Differential maturation of chloride homeostasis

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Int. J. Devl Neuroscience 25 (2007) 479–489 

www.elsevier.com/locate/ijdevneu 

Differential maturation of chloride homeostasis in primary 

afferent neurons of the somatosensory system 

Daniel Gilbert a,1 , Christina Franjic-Würtz a , Katharina Funk a , 

Thomas Gensch b , Stephan Frings a, *, Frank Möhrlen a 

a Department of Molecular Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany 

b Institute for Neuroscience and Biophysics 1, Juelich Research Center, Leo-Brand-Strasse, 52425 Juelich, Germany 

Received 16 June 2007; received in revised form 23 July 2007; accepted 6 August 2007 

Abstract 

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their 

afferent signals in the spinal cord are subject to modulation by Cl currents. The underlying Cl homeostasis of nociceptive neurons, in particular 

its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl concentration, [Cl ] i , of somatosensory neurons in 

intact dorsal root ganglia of mice. Using two-photon fluorescence-lifetime imaging microscopy, we determined [Cl ] i in newborn and adult 

animals. We found that the somatosensory neurons undergo a transition of Cl homeostasis during the first three postnatal weeks that leads to a 

decline of [Cl ] i in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation– 

chloride co-transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl accumulation and extrusion is present. RT-PCR 

analysis showed that the transcription pattern of electroneutral Cl co-transporters does not change during the maturation process. 

These findings demonstrate that dorsal root ganglion neurons undergo a developmental transition of chloride homeostasis during the first three 

postnatal weeks. This process parallels the developmental ‘‘chloride switch’’ in the central nervous system. However, while most CNS neurons 

achieve homogeneously low [Cl ] i levels – which is the basis of GABAergic and glycinergic inhibition – somatosensory neurons maintain a 

heterogeneous pattern of [Cl ] i values. This suggests that Cl currents are excitatory in some somatosensory neurons, but inhibitory in others. Our 

results are consistent with the hypothesis that Cl homeostasis in somatosensory neurons is regulated through posttranslational modification of 

cation–chloride co-transporters. 

# 2007 ISDN. Published by Elsevier Ltd. All rights reserved. 

Keywords: Pain; Nociceptors; Dorsal root ganglia; Chloride homeostasis; Chloride transporters; Fluorescence-lifetime imaging 

1. Introduction 

Neuronal activity can be strongly influenced by Cl currents. 

Inhibitory Cl currents mediate GABAergic and glycinergic 

inhibition in the CNS. Excitatory Cl currents occur in neurons 

which accumulate Cl , as described for immature neurons of the 

CNS (Ben-Ari, 2002), for olfactory sensory neurons (Kaneko 

Abbreviations: 2P-FLIM, two-photon fluorescence-lifetime imaging 

microscopy; [Cl ] i , intracellular chloride concentration; CGRP, calcitoningene 

related peptide; MQAE, N-6-methoxyquinolinium acetoethylester; 

TRPV1, transient receptor potential vanilloid receptor type 1. 

* Corresponding author. Tel.: +49 6221 54 5661; fax: +49 6221 54 5627. 

E-mail address: s.frings@zoo.uni-heidelberg.de (S. Frings). 

1 Current address: Department of Physiology and Pharmacology, University 

of Queensland, Brisbane, QLD 4072, Australia. 

et al., 2004), for neurons in spinal and autonomic ganglia (review, 

Frings et al., 2000), as well as for neurons challenged by 

ischemia, inflammation, or neurological disorders (Payne et al., 

2003; Pond et al., 2006; De Koninck, 2007). The balance 

between inhibitory and excitatory Cl effects is determined by 

Cl uptake and Cl extrusion pathways active in each 

cell. Studies of neuronal Cl homeostasis have indicated that 

the Na + –K + –2Cl co-transporter NKCC1 provides the main 

route for Cl uptake, while KCC2 couples Cl extrusion to K + 

efflux. It is generally held that NKCC1 expression dominates in 

immature neurons of cortex and hippocampus, and that KCC2 

expression is only induced after birth (Lu et al., 1999; Rivera 

et al., 1999; Stein et al., 2004). This developmental transition – 

often termed ‘‘chloride switch’’ – shifts the Cl -equilibrium 

potential, E Cl , from values near 40 mV to values near the 

resting voltage. Once this transition is completed (in rats and 

0736-5748/$30.00 # 2007 ISDN. Published by Elsevier Ltd. All rights reserved. 

doi:10.1016/j.ijdevneu.2007.08.001


480 

D. Gilbert et al. / Int. J. Devl Neuroscience 25 (2007) 479–489 

mice 2 weeks after birth), the opening of Cl channels stabilizes 

the resting voltage. 

In contrast to CNS neurons, primary afferent neurons of the 

peripheral nervous system (e.g. somatosensory neurons and 

olfactory sensory neurons) are believed to mature without 

undergoing a transition of chloride homeostasis, and to support 

depolarizing Cl currents throughout the adult life. In olfactory 

sensory neurons, E Cl is maintained near 0 mV in adult animals, 

and a depolarizing Cl efflux carries the main part of the odorinduced 

receptor current (Reuter et al., 1998; Kaneko et al., 

2004). A similar role is assumed for Cl currents in 

somatosensory neurons, based on reports of elevated Cl 

levels (30–50 mM) in dorsal root ganglion neurons (Gallagher 

et al., 1978; Alvarez-Leefmans et al., 1988; Kenyon, 2001; 

Kaneko et al., 2002). Furthermore, somatosensory neurons can 

respond to GABA with depolarization (Rudomin and Schmidt, 

1999; Sung et al., 2000). Finally, the expression of NKCC1 

(Alvarez-Leefmans et al., 2001; Kanaka et al., 2001), combined 

with the hardly detectable levels of KCC2 mRNA (Rivera et al., 

1999; Kanaka et al., 2001), is interpreted as indicative of Cl 

accumulation. However, two observations are not easily 

reconciled with the general concept of excitatory Cl currents 

in somatosensory neurons: (1) the measured E Cl values (around 

40 mV) limit the excitatory effect of Cl currents and may 

even oppose strong depolarization and excitation; (2) NKCC1 

mRNA was detected in only 50% of somatosensory neurons in 

dorsal root ganglia and trigeminal ganglia, where it is colocalized 

with TRPV1 (Price et al., 2006). This expression 

pattern may indicate that excitatory Cl currents are restricted 

to certain modalities of the somatosensory system. 

