Eur J Neurosci. 22:2708-22.

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Eur J Neurosci. 22:2708-22.

European Journal of Neuroscience, Vol. 22, pp. 2708–2722, 2005 ª Federation of European Neuroscience Societies

Shift from depolarizing to hyperpolarizing glycine action

occurs at different perinatal ages in superior olivary

complex nuclei

Stefan Löhrke, Geetha Srinivasan, Martin Oberhofer, Ekaterina Doncheva and Eckhard Friauf

Animal Physiology Group, Department of Biology, University of Kaiserslautern, POB 3049, D-67653 Kaiserslautern, Germany

Keywords: auditory brainstem, chloride regulation, glycine reversal potential, voltage-sensitive dyes, gramicidin perforated-patch

Abstract

The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D ⁄ H-shift). To

analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage-sensitive dye recordings in

auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated-patch recordings. Our results show a

differential timing of the D ⁄ H-shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day

(P) 5–9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D ⁄ H-shift was observed in the

medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed

glycine-induced depolarizations between P0–9. While no regional differences regarding the D ⁄ H-shift were found within the MSO, SPN,

and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D ⁄ H-shift at P4–5. However, in

the high-frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the lowfrequency

regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D ⁄ H-shifts in

SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane-associated immunoreactivity of the Cl –

outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the

mere presence of KCC2 does not correlate with functional Cl – outward transport.

Introduction

Glycine and GABA are prominent inhibitory neurotransmitters in the

mammalian CNS. Early in development, the action of glycine and

GABA is depolarizing in various neuronal systems, e.g. hippocampus

(Cherubini et al., 1990), hypothalamus (Chen et al., 1996), lateral

superior olive (Kandler & Friauf, 1995a), neocortex (Luhmann &

Prince, 1991), retina (Huang & Redburn, 1996), and spinal cord (Wu

et al., 1992). Glycine ⁄ GABA-mediated depolarizations can cause an

increase in the intracellular Ca 2+ concentration and thus, may act as

trophic signals (reviewed in Ben-Ari, 2002). By the end of the second

postnatal week (rats and mice), the glycine ⁄ GABA action has shifted

from depolarization to hyperpolarization (D ⁄ H-shift).

The lateral superior olive (LSO), an auditory brainstem nucleus

involved in sound localization by computing bilateral intensity differences,

is a suitable model for studying the D ⁄ H-shift. The glycinergic

input from the medial nucleus of the trapezoid body (MNTB) is

anatomically and electrophysiologically well characterized (Fig. 1A;

reviewed in Sanes & Friauf, 2000), and easily accessible in brainstem

slices. The D ⁄ H-shift in the rat LSO was originally observed with sharp

intracellular microelectrodes (Kandler & Friauf, 1995a). Later on,

whole-cell patch-clamp and gramicidin perforated-patch recordings

revealed that the D ⁄ H-shift is due to age-dependent Cl – regulation

(Ehrlich et al., 1999; Kakazu et al., 1999). The glycine reversal potential

(EGly ECl–) decreases significantly between postnatal day (P) 1 and

Correspondence: Dr Stefan Löhrke, as above.

E-mail: loehrke@rhrk.uni-kl.de

Received 10 May 2005, revised 19 September 2005, accepted 21 September 2005

doi:10.1111/j.1460-9568.2005.04465.x

P11 in LSO neurons, in line with a reduction of intracellular chloride

concentration, [Cl – ]i, during this period (Fig. 1B). As a consequence of

the high [Cl – ]i, glycine and GABA responses are often excitatory

(Ben-Ari, 2002; see also Fig. 1C) and can cause an increase in

intracellular Ca 2+ concentration (Kullmann et al., 2002). Developmental

lowering of [Cl – ]i in LSO neurons is achieved through the action of the

K-Cl cotransporter KCC2 (Balakrishnan et al., 2003). KCC2 is a

secondary-active cotransporter, which is fuelled by the outward-directed

K + -gradient and coexports Cl – from the cytosol (Delpire, 2000).

Aside from the LSO, other superior olivary complex (SOC) nuclei

also receive inhibitory input from the MNTB, e.g. the medial superior

olive (MSO; Grothe & Sanes, 1994; Smith et al., 2000) and the

superior paraolivary nucleus (SPN; Behrend et al., 2002). Moreover,

MNTB neurons themselves receive inhibitory input via recurrent

collaterals from MNTB neurons and from neurons in the ventral

nucleus of the trapezoid body (reviewed in Thompson & Schofield,

2000). In contrast to the LSO, the perinatal development of inhibition

in MNTB, MSO, and SPN (i.e. the presence of a D ⁄ H-shift), has not

been studied so far.

