• KSBNS 2024


Short Communication

Exp Neurobiol 2024; 33(2): 68-76

Published online April 30, 2024

© The Korean Society for Brain and Neural Sciences

NKCC1 in Neonatal Cochlear Support Cells Reloads Ions Necessary for Cochlear Spontaneous Activity

Kwon-Woo Kang1†, Kushal Sharma1†, Shi-Hyun Park1†, Jae Kwang Lee2, Justin C. Lee3 and Eunyoung Yi1*

1College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan 58554,
2Division of Functional Food Research, Korea Food Research Institute, Wanju 55365,
3Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34141, Korea

Correspondence to: *To whom correspondence should be addressed.
TEL: 82-61-450-2683, FAX: 82-61-450-2689
These authors contributed equally to this article.

Received: February 29, 2024; Revised: March 14, 2024; Accepted: March 28, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the auditory system, the spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from inner supporting cells (ISCs). This ATP release sets off a cascade, activating purinergic autoreceptors, opening of Ca2+-activated Cl- channel TMEM16A, Cl- efflux and osmotic cell shrinkage. Then, the shrunken ISCs efficiently regain their original volume, suggesting the existence of mechanisms for refilling Cland K+, priming them for subsequent activity. This study explores the potential involvement of NKCCs (Na+-K+-Cl- cotransporters) and KCCs (K+-Cl- cotransporters) in ISC spontaneous activity, considering their capability to transport both Cl- and K+ ions across the cell membrane. Employing a combination of immunohistochemistry, pharmacological interventions, and shRNA experiment, we unveiled the pivotal role of NKCC1 in cochlear spontaneous activity. Immunohistochemistry revealed robust NKCC1 expression in ISCs, persisting until the 2nd postnatal week. Intriguingly, we observed a developmental shift in NKCC1 expression from ISCs to synaptophysin-positive efferent terminals at postnatal day 18, hinting at its potential involvement in modulating synaptic transmission during the post-hearing period. Experiments using bumetanide, a well-known NKCC inhibitor, supported the functional significance of NKCC1 in ISC spontaneous activity. Bumetanide significantly reduced the frequency of spontaneous extracellular potentials (sEP) and spontaneous optical changes (sOCs) in ISCs. NKCC1-shRNA experiments conducted in cultured cochlear tissues further supported these findings, demonstrating a substantial decrease in event frequency and area. Taken together, we revealed the role of NKCC1 in shaping the ISC spontaneous activity that govern auditory pathway development.

Keywords: Cochlea, Spontaneous activity, NKCC1, KCC4, Inner supporting cell

Developing mammalian cochlea before hearing onset exhibits spontaneous activity that facilitates maturation of auditory pathway [1]. The phenomenon is initiated by spontaneous ATP release from ISCs. ISC-released ATP evokes action potential bursts in neighboring IHCs and auditory afferent fibers [2]. The bursts propagate to the brain auditory centers and strengthen neuronal connection. The ATP released from ISC also stimulates purinergic receptors in nearby ISCs and evokes peculiar morphologic changes in these ISCs. Activated purinergic receptor subsequently causes an increase in intracellular Ca2+ concentration, opening of a Ca2+-activated Cl- channel TMEM16A, and Cl- efflux from ISCs [2, 3]. Like in many secretory gland cells the Cl- efflux from ISCs is accompanied by K+ and water efflux to maintain ionic and osmotic gradients, which leads to shrinking of ISCs and expansion of the extracellular space between IHC and ISC. Cl- efflux and shrinking of ISCs appeared to be critical in modifying excitability of nearby IHCs. When Cl- efflux was genetically disturbed by TMEM16A knock-out the spontaneous activity of ISC was abolished and burst firing pattern of IHCs and auditory afferent fibers were significantly altered despite intact ATP release and responsiveness to ATP [4].