To approach the question of Cl accumulation and its role in 

the somatosensory system, we examined the maturation of Cl 

homeostasis after birth. We directly measured [Cl ] i in intact 

dorsal root ganglion neurons of early postnatal and adult mice 

using two-photon fluorescence-lifetime imaging microscopy 

(2P-FLIM). We looked at the expression of NKCC1 and KCC2 

protein in rat dorsal root ganglia. To assess possible 

contributions of other Cl transporters to the maturation of 

Cl homeostasis, we examined the transcription pattern of all 

genes that encode electroneutral cation–chloride co-transporters 

in newborn and adult rats. 

2. Experimental procedures 

2.1. Isolation of somatosensory neurons for calibration 

All experimental procedures were performed in accordance with the Animal 

Protection Law and the guidelines and permissions of Heidelberg University. 

Adult female NMRI (Naval Medical Research Institute, USA) mice were 

anesthetized and killed by cervical transsection. The spinal column was 

prepared and opened sagittally with scissors. Thirty to forty dorsal root ganglia 

were dissected from the lumbar, thoracic and cervical region. Ganglia were cut 

into small pieces and incubated in 2 ml of collagenase solution (0.3% collagenase, 

C-9891, Sigma, Germany, in DMEM) for 1 h at 37 8C. After 

centrifugation (200 g, 20 min) the supernatant was discarded, and 2 ml of 

trypsin solution (0.25% trypsin, T-1426, Sigma, in MEM) was added to the 

pellet. The pellet was resuspended by trituration with a truncated, fire-polished 

Pasteur pipette (3 mm opening). The ganglia were kept in trypsin solution for 

30 min at 37 8C, and then centrifuged for 20 min at 200 g. The supernatant 

was discarded and 4 ml of culture medium (DMEM + 10% FCS, 10106-169, 

and 1% antibiotics/antimycotics, 15240-062, Gibco Life Technologies, Invitrogen, 

Karlsruhe, Germany) was added to the pellet. The pellet was triturated, and 

then filtered through a 150 mm nylon mesh (Typ 1110, Bückmann, Germany). 

This step removes a substantial amount of myelin debris and non-dissociated 

fragments of ganglia, which are retained on the nylon mesh. The pooled cells 

were plated on coverslips (coated with 100 mg/ml poly-L-lysine, P-1399, 

Sigma, and 20 mg/ml laminin, L-2020, Sigma) and cultured at 37 8C in culture 

medium under an atmosphere containing 5% CO 2 . Nerve growth factor (NGF-b 

from rat, N-2513, Sigma) was added 1 h after plating at a final concentration of 

50 ng/ml. Cultures were used within 24 h after plating. Cells were incubated in 

standard extracellular solution (Table 1) containing 5 mM MQAE (N-6-methoxyquinolinium 

acetoethylester; Molecular Probes, Invitrogen) for at least 

30 min in the dark, at 37 8C. The dye progressively accumulates within the 

cells as the molecule is rendered membrane impermeable by cytosolic esterases 

(Koncz and Daugirdas, 1994) with only minor effects on its fluorescence 

properties (Kaneko et al., 2002). After incubation, extracellular MQAE was 

washed away and coverslips were transferred to the FLIM instrument. 

2.2. Tissue preparations for 2P-FLIM measurements 

Newborn or adult female NMRI mice were anesthetized and killed by 

cervical transsection. The backbone was prepared and vertebrae containing the 

spinal cord with attached dorsal root ganglia were isolated with a razor blade 

from the spinal column. All preparations were done in cooled (4 8C) standard 

extracellular solution (Table 1). Only those vertebrae were used for 2P-FLIM 

measurements whose dorsal root ganglia had an intact dura mater and which 

were connected to undamaged dorsal roots and spinal nerves. Preparations were 

incubated in standard extracellular solution containing 5 mM MQAE for 3.5 h 

at room temperature (20 8C). After incubation, the dye solution was replaced by 

standard extracellular solution and the preparations were stored at 4 8C until the 

measurement. For 2P-FLIM recordings, MQAE-loaded preparations were 

immobilized with low melting agarose (USB Corporation, USA) at the bottom 

of 6 cm dishes (Greiner, Germany). All recordings were done at room temperature 

in standard extracellular solution. To test the viability of neurons in the 

preparation during the time of the 2P-FLIM experiments, we performed 

viability tests with 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 

bromide; Molecular Probes), and with 0.4% trypan blue (Gibco 

Life Technologies). Following the preparation, the tissues were incubated at 

room temperature in standard extracellular solution and were subsequently 

loaded with either MTT (60 min) or trypan blue (10 min) at various times. 

Using this method, we established a time window of 5 h post mortem during 

which the viability of DRG neurons was good and the tissue could be used for 

2P-FLIM analysis. 

2.3. Two-photon fluorescence-lifetime imaging microscopy (2P- 

FLIM) 

MQAE was used as a fluorescent probe for intracellular Cl ([Cl ] i ) 

(Verkman, 1990). MQAE molecules reach the excited state upon absorption 

of a single ultraviolet photon (l = 375 nm) or, alternatively, the simultaneous 

absorption of two infrared photons (l = 750 nm). We used two-photon excita- 

Table 1 

Solutions (in mM) 

Na K Ca Mg Cl NO 3 

Standard extracellular solution 140 5 2.5 1 151 

15 Cl standard solution 150 15 105 





Solutions contained 10 mM glucose; pH 7.4 was buffered with 10 mM HEPES. 

The cell isolation solution was buffered with phosphate (in mM: 1.9 NaH 2 PO 4 , 

8.1 Na 2 HPO 4 ).


D. Gilbert et al. / Int. J. Devl Neuroscience 25 (2007) 479–489 481 

tion to achieve an optical resolution of 0.5 mm(x-/y-plane) and 1 mm(z-axis). 

The infrared light used for two-photon excitation caused no detectable photodamage, 

even with the relatively long observation times of multiple images with 

1 min of illumination per image. For the MQAE molecule, the dwell time in the 

excited singlet state (the fluorescence lifetime, t) is near 30 ns in water 

containing 50 mM MQAE and is reduced by anions through collisional quenching. 