Here, we investigated the presence of a D ⁄ H-shift in the MNTB, the

MSO, and the SPN by using optical imaging with the fast voltagesensitive

dye RH795 (for method review, see Ebner & Chen, 1995), as

well as gramicidin perforated-patch recordings. With both methods,

the cell interior is not affected and therefore glycine responses are

obtained at the native [Cl – ]i, a prerequisite for investigating the

development of Cl – regulation. Voltage-sensitive dye recordings have

been recently established for the SOC (Srinivasan et al., 2004). The

capability of measuring electrical activity from cell ensembles with


Fig. 1. Inhibitory circuits within the SOC, which originate from the MNTB. (A) Schematic representation of the inhibitory, glycinergic inputs from the MNTB

(arrows) to various SOC nuclei, namely the LSO, MSO, and the SPN. The MNTB receives inhibitory input via its own collaterals. The tonotopic organization of

each nucleus, from high-frequency (hf) to low-frequency (lf), is illustrated by the shading from dark grey to white. d, dorsal; l, lateral; cl, centre line.

(B) Gramicidin perforated-patch recordings (illustrated by a cell-pipette symbol here and subsequently in Figs 5, 6 and 8) from LSO neurons show a negative shift

of EGly (open symbols) between P1 and P11, while VR (horizontal broken lines) remains nearly unchanged during this time period ()58 ± 1 mV at P1–4, n ¼ 24;

)63 ± 2 mV at P5–8, n ¼ 9; )63 ± 1 at P9–11, n ¼ 15). Before P5, EGly was usually more positive than VR, whereas after P7, EGly was usually more negative than

V R, demonstrating the D ⁄ H-shift in glycine action between P5 and P8. Modified from Ehrlich et al. (1999). (C) Glycine and ⁄ or GABA-mediated action potentials

of a P3 LSO neuron, elicited by electrical stimulation of the MNTB input (0.6 mA, 100 ls, 0.2 Hz) in the presence of the glutamate receptor antagonists CNQX

(20 lm) and APV (30 lm). In four out of five stimulus presentations, the responses are supra-threshold, giving rise to a single spike. (D) Schematic representation

of the S-shaped LSO in a P10 slice, scanned by the hexagonally arranged 464-photodiode array with a 20· objective (each square depicts one photodiode). At this

magnification, the entire LSO is captured by the photodiode array (with approximately 265 photodiodes covering the nucleus). In the present study, the LSO was

divided into four regions from dorsomedial (1) to ventrolateral (4) to search for possible regional differences in the VSD responses.

high spatio-temporal resolution (usage of a 464-photodiode array)

represents a methodological strength of the optical imaging method. It

enables the analysis of region-specific response patterns within a given

nucleus and across different nuclei in the same experiment (cf.

Fig. 1D). The availability of voltage-sensitive dye recordings prompted

us to reinvestigate the D ⁄ H-shift in the LSO, where developmental

differences regarding the tonotopic organization were controversially

discussed in the past. Kotak et al. (1998) reported depolarized IPSC

reversal potentials at P3–5 in medial limb neurons (high-frequency),

yet not in lateral limb neurons (low-frequency). In contrast, Ehrlich

et al. (1999) found depolarized EGly values throughout the LSO at P1–

4. Finally, we investigated the distribution of KCC2 immunoreactivity

in the SOC nuclei between P0 and P8 in order to assess whether the

expression of this Cl – extrusion protein correlates with the occurrence

of the D ⁄ H-shift.

Materials and methods

All protocols were approved by the animal care and use committees

responsible for our institution and adhered to the National Institutes of

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722

D ⁄ H-shift in various SOC nuclei 2709

Health Guide for the Care and Use of Laboratory Animals. The

number of animals were reduced to the possible minimum.

Voltage-sensitive dye recordings

Brainstem slices were prepared from Sprague–Dawley rat pups of both

gender between embryonic day (E) 18 and postnatal day (P) 10 (the

day of birth was taken as P0). The animals were bred and housed in

our facility in accordance with the current German animal protection

law. Fetal animals were obtained from time-pregnant rats deeply

anaesthetized with an intraperitoneal (i.p.) injection of ketamine

(0.1 mg ⁄ g body weight), xylazine hydrochloride (0.005 mg ⁄ g body

weight), and acepromazine maleate (0.01 mg ⁄ g body weight). Prenatal

and postnatal animals were deeply anaesthetized with an overdose

of ketamine (1 mg ⁄ g body weight i.p.), decapitated, and their brain

dissected. Coronal brainstem slices containing the SOC (300-lm

thickness) were cut in ice-cold preparation solution [composition

(mm): NaHCO3, 25; NaH2PO4, 1.25; KCl, 2.5; MgCl2, 1; CaCl2,2; d-glucose, 260; Na-pyruvate, 2; myo-inositol, 3; and kynurenic acid,