Not surprisingly, the once shrunken ISCs regain the original volume soon, suggesting that the ISCs have certain Cl- and K+ refilling mechanisms enabling the ISCs for the next wave of activity. Here, we investigated whether NKCCs and KCCs play a role in the ISC spontaneous activity because 1) they are capable of transporting both Cl- and K+ ions across cell membrane [5] and 2) genetic mutations in some isotypes are associated with hearing deficit [6, 7].


Sprague Dawley rats were euthanized (sevoflurane inhalation followed by decapitation) and cochlear tissues were collected for experiments. All procedures were performed in accordance with the animal protocol approved by the Mokpo National University Animal Care and Use Committee (MNU-IACUC- 2021-016).


The procedures were adapted from the previously described [8]. The cochlear tissue was fixed (ice-cold 4% paraformaldehyde, for 1 h), immersed in blocking/permeabilizing buffer (PBS supplemented with either 5% normal goat or donkey serum and 0.25% Triton X-100, pH 7.4, for 1 h at room temperature), and then, incubated with the primary antibodies (overnight at 4°C). After 3 rinses, the sample was reacted with fluorescence-labeled secondary antibodies (for 1 h at room temperature) and was mounted on slideglass using Fluorsave® mounting medium (Calbiochem, 345789). Specific immunolabeling was initially examined under epifluorescence microscope (CKX53, Olympus) and high-resolution imaging was performed using a confocal laser scanning microscope (LSM710, Zeiss). Confocal images were further processed using image viewing software provided by the microscope manufacturers (Zeiss Zen), Imaris (version 7.3.0, Bitplane, Switzerland) and ImageJ (NIH). To test for specificity of labeling, control preparation in which primary antibody was omitted was examined under comparable conditions. Primary antibodies used in this study were: rabbit anti-calretinin (1:500, EMD Millipore Corporation, AB5054), mouse anti-calretinin (1:500, EMD Millipore Corporation, MAB1568), goat anti-parvalbumin (1:250, Swant, PVG-213), mouse anti-synaptophysin (1:500, EMD Millipore Corporation, MAB5258), rabbit anti-NKCC1 (1:500, EMD Millipore Corporation, ab3560P), goat anti-KCC4 (1:100, Abcam, ab78912), and mouse anti-connexin 26 (1:250, Thermofisher, 13-8100). All secondary antibodies (1:1000) were from Thermo Fisher Scientific Inc.

Simultaneous transmitted light imaging and electrophysiological recording

A freshly isolated or cultured cochlear tissue was transferred into a recording chamber under an upright microscope (Axio Examiner, Zeiss) with differential interference contrast (DIC) optics and a charge-coupled device camera (EC-50, Sony). The tissue was continuously perfused with the physiological buffer containing (in mM): 5.8 KCl, 144 NaCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 Glucose, 10 HEPES (300 mOsm, pH 7.4). All recordings were performed at room temperature (22~25°C). Drugs were prepared from frozen stocks daily, diluted in the buffer to their final concentrations and were applied by bath perfusion.

Optical images were acquired at 1 frame per s using SKYHD CAPTURE (Sky Digital Corporation). Image stacks were then created for each condition. Differential images were created by subtracting images captured at times tn and tn-5 using ImageJ (NIH) and the area and frequency of ISC shrinking was monitored. Also, an index of optical change (sOC) over time was generated. A threshold (3 standard deviations above the mean) was applied to count pixels that changed (in arbitrary units, AU).

The recording electrodes were prepared from 1 mm borosilicate glass capillaries (1B100F-4, WPI) using a multistep horizontal puller (P97, Sutter) and a microforge (MF200, WPI). The final electrode tip resistance was 5-7 MΩ. The recording electrode was filled with the buffer used for bath perfusion and was placed near the ISCs. Spontaneous extracellular potential (sEP) was recorded using pClamp 10.4 software in conjunction with Multiclamp 700B amplifier (Molecular Devices), low-pass filtered at 1 kHz, and digitized at 2 kHz with a Digidata 1440A. Baseline noise was determined by calculating root mean square (RMS, 0.05 mV). sEP with an amplitude above 0.2 mV (4 times of RMS) was counted as an event. Plots of the sOC over time were aligned to sEP recordings.