The Cl dependence of t is described by the Stern–Volmer relation 

(t 0 /t =1+K SV [Cl ] i ), where t 0 is the fluorescence lifetime in 0 mM Cl 

and K SV , the Stern–Volmer constant, is a measure of the Cl sensitivity of 

MQAE. K SV has a value of 185 M 1 in water but only 3–20 M 1 inside cells 

(Lau et al., 1994; Bevensee et al., 1997; Maglova et al., 1998; Eberhardson et al., 

2000; Kaneko et al., 2002). This reduced sensitivity of intracellular MQAE may 

result in part from interactions of the dye with other soluble anions and from 

self-quenching of MQAE at concentrations >100 mM(Kaneko et al., 2002). For 

2P-FLIM measurements, the tissues or cells were placed on the stage of an 

upright fluorescence microscope (BX50WI; Olympus Optical, Japan) and 

observed through a 60 water-immersion objective (n.a. = 0.9; Olympus 

Optical). Fluorescence was excited with 150 fs light pulses (l = 750 nm) 

applied at sufficient intensity to generate two-photon excitation. Light pulses 

were generated at a frequency of 75 MHz by a mode-locked Titan-Sapphire 

laser (Mira 900; output power >500 mW; Coherent, Santa Clara, USA), which 

was pumped by the frequency-doubled output (532 nm) of a Nd–vanadate laser 

(Verdi; Coherent). The laser light was directed through the objective to the 

tissue surface at reduced power (2.5 mW) using a beam scanner (TILL 

Photonics, Munich, Germany). Fluorescence was recorded by photomultipliers, 

and lifetime analysis was performed using electronics (SPC-730; Becker & 

Hickl, Berlin, Germany) and software (SPC7.22; Becker & Hickl) for timecorrelated 

single-photon counting (Lakowicz, 1999). Lifetime images were 

analyzed using SPCImage 1.8 and 2.6 (Becker & Hickl) and a self-made image 

analysis software. A detailed description of the instrument and the calibration 

procedure is described in Kaneko et al. (2002). Images were obtained by 

scanning the excitation light focus through tissue layers 1–3 cells below the 

dura mater. Mean values of Cl concentrations are given with standard 

deviations (S.D.). 

2.4. Single cell-based quantitative analysis of 2P-FLIM data 

For quantification of cytosolic fluorescence lifetimes, we developed a 

software that enables single cell-based analysis in 2P-FLIM z-scan images. 

The cytosolic MQAE signal of a single neuron was analyzed at the equatorial 

layer, i.e. the optical section in the image stack, where the total area of the cell 

was largest. The cytosolic region was outlined by defining manually two regions 

of interest, delimiting the margins of the nucleus and of the cell, respectively. 

The quantitative parameters, mean fluorescence lifetime (in ns) and cell 

diameter (in mm), were extracted by the software. The cell diameter, d, was 

calculated according to d = H(4A/p), with A = area of the cell, assuming 

circular cell morphology. The diameter is an important parameter, because 

sensory modalities of dorsal root ganglion neurons are often correlated with this 

value (e.g. Petruska et al., 2002). 

2.5. Immunohistochemistry 

To examine the expression of Cl transport proteins, we used rat instead of 

mouse dorsal root ganglia because the available antibodies were better suited 

for rat than for mouse tissue. Four dorsal root ganglia were dissected from the 

thoracic spinal cord of two adult Wistar rats and immersed in 4% paraformaldehyde 

in PBS (8.1 mM Na 2 HPO 4 , 1.9 mM NaH 2 PO 4 , 130 mM NaCl, pH 7.4) 

for 25 min. After three washing steps in PBS, the tissue was sequentially 

dehydrated in 10–30% sucrose overnight. Tissue was then embedded in Tissue- 

Tek (Leica, Nussloch, Germany) and frozen onto the cryostate stage (Leica CM 

3050 S). Four to eight cryosections (12 mm) were cut and collected on coated 

slides (SuperFrost 1 Plus, Menzel, Braunschweig, Germany). Sections were airdried 

for 30 min, post-fixed in 4% paraformaldehyde in PBS, washed three 

times in PBS and blocked in 5% ChemiBLOCKER TM (Millipore, Billerica, 

USA), 0.5% Triton X100 in PBS. Primary antibodies were diluted in the same 

buffer and incubated for 2 h. The following antibodies and dilutions were used: 

mouse anti-NKCC1 1:20 (T4, monoclonal antibody, obtained from the Developmental 

Studies Hybridoma Bank, The University of Iowa), goat anti-NKCC1 

1:20 (N-16; Santa Cruz Biotech, Heidelberg, Germany, #sc-21545), goat anti- 

KCC2 1:20 (Santa Cruz Biotech, #sc-19420). After three washing steps in PBS, 

sections were incubated for 90 min in fluorescent-labeled donkey anti-mouse 

Alexa594 (Invitrogen, A-21203, 1:500) or donkey anti-goat Alexa488 (Invitrogen, 

A-21206, 1:500) secondary antibodies. After three washing steps in PBS, a 

0.3 mM DAPI solution (Fluka, #32670) was used to stain the nuclei. Sections 

were coverslipped with Aqua Poly/Mount (Polysciences, Warrington, USA) and 

analyzed using a Nikon Eclipse 90i upright automated microscope equipped 

with a Nikon DS-1QM CCD camera. The instrument was used at the Nikon 

Imaging Center at the University of Heidelberg. No fluorescence signal was 

observed upon omission of primary antibodies. To test for the specificity of 

primary antibodies, preadsorption controls were performed for the NKCC1 (N- 

16) and the KCC2 polyclonal antibodies using a fivefold excess of blocking 

peptides (0.05 mg/ml) according to the manufacturer’s specifications. No 

immunosignal was observed after preadsorption. The specificity of the monoclonal 

T4 antibody for NKCC1 was demonstrated by Chen et al. (2005) through 

the absence of T4 immunosignals in brain tissue of NKCC1 / mice. Two slices 

were evaluated for each test. To avoid double counting of cells, every fourth to 

sixth section was evaluated, ensuring a minimum distance of 24–48 mm 

between individual sections. 

