indicate a marked depolarizing driving force of ∼13 mV for the GABAergic AIS responses from the resting membrane potential of −72.7 ± 2.6 mV (n = 14). The dendritic responses reversed at a more negative level (by ∼5 mV) than the somatic ones, which shows that, in addition to the axo-somatic E GABA gradient (ΔE GABA of ∼6.5 mV), a somato-dendritic ΔE GABA is also maintained by the DGCs. Assuming an intracellular pH of ∼7.2, the intracellular levels of Cl− ([Cli]) calculated on the basis of the above E GABA values (Kaila et al., 1993; Farrant and Kaila, 2007) are 11, 7.9, and 6.0 mm for the AIS, soma, and dendrite, respectively. A set of control experiments showed that the currents induced by GABA uncaging were fully blocked by 10 μm bicuculline (data not shown).

An external file that holds a picture, illustration, etc. Object name is zns0180847720001.jpg

Gramicidin patch-clamp recordings of GABAA receptor-mediated currents and E GABA in EGFP-expressing DGCs in mouse hippocampal slices. A, Confocal image of a DGC (inverted gray scale) with the three uncaging spots indicated by circles and flash symbols. B, Recordings of uncaging-induced GABAA-mediated currents and E GABA at the soma, AIS, and dendrite (at a distance of 50 μm from the soma). Uncaging flash indicated by horizontal bars in this and subsequent figures. C, I–V curves based on the specimen recordings shown in B and normalized with respect to peak I GABA at the most negative holding potential (V h) tested. D, The mean values demonstrate a steep E GABA gradient with a markedly more positive E GABA at the AIS versus soma, whereas the dendritic responses reverse at a more negative potential. *p = 0.001, # p = 0.014, based on the Wilcoxon's signed-rank test. The values for n are shown in brackets. Error bars in this and subsequent figures represent SEM.

To examine whether the Na–K–2Cl transporter NKCC1 (Payne et al., 2003) is responsible for the depolarizing GABAergic response in the AIS, we made gramicidin perforated patch recordings from DGCs of NKCC1 −/− mice (Pace et al., 2000) and their WT littermates. Cells were visualized using electroporation for staining with Alexa Fluor 488 (Fig. 2). The WT neurons showed an axo-somatic ΔE GABA that was similar (∼5 mV) to the one described above in the Thy1–mGFP neurons with AIS, somatic, and dendritic E GABA values of −61.3 ± 2.4 mV (n = 14), −66.0 ± 1.6 mV (n = 22), and −71.0 ± 2.0 mV (n = 16), respectively. Notably, in identical recordings from the NKCC1−/− neurons, there was no axo-somatic E GABA gradient; the values for E GABA at the AIS and soma were similar, −70.5 ± 1.5 mV (n = 17) and −70.3 ± 1.5 mV (n = 18), respectively. However, a somato-dendritic ΔE GABA similar to that in the WT neurons was still observed (dendritic E GABA of −74.6 ± 1.7 mV; n = 15). These data indicate a key role for NKCC1 in the generation and maintenance of the chloride gradient that results in depolarizing GABA responses in the AIS of mouse DGCs and suggest that NKCC1 has no significant influence on dendritic E GABA. Interestingly, the above observations are not compatible with the idea that the somato-dendritic E GABA gradient is set by NKCC1 and KCC2. The putative transport mechanism underlying this gradient is not in the focus of the present work, but preliminary observations point to a role of a bicarbonate-dependent somatic transporter that accumulates chloride in the DGCs (our unpublished data).

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Gramicidin patch-clamp recordings of E GABA gradients in DGCs in slices from NKCC1 −/− mice and from their WT littermates. A, Confocal image of an NKCC1 −/− DGC (inverted grayscale) with the three uncaging spots indicated by flash symbols. B, Recordings of GABAA-mediated currents and E GABA in an NKCC1 −/− neuron. C, I–V curves based on the recordings shown in B. D, The mean values demonstrate that, in the NKCC1 −/− neurons, there is no E GABA gradient (ΔE GABA) between the AIS and the soma. In line with this, there is a significant difference of 5 mV in the AIS ΔE GABA between neurons from the NKCC1 −/− mice and their WT littermates (*p ≪ 0.001) based on the t test with the values for n shown in brackets. The corresponding dendritic ΔE GABA values are not statistically different (p = 0.2).

To examine possible species- and cell-specific differences in the generation of depolarizing AIS GABA responses, we performed whole-cell recordings of the above type in rat DGCs as well as in rat neocortical layer 2/3 pyramidal neurons. Previous work has shown that plasmalemmal ion transporters are able to maintain Cl− gradients and consequent E GABA gradients along the dendrites during a somatic Cl− load under whole-cell clamp conditions (Jarolimek et al., 1999; Khirug et al., 2005). A robust axo-somatic E GABA gradient (∼7 mV) was seen in rat DGCs, with AIS, somatic, and dendritic E GABA values of −67.2 ± 1.7 mV (n = 14), −74.2 ± 1.2 mV (n = 14), and −78.8 ± 1.1 mV (n = 14), respectively (Fig. 3 A–C). Chloride uptake mediated by NKCC1 can be selectively blocked using low concentrations of bumetanide (Payne et al., 2003). A series of experiments showed that the axo-somatic ΔE GABA in slices exposed to 10 μm bumetanide was collapsed, with E GABA in the AIS at −75.8 ± 1.4 mV (n = 7) and −73.7 ± 1.8 mV (n = 6) in the soma (Fig. 3 D). However, in agreement with the data on the NKCC1 −/− DGCs, the somato-dendritic ΔE GABA (∼5 mV) was not affected by bumetanide (dendritic E GABA of −78.6 ± 2.6 mV; n = 6).