Organotypic culture

Neonatal rat (postnatal day 4) cochlear turns were isolated in ice-cold sterile Hanks’ Balanced Salt Solution. Cochlear segments were attached to the Cell-Tak (BD Biosciences) coated coverslips and maintained overnight in a humidified incubator (37°C, 5% CO2) in DMEM/F12 medium supplemented with 10% fetal bovine serum, 5 µg/ml ampicillin, B27 and N2. Upon confirming stable attachment, the tissues were treated with either scrambled or NKCC1-shRNA diluted in culture medium (1:500) for 48 h. The medium was then replaced with fresh medium every 48 h.


NKCC1-shRNA and control scrambled RNA were obtained from Korean Institute of Science and Technology (KIST) Virus faculty. mCherry and shRNA oligonucleotides were inserted into the pSicoR system using HpaI/XhoI sites. The target sequences of NKCC1-shRNA were 5ʹ-ACACACTTGTCCTGGGATT-3ʹ. shRNA transduction and protein knock-down were confirmed using immunohistochemistry.

Ion transporter expressed in neonatal ISC

The ISC spontaneous activity is observed in neonatal cochlea and rapidly wanes after the hearing onset (~P11-12 in rat). We tested if any of NKCC and KCC isotypes show such temporal expression pattern in neonatal ISC. Using immunolabeling technique, rat cochleae were examined at postnatal day 2, 9, 12, 18, 23, and 42.

First, the cochleae were double-immunolabeled with anti-NKCC1 and anti-calretinin antibodies. Calretinin is a Ca2+ -binding protein found in the cytosol of hair cells and a subset of cochlear afferent fibers [8] and therefore, can be a useful marker to test the cellular identity of NKCC1 expressing structure (hair cells and cochlear afferent fibers vs supporting cells surrounding them). Fig. 1 shows NKCC1-immunoreactivity (IR) in the organ of Corti at P2, 9, and 12. At P2, a very strong NKCC1-signal was found in interdental cells and Hensen’s cells and a relatively weaker signal was detected in supporting cells surrounding calretinin-positive hair cells and cochlear afferent fibers. NKCC1-signal near IHC row got stronger at P9. At P12, the NKCC1-positive area seems more restricted to the immediate vicinity of IHCs. Orthogonal view of the confocal z stack shows no significant overlap of NKCC1-signal with calretinin-IR, indicating the expression of NKCC1 in ISCs but not in hair cells (Fig. 1C).

At P18 or later, NKCC1-signal was no longer present in ISCs (Fig. 2A). Instead, NKCC1-signal was found in nerve ending-like structure near the basal pole of IHCs. To reveal the cellular identity of NKCC1-positive structure in older cochleae co-immunolabeling with anti-NKCC1, anti-synaptophysin and anti-parvalbumin antibodies was performed. Synaptophysin is a vesicle associated protein found in synaptic vesicle containing efferent nerve terminals [8]. Parvalbumin is a Ca2+-buffering protein present in the cytosol of hair cells and the majority of cochlear afferent fibers [8]. Neither parvalbumin-IR nor calretinin-IR showed colocalization with NKCC1-positive structure (Fig. 2A, B), which lead us to conclude that NKCC1-positive nerve ending-like structures were not cochlear afferent terminals. Most of the NKCC1-IR did overlap with synaptophysin-signal (Fig. 2A), suggesting NKCC1 expression in lateral efferent fiber endings [8] in post-hearing cochlea.

Unlike NKCC1, KCC4-IR was only detected in post-hearing organ of Corti (Fig. 2C). KCC4-signals were detected as small puncta juxtaposing connexin 26-IR in ISCs as well as in the OSCs. We concluded that KCC4 was less likely to be involved in the ISC spontaneous activity.