2.6. Expression of electroneutral cation–chloride co-transporter 

genes 

Dorsal root ganglia and, for controls, kidney and brain were dissected from 

adult and newborn (P1) Wistar rats. Total RNA was extracted following the 

protocol of Chomczynski and Sacchi (1987). After DNase I treatment (RNasefree, 

Fermentas, StLeon-Rot, Germany) cDNA was synthesized from 5 mg total 

RNA using an oligo-dT primer and Superscript TM III Reverse Transcriptase 

(Invitrogen) according to the manufacturer’s instructions. The cDNA was 

quantified for normalization using PCR (16, 20, 24, 28 and 32 cycles) with 

b-actin and ATPase primers. The annealing temperature for all primers was 

58 8C. Semi-quantitative PCR amplification was performed on 0.5 ml singlestranded 

cDNA product with 2U Taq DNA polymerase (Fermentas). The 

cycling conditions were 94 8C for 3 min, 94 8C for 30 s, 58 8C for 30 s, 

72 8C for 1 min for 28 and 32 cycles, respectively, and 72 8C for 8 min. For 

each cation–chloride co-transporter gene, primer pairs were: CCC9/F, 5 0 -TCA 

CTG TGT TTG GGG TGT TC-3 0 ; CCC9/R, 5 0 -GGA GAG GGC GAA GTA 

AGA GTA G-3 0 ; CCC6/F, 5 0 -GGT TCA ACG GAA GCA CCC TAA-3 0 ; CCC6/ 

R, 5 0 -GTG ACC ACA GCA GCC AAT GT-3 0 ; KCC1/F, 5 0 -TGA CCC TAG 

TGG TGT TTG TCG G-3 0 ; KCC1/R, 5 0 -GTT CCT GCT GAC GCC ATC A-3 0 ; 

KCC2/F, 5 0 -GTC TCT GGG CCC GGA GTT T-3 0 ; KCC2/R, 5 0 -GGC ATC CCG 

CAG GTC TC-3 0 ; KCC3/F, 5 0 -TGC TGC TGT ACA ATG TTA ACT GCC-3 0 ; 

KCC3/R, 5 0 -TAG CCA ACC CTG GAATGC C-3 0 ; KCC4/F, 5 0 -TGC TTT CTA 

TCC TGG CCA TCT ATG-3 0 ; KCC4/R, 5 0 -GCC TCC CCA AAC TTA TCT 

CGC-3 0 ; NKCC1/F, 5 0 -CGA ATTATT GGA GCC ATTACA GT-3 0 ; NKCC1/R, 

5 0 -ACATCT GGA AAG CTG GGT AGATA-3 0 ; NKCC2/F, 5 0 -ATT CAATGA 

TGG TGG ATC CAA C-3 0 ; NKCC2/R, 5 0 -CGG CGATGA GAATGA ATG C- 

3 0 ; TSC/F, 5 0 -GTA GAC CCC ATC AAT GAC ATC C-3 0 ; TSC/R, 5 0 -AAG CCA 

ATC AGA GGG TAC AGC-3 0 . For positive controls and normalization b-actin 

and Na + /K + -ATPase primer pairs were: Actin/F, 5 0 -GGT CAT CAC TAT CGG 

CAA TGA GC-3 0 ; Actin/R, 5 0 -GGA CAG TGA GGC CAG GAT AGA GC-3 0 ; 

ATPase/F, 5 0 -AGT GAG CTG AAA CCC ACG TAC C-3 0 and ATPase/R, 5 0 - 

CCC CTC TTT GTA GCC GTA GGA TT-3 0 . The resulting PCR products were 

cloned into pGMT vector (Promega, Mannheim, Germany) and sequenced. 

3. Results 

In a previous study of olfactory sensory neurons, we 

observed that these neurons were incapable of maintaining 

elevated [Cl ] i after cell isolation (Kaneko et al., 2004). In the 

present study we, therefore, measured [Cl ] i without isolating 

the somatosensory neurons from their ganglia. Moreover, the 

protein expression pattern in isolated somatosensory neurons 

changes during dedifferentiation processes in culture (Scott and 

Edwards, 1980), and this may alter the control of [Cl ] i .To


482 


obtain information about [Cl ] i values in situ, we performed 

chloride imaging in dorsal root ganglia with intact dura mater 

and intact axonal connections with the spinal cord. Isolated 

neurons were only used for calibrating the 2P-FLIM signal, as 

the manipulation of [Cl ] i by ionophores was more efficient 

when the neurons were dissociated. 

3.1. Somatosensory neurons undergo a postnatal chloride 

transition 

The neurons in MQAE-loaded dorsal root ganglia displayed 

different fluorescence intensities when illuminated with the 

excitation light (Fig. 1a, black-and-white image). The fluorescence 

intensity, however, does not report [Cl ] i because it is 

co-determined by the unknown dye concentration in each cell. 

Fluorescence originating from the nuclei was weak. This may 

be caused by poor dye loading of the nuclei or by different 

quenching conditions in the nucleoplasm. The nuclear signals 

where excluded from further analyses. The fluorescence 

lifetime, t, which is proportional to [Cl ] i and can be used 

as intracellular Cl reporter (Koncz and Daugirdas, 1994; 

Kaneko et al., 2002), was colour-coded and is displayed as 2P- 

FLIM image for the same cells (Fig. 1a, colour image). In the 

2P-FLIM images, warmer colours indicate higher levels of 

[Cl ] i . To establish the quantitative relation between 2P-FLIM 

signals and [Cl ] i in MQAE-loaded dorsal root ganglion 

neurons, we set [Cl ] i in isolated neurons to 1–5 levels (15, 30, 

45, 60, 75 mM) using a double ionophore technique. Isolated 

neurons in primary culture were used because the manipulation 

of [Cl ] i was more efficient in single cells than in intact ganglia. 

The neurons were kept in a solution containing the required 

Cl concentration (Table 1) as well as 40 mM tributyltin (a Cl / 

OH exchanger) and 20 mM nigericin (a K + /H + exchanger). 

The combination of these ionophores has been shown to 

dissipate Cl gradients across the plasma membrane (Chao 

et al., 1989). Fluorescence lifetimes decreased from 3.7 to 

3.1 ns when [Cl ] i was raised from 15 to 75 mM within the 

cytosol (15 mM: 3.73 0.17 ns, n = 16; 30 mM: 3.66 

0.09 ns, n = 4; 45 mM: 3.45 0.14 ns, n =5; 60mM: 

3.43 0.07 ns, n = 13; 75 mM: 3.14 0.13 ns, n = 10). A 

Stern–Volmer plot of the calibration data (Fig. 1b) yielded a 

quenching constant of 3.05 M 1 , which was used in all further 

experiments to calculate [Cl ] i from the measured lifetimes. 

We have previously tested the reliability of this calibration 

procedure in primary afferent neurons of the olfactory system. 