An external file that holds a picture, illustration, etc. Object name is zns0180847720003.jpg

Whole-cell patch-clamp recordings of GABAA-mediated currents and E GABA gradients in rat DGCs. A, Sample recordings. B, Normalized I–V curves based on the recordings shown in A. C, The mean values reveal a robust axo-somato-dendritic E GABA gradient. *p = 0.001, # p = 0.001, based on the Wilcoxon's signed-rank test. D, In the presence of 10 μm bumetanide, E GABA at the AIS shifts to the level at the soma, whereas the somato-dendritic ΔE GABA is not affected. *p = 0.005, based on the t test. The values for n are shown in brackets.

The whole-cell recordings in rat neocortical layer 2/3 pyramidal neurons (Fig. 4) also revealed an E GABA gradient between the AIS and soma of ∼7 mV, with AIS, somatic, and dendritic E GABA levels of −69.2 ± 1.7 mV (n = 4), −76.1 ± 0.9 mV (n = 9), and −80.7 ± 1.0 mV (n = 6), respectively. In two of two slices tested (data not shown), the axo-somatic gradient was abolished by bumetanide (ΔE GABA of <1 mV), with no observable effect on the somato-dendritic ΔE GABA.

An external file that holds a picture, illustration, etc. Object name is zns0180847720004.jpg

Whole-cell patch-clamp measurements of GABAA receptor-mediated currents and E GABA in layer 2/3 neocortical neurons. A, Confocal image of pyramidal cell (inverted grayscale) with the three uncaging spots indicated by flash symbols; patch pipette containing Alexa Fluor 488 shown at the bottom right of the image. B, Sample recordings. C, Normalized I–V curves based on the recordings shown in B. D, The mean values demonstrate an axo-somato-dendritic E GABA gradient. *p = 0.02, # p = 0.03, based on the Wilcoxon's signed-rank test. The values for n are shown in brackets.

Discussion

There is much evidence that the strongly depolarizing action of GABA in immature cortical pyramidal neurons is attributable to Cl− uptake by NKCC1 (Yamada et al., 2004; Sipilä et al., 2006; Achilles et al., 2007; Brumback and Staley, 2008). During the first 2 postnatal weeks, the developmental downregulation of Cl− uptake by NKCC1 and the upregulation of Cl− extrusion by KCC2 produce a negative shift in E GABA that leads to hyperpolarizing GABAA responses (Rivera et al., 1999; Payne et al., 2003; Yamada et al., 2004). However, recent work has demonstrated depolarizing actions of GABA in presynaptic terminals, which are based on a higher [Cl−]i in the presynaptic axonal compartment than in somata (Price and Trussell, 2006; Stell et al., 2007). Furthermore, selective activation of AACs has been shown to evoke a depolarizing GABAA response at the AIS of mature neocortical pyramids (Szabadics et al., 2006).

Our work demonstrates that the depolarizing action of AACs at the AIS of cortical pyramidal neurons is attributable to Cl− uptake mediated by NKCC1. In view of the high expression levels of KCC2 in the somata and dendrites and absence in the AIS (Szabadics et al., 2006) (see also Gulyás et al., 2001), the axo-somatic Cl− gradient is readily explained on the basis of the distinct subcellular expression patterns of these two transporters. Interestingly, our work also points to the presence of a somatic Cl− uptake mechanism that generates the somato-dendritic gradient. Preliminary experiments suggest a bicarbonate-dependent mechanism of chloride uptake (our unpublished data), which will be a subject of future studies. Notably, however, the present results indicate that this putative transporter does not play a role in the NKCC1-dependent depolarizing responses generated by the AIS.

In view of the similarity of our observations in hippocampal DGCs (both mouse and rat) and neocortical layer 2/3 pyramidal neurons, it appears that the presence of a prominent steady-state axo-somato-dendritic E GABA gradient is a common property of most if not all types of cortical principal neurons (for data on cortical interneurons, see Martina et al., 2001; Banke and McBain, 2006). This is an important conclusion with several implications. First of all, it is obvious that the widely used manner of assigning a “typical” E GABA value to a given principal neuron is not correct. In view of the high subcellular target specificity of interneurons onto principal neurons (Freund and Buzsaki, 1996), a more appropriate approach is to specify the E GABA level of a given interneuronal input in the postsynaptic neuron. This also points to a need to reexamine the “developmental switch” (or rather “shift”) (Farrant and Kaila, 2007) from depolarizing to hyperpolarizing GABA transmission (Rivera et al., 1999; Payne et al., 2003) at the subcellular level. Such information is necessary for understanding the various modes of functionally inhibitory and excitatory actions of GABAergic transmission in the generation of early endogenous activity (Ben-Ari, 2002; Sipilä and Kaila, 2007), as well as of sharp-wave ripple complexes (Klausberger et al., 2003) and ongoing oscillations (Bartos et al., 2007; Mann and Paulsen, 2007) in the mature brain.

Footnotes

This work was supported by the Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Cultural Foundation. K.K. is a member of the Nordic Center of Excellence for Water Imbalance-Related Disorders and of the Finnish Center of Excellence in Molecular and Integrative Neuroscience Research. We thank Dr. Mark Farrant for discussions and comments on an early version of this manuscript and Dr. Peter Blaesse for providing expertise on electroporation experiments and helpful discussions. We are grateful to Dr. Beverly H. Koller (University of North Carolina, Chapel Hill, NC) and to Dr. Pico Caroni (Friedrich Miescher Institut, Basel, Switzerland) for providing the transgenic mice used in this study.

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