NKCC inhibitor reduced spontaneous activity in ISC

Since the temporal pattern of NKCC1 expression is consistent with spontaneous activity in the developing cochlea we proceeded to test a role of NKCC1 in the spontaneous activity of neonatal ISC. Morphological changes accompanying the ISC spontaneous activity were optically monitored in freshly isolated cochlear tissue from neonatal rat. When the cochlear tissue was perfused with the standard physiological buffer, periodic morphologic changes in ISCs were detected. Fig. 3A shows the extent and the timing of each event during a 5 min recording period. The frequency and average area of ISC shrinking was 4.20±1.30 events/min and 2630.46±1032.72 µm2 (n=4). We did not observe any significant changes in the frequency and area of the event up to 1~2 hours of experiment under the same condition. When a NKCC inhibitor bumetanide (10~40 µM) was applied, however, the frequency of ISC shrinking decreased significantly (1.53±1.08 events/min). The average area of event was slightly smaller compared to the control but did not reach statistical significance (2429.85±884.34 µm2). The effect of bumetanide was reversible upon washout (frequency 3.45±2.46 events/min, area 2564.16±1499.61 µm2). In a subset of these tissues, we also recorded ISC shrinking and extracellular potential (sEP) simultaneously. Most of ISC shrinking (sOC) coincided with changes in extracellular potential (Fig. 3B). Bumetanide treatment significantly decreased the frequency of sEP as well (from 6.50±2.09 events/min to 2.83±1.13 events/min, n=3) and the effect on sEP was also reversible upon washout (5.71±0.83 events/min). These results support NKCC involvement in the ISC spontaneous activity.

NKCC1-shRNA inhibited NKCC1 expression and spontaneous activity in ISC

Bumetanide is known to block both NKCC1 and NKCC2 [9]. Thus, further experiment, using a more selective NKCC1 modulator, was sought although we did not detect NKCC2-IR in neonatal ISCs (data not shown). Due to limited specificity of conventional drugs we chose to employ NKCC1-shRNA and cultured cochlear tissue. Compared to naive or scramble RNA treated tissues, significantly lower level of NKCC1-signal was detected in NKCC1-shRNA treated cochlea (Fig. 4A). Having confirmed that NKCC1-shRNA decreased NKCC1 expression, we next optically monitored the ISC shrinking in naïve, scramble RNA, and shRNA treated cochlear tissues. The frequency of event was significantly lower and the average area of event smaller in NKCC1-shRNA treated tissue (0.53±0.54 events/min, 304.81±22.35 µm2, n=5) compared to the naïve (1.50±0.56 events/min, 1084.49±278.66 µm2, n=5) or scramble RNA treated groups (1.51±0.67 events/min, 685.90±33.86 µm2, n=7) (Fig. 4E, F). In addition, simultaneous sOC and sEP recordings were performed in a subset of these cultured cochlear tissues. As seen in the freshly isolated cochleae, most sOCs closely coincided with sEPs (Fig. 4D). The frequency of sEP in NKCC1-shRNA treated tissues (0.24±0.14 events/min) was significantly lower compared to the naïve (2.05±0.58 events/min) or scramble RNA treated groups (1.91±0.54 events/min). These results suggest that NKCC1 indeed plays a significant role in ISC spontaneous activity.

The combined evidence from immunohistochemistry, pharmacology, and shRNA experiments strongly and consistently supports the central role of NKCC1 in the spontaneous activity of ISCs in the neonatal cochlea. The immunohistochemical findings unveiled a distinct temporal expression pattern of NKCC1 in ISCs during early postnatal stages. Our previous work, however, reported NKCC1-signal in interdental cells of P9 mouse cochlea with no apparent signal in the ISCs [10]. We concluded that the use of the primary antibody with certain selectivity concerns [11] (monoclonal anti-NKCC1 antibody T4, hybridoma bank) and different animal species might have misled us previously. In the current study, a robust NKCC1-IR was detected in ISCs, persisting until the 2nd postnatal week. Results from shRNA experiments presenting a significant suppression of NKCC1-IR in NKCC1-shRNA treated cochlea tissues supported the detection capability of NKCC1 antibody used in the current study.