In a comparison of 2P-FLIM analysis (Kaneko et al., 2004) and 

chloride measurements by energy-dispersive X-ray microanalysis 

(Reuter et al., 1998), the measured [Cl ] i values differed 

Fig. 1. Determination of [Cl ] i in somatosensory neurons by 2P-FLIM. (a) Comparison of fluorescence intensity (left) and lifetime (right) images from the same 

dorsal root ganglion. The intensity image shows strong signals from the cytosol and weak signals from the nuclei. In the 2P-FLIM image, the fluorescence lifetime, t, 

is colour-coded with warmer colours representing higher [Cl ] i levels. (b) Calibration of 2P-FLIM signals in isolated dorsal root ganglion neurons with [Cl ] i set to 

the indicated values using the two-ionophore method. The solid line represents a least-squares fit to the data using the Stern–Volmer equation t 0 /t =1+K SV [Cl ] i 

with K SV = 3.05 M 1 . The colour scale illustrates the false-colour representation of [Cl ] i in the following 2P-FLIM images. (c) 2P-FLIM images illustrating [Cl ] i 

levels in somatosensory neurons from newborn (P1–P4) and adult (third week) mice. The calibration established in (b) was used for false-colour representation of 

[Cl ] i . Newborn neurons show almost uniformly high [Cl ] i (70 mM). During maturation, most somatosensory neurons decrease their [Cl ] i to some extent, 

resulting in a heterogeneous mosaic of [Cl ] i levels in the ganglia of mature animals.



by less than 20%. For this reason, we give all absolute Cl 

concentrations in the present study with a confidence range 

of 20%. 

We determined [Cl ] i in somatosensory neurons in intact 

ganglia by focusing the excitation light of the 2P-FLIM 

instrument through the dura mater into cells 20–100 mm deep 

inside the ganglion. Deeper cell layers were not accessible for 

analysis because the dye did not sufficiently load the cells at 

distances > 100 mm from the dura mater. To determine [Cl ] i 

in neurons of newborn mice, preparations of 1–4 days old 

animals (P1–P4) were examined by 2P-FLIM. Since the 

chloride transition develops between P6 and P14 (Rivera et al., 

2005), we expected to find in the P1–P4 animals the high levels 

of [Cl ] i characteristic for embryonal and early postnatal 

neurons. Indeed, the somatosensory neurons exhibited uniformly 

high levels of [Cl ] i (Fig. 1c, P1–P4). Single cell-based 

quantitative analysis of 975 neurons (pooled from P1 to P4) 

yielded a distribution of [Cl ] i between 35 and 120 mM with a 

mean [Cl ] i of 77.2 mM (S.D. = 13.6 mM) (Fig. 2). 

2P-FLIM measurements in the third postnatal week revealed a 

decrease of [Cl ] i in most cells, and a heterogeneous distribution 

of [Cl ] i levels among different neurons, ranging from 10 to 

>80 mM [Cl ] i (Fig. 1c). The general decline of [Cl ] i indicates 

a profound change in chloride homeostasis with reduced Cl 

uptake and/or enhanced Cl extrusion. In adult animals (12–15 

weeks), the heterogeneous distribution was maintained (Fig. 1c). 

A histogram of [Cl ] i values obtained from 818 neurons in eight 

ganglia from six adult animals is shown in Fig. 2. The mean value 

is significantly lower than in P1–P4 animals (Student’s t-test: 

p 0.0005), and the distribution is broad (mean: 61.8 mM, 

S.D. = 19.7 mM). Both postanatal and adult data could be 

Fig. 2. Distribution of [Cl ] i values in dorsal root ganglia of newborn and adult 

mice. The histograms show the distribution of measured [Cl ] i levels in 

newborn (closed circles) and adult (open circles) animals. During the course 

of maturation, the distribution of [Cl ] i shifts towards lower levels. The lines 

compare global fits to the data with a single Gaussian distribution (dashed lines) 

and with three Gaussian distributions (solid lines). The three populations 

contributing to the latter fit are characterized by mean [Cl ] i levels of 

37.8 mM, 58.7 mM, and 80.5 mM, and their relative contributions change 

during maturation from 0 to 18.9%, from 21 to 55.2%, and from 79 to 

25.9%, respectively. 

tentatively described by a sum of three Gaussian distributions 

with mean values of 37.8 mM, 58.7 mM and 80.5 mM [Cl ] i , 

respectively (Fig. 2). During the developmental transition, the 

percentage of ‘‘high-chloride cells’’ decreased from 79 to 26%, 

that of ‘‘medium-chloride cells’’ increased from 21 to 55%, and 

that of ‘‘low-chloride cells’’ from 0 to 19%. Our data demonstrate 

that the somatosensory neurons in an individual ganglion of an 

adult animal exhibit a differential pattern of chloride homeostasis. 

Most neurons undergo a transition of the chloride 

homeostasis during maturation, while roughly a third of the cells 

maintain the high [Cl ] i levels of the early postnatal days. 

3.2. Lack of correlation between [Cl ] i and cell size 

The size of somatosensory neurons can be indicative of their 

sensory modalities. Small and medium-sized somata (B-cells; 

mean volume: 10,700 mm 3 ; Tandrup, 2004) are often polymodal 

nociceptors or heat-sensitive cells with unmyelinated C- 

fibres. Large-diameter neurons (A-cells; mean volume: 

57,200 mm 3 ; Tandrup, 2004) mediate proprioception, touch 

or other low-threshold sensations. While detailed information 

about protein expression patterns, conduction velocities, and 

excitation properties is required to establish the specific 

modality of a somatosensory neuron (Guo et al., 1999; Lawson, 

2002; Fang et al., 2006), the cell size can serve as a first 

indication for the sensory system involved. To find out whether 

the different [Cl ] i levels in adult neurons correspond to any 

specific cell size, we measured the diameters of all neurons 

analyzed for Fig. 2, and related them to the measured [Cl ] i 

values. Since the 2P-FLIM images represent optical slices of 

about 1 mm thickness, an individual image may show either the 

maximal (equatorial) diameter of a cell or, alternatively, a 

smaller (non-equatorial) diameter. For our analysis, we 

determined the maximal diameter for each individual cell. 

[Cl ] i was determined in the cytosolic region of each neuron, 

excluding the nucleus (Fig. 3a, arrows). The plot of [Cl ] i 

versus cell diameter does not reveal any correlation between the 

two parameters in adult neurons (Fig. 3b, black dots; linear 

regression analysis: r 2 = 0.019, n = 818). Both small-diameter 

cells (15–25 mm) and larger neurons cover the entire range of 

measured [Cl ] i levels. The mean values (Fig. 3c) illustrate an 

increase of the mean cell diameter from 17.5 2.8 to 

25.8 6.3 mm and a decrease of mean [Cl ] i from 

77.2 13.6 to 61.8 19.7 mM during maturation. However, 

the chloride transition appears not to be restricted to a cell 

population with a distinct size. This finding suggests that a 

change in the regime of chloride handling occurs during 

postnatal maturation in somatosensory neurons of various 

sensory modalities. 

3.3. Expression of NKCC1 and KCC2 proteins 

A possible explanation for the mosaic pattern of [Cl ] i levels 

in adult dorsal root ganglia could be that some cells express 

predominantly NKCC1 while others express mainly KCC2. To 

test this hypothesis, we double-stained cryosections of dorsal 

root ganglia with antibodies raised against the two proteins. To


484 


Fig. 3. Heterogeneous [Cl ] i levels in mouse somatosensory neurons of various sizes. (a) 2P-FLIM images show the nuclear region in many cells with fluorescence 

signals distinct from the cytosol. To determine [Cl ] i , the fluorescence lifetime was evaluated in the cytosolic region, illustrated as the area between two circles 

(arrows). To determine the cell size, a series of images (z-stack) was analyzed, and the maximal diameter of each individual cell was measured. (b) Dot plot relating 

[Cl ] i to cell diameter for 975 neurons from early postnatal animals (red) and 818 neurons from adult animals (black). No correlation between cell size and [Cl ] i is 

detectable. (c) Mean [Cl ] i decreased from 77.2 13.6 to 61.8 19.7 mM, while the mean cell diameter increased from 17.5 2.8 to 25.8 6.3 mm during 

maturation. 

detect NKCC1 expression, we used two different antibodies: 

the monoclonal T4 antibody (raised against a C-terminal 

epitope of NKCC1; Lytle et al., 1995) which was demonstrated 

to be appropriate for somatosensory neurons (Alvarez-Leefmans 

et al., 2001), and the polyclonal N-16 antibody (raised 

against an N-terminal epitope of NKCC1, Santa Cruz). Of 534 

cells, 450 (84%) were stained with the T4 antibody. Co-staining 

with T4 and N-16 antibodies revealed a highly consistent 

pattern (Fig. 4a–c). Two hundred and forty-nine small- and 

medium-diameter neurons were examined. Eighty-one percent 

of these (202 cells) were positive for both antibodies, 5% (12 

cells) showed only a T4 signal, and 14% (35 cells) were not 

stained. Thus, 94% of T4-positive neurons were also N-16 

positive, which represents strong evidence for NKCC1 

expression in most somatosensory neurons. Immunosignals 

from the KCC2 antibody were significantly weaker than those 

from NKCC1. Seventy-six percent of 285 small-to-medium 

sized cells were immunopositive for KCC2, and 91% of these 

were also stained with the T4 antibody (Fig. 4d–f). In summary, 

expression of KCC2 appears to be distinctly weaker than that of 

NKCC1. While most of the somatosensory neurons coexpressed 

NKCC1 and KCC2, a subpopulation of 20% of 

the small-to-medium size neurons express NKCC1 without 

detectable KCC2 expression. 

3.4. Invariant transcription of electroneutral cation– 

chloride transporters during maturation 

The change of [Cl ] i during postnatal development may be 

the consequence of altered expression of Cl transporters in the 

somatosensory neurons. To test whether an altered transcription 

program causes the Cl transition, we examined the mRNA



Fig. 4. Expression of electroneutral cation–chloride co-transporters in rat somatosensory neurons. (a–c) Immunohistochemistry with two antibodies directed against 

NKCC1. The immunosignals of the antibodies T4 (C-terminal) and N-16 (N-terminal) match, with only few neurons missing the N-16 signal (red cells in c). (d–f) 

Expression of KCC2 is detectable but weak in most somatosensory neurons. No immunostain was observed after preadsorption of the polyclonal antibodies (N-16 and 

KCC2) with their respective immunization peptides (not shown). Nuclei are stained blue with DAPI. 

expression of all nine electroneutral cation–chloride cotransporters 

present in the rat genome (synonym: solute carrier 

family 12, SLC12; review, Gamba, 2005) by semi-quantitative 

RT-PCR. We made first-strand cDNA from dorsal root ganglia 

of newborn (P1) and adult animals. The relative concentrations 

of the cDNAs in the RT-PCR assays were adjusted using the 

highly abundant b-actin and the less abundant Na + /K + -ATPase 

as internal standards (Fig. 5a). The cDNAs were amplified with 

primers specific for CCC6, CCC9, KCC1-4, NKCC1-2 and 

TSC. CCC6 and CCC9 are orphan members of the SLC12 

family with unknown function, while TSC is the thiazidesensitive 

Na + –Cl co-transporter (for gene identification see 

legend to Fig. 5). Cycling conditions were adjusted to 28 and 32 

cycles. Fig. 5b demonstrates that the mRNA of five Cl cotransporters 

(CCC6, KCC1, KCC3, KCC4, NKCC1) could be 

detected by 28 PCR cycles from both P1 and adult animals. 

Fig. 5c indicates that 32 PCR cycles reached the amplification 

plateau for the five co-transporters listed above. In addition, a 

diagnostic signal of CCC9, KCC2 and TSC was observed, 

while no NKCC2 signal could be detected. Generating PCR 

amplification products under these conditions may be 

influenced by template abundance or by primer efficiency. 

To distinguish between these alternatives, we amplified KCC2 

from normalized brain cDNA, as well as NKCC2 and TSC from 

normalized kidney cDNA. Strong signals were detected by 32 

PCR cycles (Fig. 5a, right). Since no tissue-specific expression 

data were available for CCC9, the respective control was 

performed on adult dorsal root ganglia cDNA only and showed 

a strong signal with 36 cycles (Fig. 5a, right). These data 

indicate that each of the primer pairs used in this study 

exhibited similar efficiency. Therefore, the yield of the PCR 

amplification products depended only on the level of input 

template. All primer pairs were designed to span intron/exon 

boundaries, to reveal possible genomic DNA contamination. 

None of the PCR reactions showed amplification of genomic 

DNA which would have given rise to correspondingly larger 

DNA fragments. For unambiguous identification, all PCR 

products were eluted from agarose gels, cloned into the vector 

pGMT and sequenced. 

In summary, we found high expression levels of CCC6, 

KCC1, KCC3, KCC4 and NKCC1, much lower expression 

levels for CCC9, KCC2 and TSC, while no expression for 

NKCC2 mRNA was observed. Most importantly, the mRNA 

expression patterns of P1 and adult animals could not be 

distinguished on the grounds of the semi-quantitative PCR data. 