Interestingly, the timing of NKCC1 expression in ISC coincides with the previously investigated expression pattern of TMEM16A [3], suggesting an interplay of NKCC1 and TMEM16A in the temporal initiation and cessation of spontaneous activity in the ISCs of neonatal cochlea. The Cl- efflux via the Ca2+-activated Cl- channel TMEM16A during earlier phase of ISC spontaneous activity [3, 10, 12] necessitates restoration by a transporter capable of concentrating Cl- to maintain an equilibrium potential. Utilizing the energy stored in the Na+ gradient via Na+, K+-ATPase, NKCC1 can concentrate Cl- within cells [5]. Indeed, similar co-play system of TMEM16A and NKCC1 has been found in the interstitial cell of Cajal (ICC), the pivotal pacemaker cell in the gastrointestinal tract responsible for initiating rhythmic spontaneous electrical activity. The rhythmic activity relies on TMEM16A that induces Cl- efflux. Concurrently, NKCC1 facilitates the influx of Cl- into ICC [13, 14]. This coordinated interaction sets the foundation for rhythmic depolarizations and repolarizations, playing a critical role in regulating the smooth muscle contractions within the gastrointestinal tract. It is conceivable that a comparable coordinated interaction between NKCC1 and TMEM16A may contribute to the regulation of spontaneous activity in cochlear ISCs.

The experiments using a NKCC inhibitor bumetanide or NKCC1-shRNA provided functional validation for the involvement of NKCC1 in ISC spontaneous activity. The significant reduction of sOC and sEP by those inhibitors underscored the direct association between NKCC1 activity and the observed spontaneous events. In contrast to our findings, Babola et al. [4] did not report any significant effect of bumetanide in a recent investigation exploring the involvement of P2RY1 in the ISC spontaneous activity. Notably, they preincubated the cochlear tissue with bumetanide, and consequently, a direct comparison between the control and bumetanide treated period was not feasible. Also, the continuous perturbation of the intracellular milieu with the internal solution during whole-cell patch clamp recordings might have made ISCs less dependent on bumetanide-sensitive transporters for the reloading of ions. In addition, we observed only a partial block, with approximately 60% inhibition even at the highest concentration (40 μM) of bumetanide. This suggests that NKCC1 alone may not be the exclusive contributor to ion replenishment, leading us to consider the potential involvement of alternative mechanisms and ion transporters in influencing the observed ion dynamics within ISCs. For instance, a previous research has highlighted the expression of Na+, K+, ATPase α1 in supporting cells of the cochlea [15], indicating its potential role alongside NKCC1 in ion replenishment. Similarly, prior works identified cation channels such as TMEM43 and TASK1 in ISCs [16, 17], suggesting their potential contributions to K+ dynamics and overall ion homeostasis in the cochlea.

At postnatal day 18 (P18) or later we found a developmental shift in the expression of NKCC1 from ISCs to synaptophysin-positive nerve terminals. which implied additional role of NKCC1 in lateral efferent synaptic transmission during the post-hearing period. Previously, NKCC1 expressed at sensory synaptic terminals has been shown to modulate synaptic connectivity and sensory processing. For instance, NKCC1 in photoreceptor terminals not only contributed to synaptogenesis between the photoreceptors and the next order neurons (horizontal and bipolar cells) in developing retina but also regulated neurotransmitter release from photoreceptor terminals in adult animals [18, 19]. In rodent spinal dorsal horn, NKCC1 was found in spinal axon terminals of peptidergic nociceptive primary afferents but was absent in nonpeptidergic nociceptive axon terminals [20], proposing its discriminating role in processing of pain information. Likewise, NKCC1 in lateral efferent terminals might contribute to the fine-tuning of synaptic connections around IHCs, and ultimately to the establishment of proper neural circuits for the auditory processing.

Unlike NKCC1, KCC4-signal was below our detection threshold in the neonatal cochlear ISCs before the hearing onset. However, both ISCs and OSCs of mature cochlea, as previously reported [7], displayed KCC4-signal in close proximity to connexin 26. This positioning suggests a potential role of KCC4 in facilitating the movement of K+ and Cl- ions between these supporting cells through gap junction. This becomes particularly significant in the context of maintaining constant homeostatic ion movement around cochlear hair cells, in response to ambient sound stimuli encountered in everyday life.

Author contributions: KS, K-WK, S-HP performed experiments and analyzed the data, J-KL and CJL provided reagents critical for the study. EY designed and supervised the study. All authors contributed to writing and revising the manuscript.

This work was supported by Research Funds of Mokpo National University in 2021.

Fig. 1. NKCC1 expression in neonatal cochlea. (A~C) Image of organ of Corti double-immunolabeled with anti-NKCC1 (green) and anti-calretinin (red) at postnatal day 2 (A), 9 (B), and 12 (C). NKCC1 signal is found in inner and outer supporting cells of developing cochlea. At all ages tested, NKCC1-IR was not detected in calretinin-positive cells as shown in the orthogonal views (YZ, ZX). Scale bar: 10 µm. For orientation, a few inner and outer hair cells are outlined by dotted lines.
Fig. 2. NKCC1 and KCC4 in mature cochlea. (A) Image of organ of Corti triple-immunolabeled with anti-NKCC1 (green), anti-synaptophysin (red) and anti-parvalbumin (magenta). Scale bar: 10 µm. (B) Image of organ of Corti double-immunolabeled with anti-NKCC1 (green) and anti-calretinin (red). Scale bar: 10 µm. (C, D) Images of organ of Corti (P42) double-immunolabeled with anti-KCC4 (green) and anti-connexin 26 (red). Small KCC-4 puncta juxtaposing connexin26-signals are found in inner supporting cells (C) as well as outer supporting cells immediately radial to the outer hair cells (D, Hensen’s cells). Scale bar: 5 µm.
Fig. 3. Bumetanide, a NKCC blocker, inhibited spontaneous activity of ISC. (A) Optical imaging of organs of Corti before, during and after 10 µM bumetanide treatment. The maxium size of spontaneous activities are outlined by color. The time of occurrence is indicated by color-coded timelines below. For orientation, some IHCs are outlined by dotted lines. Scale bar: 20 µm. (B) Simultaneous recordings of optical changes (blue, sOC) and extracellular potential (black, sEP). (C) Summary plot of the frequency of sOC. (D) Summary plot of the area of sOC. (E) Summary plot of the frequency of sEP. Data are mean±SD. *p<0.05 (One-way ANOVA with repeated measures followed by Tukey test).
Fig. 4. NKCC1-shRNA inhibited spontaneous activity of ISC. (A) Cultured organs of Corti immunolabeled with anti-NKCC1 antibody. For orientation, IHCs are outlined by dotted lines. Scale bar: 5 µm. (B) Summary plot of NKCC1-immunofluorescence intensity. (C) Optical imaging of cultured organs of Corti. The maxium size of spontaneous optical changes are outlined by color. The time of occurrence is indicated by color-coded timelines below. For orientation, some IHCs are outlined by dotted lines. Scale bar: 20 µm. (D) Simultaneous recording of optical changes (blue, sOC) and extracellular potentials (black, sEP) (E) Summary plot of the frequency of sOC. (F) Summary plot of the area of sOC. (G) Summary plot of the frequency of sEP. Data are mean±SD. *p<0.05 (One-way ANOVA followed by Tukey test).
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