These results clearly demonstrate that the maturation of Cl 

homeostasis cannot be attributed to changes in the transcription 

pattern of electroneutral cation–chloride co-transporters. 

4. Discussion 

In trying to assess the role of Cl accumulation in 

somatosensory signal generation, we have examined the 

question whether Cl homeostasis changes after birth. We


486 


Fig. 5. Expression of electroneutral cation–chloride co-transporters in dorsal root ganglia (DRG) of newborn (P1) and adult rats. Semi-quantitative RT-PCR assays 

were used to determine expression levels of mRNA transcripts encoding cation–chloride co-transporter 9 (CCC9; synonym: solute carrier family 12 member 8, 

SLC12A8; GenBank accession NM_153625), cation–chloride co-transporter 6 (CCC6; synonyms: cation–chloride co-transporter-interacting protein, CIP; 

SLC12A9; NM_134405), K–Cl co-transporter 1 (KCC1, SLC12A4; NM_019229), K–Cl co-transporter 2 (KCC2; SLC12A5; NM_134363), K–Cl co-transporter 

3 (KCC3; SLC12A6; XM_001066756), K–Cl co-transporter 4 (KCC4; SLC12A7; XM_001060536), Na–K–Cl co-transporter 1 (NKCC1, SLC12A2; NM_031798), 

Na–K–Cl co-transporter 2 (NKCC2, SLC12A1; NM_019134), thiazide-sensitive Na–Cl co-transporter (TSC; SLC12A3; NM_019345). The primers were specific for 

the individual subtypes of co-transporters and were designed to span several exons to control for the possible presence of genomic DNA in the RNA preparation. 

Cycles of amplification are indicated for each reaction. The figures are negative images of ethidium bromide-stained agarose gels. DNA marker (M): 100 bp ladder. 

(a) The cDNA concentrations were normalized by amplification (16, 20, 24, 28 and 32 cycles) with b-actin and ATPase primers, and 0.5 ml of cDNA product was used 

per amplification. For positive controls, KCC2, NKCC2 and TSC were amplified from kidney or brain cDNA, and CCC9 was amplified with 36 cycles from dorsal 

root ganglia cDNA of adult animals. (b) Strong signals were observed for CCC6, KCC1, KCC3, KCC4 and NKCC1 in either P1 or adult animals using 26 cycles of 

amplification. No expression was detected under these conditions for CCC9, KCC2, NKCC2 and TSC. (c) Amplification begins to plateau within 32 cycles for CCC6, 

KCC1, KCC3, KCC4 and NKCC1 in P1 and adult animals. Weak expression was detected for CCC9, KCC2 and TSC in cDNA from P1 and adult animals. No 

expression was detected for NKCC2. 

measured [Cl ] i in the somata of dorsal root ganglion neurons 

using a preparation with intact ganglia from newborn and adult 

animals. We avoided the use of cultured neurons for the 

determination of [Cl ] i because the expression of Cl 

transporters and, hence the equilibrium level of [Cl ] i , may 

change during culture. [Cl ] i in vivo may also be co-determined 

by the satellite glial cells which closely wrap each neuronal 

soma inside the ganglion (Hanani, 2005), and which are lost 

upon cell isolation. We utilized the Cl dependence of the 

fluorescence lifetime of MQAE to directly access [Cl ] i .We 

found that [Cl ] i levels were uniformly high in newborns. In the 

course of the first 3 weeks after birth, somatosensory neurons



changed their mode of Cl handling, and most neurons lowered 

their [Cl ] i level to some extent. In adult dorsal root ganglia, 

[Cl ] i varied over a wide range of concentrations. The 

distribution of [Cl ] i levels among individual neurons indicated 

an E Cl range of 70 to 20 mV. Statistical analysis pointed to 

the presence of three distinct populations of neurons with [Cl ] i 

levels near 40, 60 and 80 mM, respectively. However, the 

interpretation of this finding requires further examinations. Our 

results suggest that Cl currents are inhibitory in some 

somatosensory neurons and excitatory in others. 

What is the molecular basis of this transition? Our PCR 

study shows that five members of the SLC12-family of 

electroneutral Cl co-transporters (CCC6, KCC1, 3 and 4, 

NKCC1) show robust transcription at P1 which does not change 

during maturation. The mRNA levels of three other family 

members (CCC9, KCC2, and TSC) are lower, but persist during 

maturation. Our results are in accordance with in-situ 

hybridization studies on dorsal root ganglia from adult rats. 

Strong KCC1 and NKCC1 expression was detected with 

riboprobes, while KCC2 mRNA could hardly be detected 

(Kanaka et al., 2001). Moreover, using a combination of in situ 

hybridization and immunohistochemistry, Price et al. (2006) 

found that the NKCC1 mRNA displays 50% co-localization 

with markers of unmyelinated nociceptors (peripherin, CGRP, 

TRPV1) with diameters in the range of 15–35 mm. Only 10– 

20% co-localization was observed with a marker for the 

myelinated, low-threshold sensory neurons (N52 protein) of 

somewhat larger size (25–55 mm). Interestingly, NKCC1 

mRNA was also detected in the satellite glial cells (Price 

et al., 2006), the second major cell population in the dorsal root 

ganglia. This observation points to a caveat in the interpretation 

of our PCR data, as they do not reveal from which cell type the 

SLC12 templates originated. Nevertheless, our observation that 

mRNA levels do not change during the course of the Cl 

transition argues against the notion that transcriptional 

regulation underlies the change in Cl homeostasis in 

somatosensory neurons. 

Our immunohistochemical results indicate that most 

somatosensory neurons are equipped with NKCC1 and, hence, 

are capable of accumulating intracellular Cl . This interpretation 

rests on the results obtained with two antibodies (one N- 

terminal, one C-terminal) and is consistent with similar reports 

from cat and rat dorsal root ganglia (Alvarez-Leefmans et al., 

2001), as well as with the expression pattern of NKCC1 mRNA 

(Price et al., 2006). A recent study with different antibodies 

showed NKCC1 signals mainly or exclusively in the satellite 

glial cells (Price et al., 2006). To our knowledge, the antibodies 

in this study were not characterized as rigorously on dorsal root 

ganglia as the T4 and N-16 antibodies used here. The 

monoclonal T4 antibody stains somatosensory neurons and 

satellite glial cells, and it recognizes a single band of the 

appropriate size in a Western blot from dorsal root ganglia 

(Alvarez-Leefmans et al., 2001). Under our experimental 

conditions, staining of satellite glial cells is discernible, but 

staining of neurons is prominent. The polyclonal N-16 antibody 

appears to be specific as indicated by the preadsorption control. 

Together with the matching mRNA expression profile (Price 

et al., 2006), the immunohistochemical data thus indicate 

that somatosensory neurons in situ express NKCC1. This 

result is in good agreement with the findings of Sung et al. 

(2000) who analyzed the reversal voltage of GABA-induced 

currents in NKCC1 knockout mice (Delpire et al., 1999). In 

NKCC1 / mice, the reversal voltage was shifted to more 

negative values, indicating a loss of Cl accumulation efficacy. 

The NKCC1 / animals also showed altered pain behavior, 

pointing to an important role of NKCC1 in nociceptor 

physiology (Sung et al., 2000; Laird et al., 2004; see also 

Granados-Soto et al., 2005). 

How is [Cl ] i lowered in most adult somatosensory neurons 

– in some cells even into the 10 mM range – without change in 

the SLC12 transcription pattern? Several recent reports support 

the hypothesis that the activity of chloride–cation cotransporters 

is altered by posttranslational modification. Two 

distinct mechanisms could be involved: (1) the NKCC1 and 

KCC proteins are regulated by phosphorylation (Gagnon et al., 

2006; Giménez, 2006), and this regulation may determine the 

[Cl ] i level in each individual neuron. NKCC1 activity is 

increased by phosphorylation by the STE20-related protein 

kinases SPAK/OSR1 and WNK (Dowd and Forbush, 2003; 

Gamba, 2005; Moriguchi et al., 2005; Kahle et al., 2005; Vitari 

et al., 2006), while WNK kinases reduce Cl extrusion by 

KCC1-4 (De los Heros et al., 2006). Our data indicate that most 

somatosensory neurons are equipped with NKCC1 and the four 

KCC isoforms. Since all of these proteins are regulated by 

kinases, it is reasonable to speculate that the different [Cl ] i 

levels after maturation of Cl homeostasis result from different 

phosphorylation states of the Cl transporters. (2) The activity 

of the Cl exporters may depend on oligomerization. A recent 

study of the chloride transition in the auditory brain stem 

showed convincingly that the postnatal development of Cl 

extrusion correlates with oligomerization of KCC2 (Blaesse 

et al., 2006). Thus, KCC2 appears to be inactive as homomer in 

the neurons of newborn animals and to be turned active after 

dimerization. A similar maturation process may develop in the 

somatosensory system and may lead to enhanced Cl extrusion. 

We did not investigate contributions of Cl transporters that 

do not belong to the SLC12 family. Cl /HCO 3 exchangers of 

the SLC26 family (Mount and Romero, 2004; Shcheynikov 

et al., 2006) may be contributing to [Cl ] i in vivo, or 

transporters from the SLC4 family (Romero, 2005). However, 

the Cl /HCO 3 exchanger is conceptionally associated with 

Cl uptake rather than Cl extrusion, in particular, because 

HCO 3 can be continuously generated in the cytosol by 

carbonic anhydrase (Rivera et al., 2005). In our experiments, 

we did not supply extracellular HCO 3 . It is, therefore, unlikely 

that the decrease of [Cl ] i in our experiments is caused by Cl 

extrusion through Cl /HCO 3 exchange. However, in the 

living animal, this transporter may contribute significantly to 

setting [Cl ] i levels in somatosensory neurons. Furthermore, 

our PCR analysis showed that thiazide-sensitive Na + /Cl cotransporter 

mRNA can be detected in dorsal root ganglia, albeit 

at a comparably low level as KCC2. It will be interesting to 

establish the cellular distribution of this protein within the 

ganglia.


488 


What could be the physiological significance of the postnatal 

change in Cl homeostasis in somatosensory neurons? Recent 

studies on peripheral Cl effects indicate that depolarizing Cl 

currents amplify the primary signal in the sensory terminals of 

nociceptors. Transduction in these neurons works without 

metabotropic amplification of the primary signal. The 

transduction channels, which are directly gated by heat, 

chemical or mechanical stimuli, are invariably Ca 2+ -permeable 

(McNaughton, 2004), and most somatosensory neurons express 

Ca 2+ -activated Cl channels (Kenyon and Goff, 1998; Lee 

et al., 2005; Currie et al., 1995). Stimulus-induced Ca 2+ -influx 

thus triggers Cl currents, and those neurons which accumulate 

Cl in their sensory endings are depolarized by Cl efflux 

(Granados-Soto et al., 2005). Accordingly, pain behavior can be 

induced in animal models by excitatory Cl currents in the skin 

(Ault and Hildebrand, 1994), and can be attenuated by 

experimental inhibition of Cl accumulation into the sensory 

endings (Willis et al., 2004; Laird et al., 2004). Based on these 

observations, Price et al. (2005) proposed that increased Cl 

accumulation into sensory endings of nociceptors promotes 

hyperalgesia. Our data suggest that the Cl dependence of the 

nociceptive response reflects the actual state of Cl accumulation 

in each individual nociceptive neuron. In general, the 

sensitivity of somatosensory neurons may be co-determined by 

the efficiency of Cl accumulation, which, in turn, is regulated 

by posttranslational modification of its Cl transporters. 

Interestingly, a recent study of [Cl ] i levels in axotomized 

mouse somatosensory neurons revealed that NKCC1 phosphorylation 

and a consequent rise of [Cl ] i from 31 to 68 mM is 

associated with peripheral nerve regeneration (Pieraut et al., 

2007). Thus, a dynamic regulation of [Cl ] i levels may be 

involved in different cellular processes in somatosensory 

neurons, including signal transduction and growth. 

Taken together, this study demonstrates that Cl homeostasis 

in somatosensory neurons develops during postnatal 

maturation into a state where [Cl ] i can be regulated 

individually in each neuron. Regulation does not affect the 

transcriptional level of electroneutral cation–chloride cotransporters, 

but may exert its control via posttranslational 

modification. The efficiency of Cl accumulation sets the level 

of [Cl ] i and may, hence, control the sensitivity of the adult 

sensory neuron. 

Acknowledgements 

We thank Dr. Joe Lynch (University of Queensland) for 

helpful comments on the manuscript. This work was supported 

by the Deutsche Forschungsgemeinschaft (Fr 937/6). 

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Differential maturation of chloride homeostasis

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