2710 S. Löhrke et al.

1; pH 7.4 when bubbled with 95% O2, 5%CO2] with a vibrating

microtome (VT-1000, Leica, Bensheim, Germany) and transferred into

a chamber filled with carboxygenated (95% O 2,5%CO 2) storage

solution [composition (mm): NaCl, 125; NaHCO 3, 25; NaH 2PO 4,

1.25; KCl, 2.5; MgCl2, 1; CaCl2, 2;d-glucose, 10; Na-pyruvate, 2;

myo-inositol, 3; ascorbic acid, 0.4; pH 7.4]. After 60 min at approximately

34 °C, the slices were stored at room temperature (20–23 °C)

for another 30 min. Thereafter, they were transferred into voltagesensitive

dye (VSD) solution (composition equaled the storage

solution, except that it contained 122.5 mm NaCl and 5 mm KCl),

supplemented with 100 lm of the dye RH795 (Molecular Probes,

Leiden, the Netherlands). The increased potassium concentration

(5 mm K + instead of 2.5 mm K + ) improved the success rate of VSD

experiments (Srinivasan et al., 2004) without influencing resting

membrane potential (VR; assessed with whole-cell patch-clamp

recordings). After dye incubation for 30 min, unbound dye was

washed out for 50 min by placing the slices in the recording chamber,

which was continuously perfused with VSD solution (perfusion rate

3–4 mL ⁄ min).

The recording chamber was mounted on an upright microscope

(Axioskop2 FS, Zeiss, Jena, Germany), equipped with differential

interference contrast ⁄ fluorescent optics (Zeiss objectives: 5· Fluar,

0.25 numerical aperture (NA); 20· Achroplan, 0.5 NA; 40· Achroplan,

0.8 NA) and a camera system (CCD camera, VC 45, PCO,

Kelheim, Germany; PC frame grabber card, DT 3155, Data Translation,

Bietigheim-Bissingen, Germany). Images of the slices were

digitized, and the SOC nuclei and the border of the slice were outlined

with graphic software (CorelDraw, version 9.0, Corel, Ottawa,

Canada; cf. Fig. 1A). During optical recordings, the slices were

illuminated with light from a mercury arc light bulb (HBO103, Osram,

München, Germany), passing through a set of fluorescent filters

(excitation 546 ± 12 nm, beam splitter 580 nm, emission 590 nm;

Zeiss). Emitted light was detected by a photodiode-array (Neuroplex,

RedShirtImaging, Fairfield, CT, USA), containing 464 hexagonally

arranged diodes. The physical dimension of a single diode was

750 lm · 750 lm. Depending on the magnification of the objective

(40·)5·), the area covered by the photodiode-array ranged from

0.163 mm 2 to 10.44 mm 2 and allowed the scanning of the entire

SOC nuclei (Fig. 1D). Signals were digitized, low-pass filtered at

1 kHz, and sampled at a frequency of 1.66 kHz.

Glycine responses were induced by electrical stimulation of the

appropriate input fibers (bipolar stainless steel electrodes, Rhodes

NEX 200, Science Products; single pulse, 0.2 ms duration, 3 mA

amplitude) and pharmacologically isolated by bath-applied strychnine

(0.5 lm). In case of LSO, MSO, and SPN recordings, the stimulus

electrodes were placed in the ipsilateral MNTB. In case of MNTB

recordings, they were placed in the region of the centre line to

stimulate the axons of the globular bushy cells (Fig. 1A). In all cases,

responses obtained in the presence of strychnine were subtracted from

those obtained under control conditions to reveal the strychninesensitive

responses and, thus, the glycine-induced PSPs.

Data acquisition and analysis were performed using Neuroplex 3.01

(RedShirtImaging), Origin 5.0 (Microcal Inc., Northampton, MA,

USA), and Winstat für Excel 1999.3 (Fitch Software, Zierenberg,

Germany). Improvement of signal-to-noise ratio was obtained by

temporal averaging, i.e. signals from a single diode represent the

average of ten subsequent recordings (20-s intervals). Regional

differences within a given nucleus were assessed by comparing

signals from defined nucleus regions. Region selection was performed

according to the tonotopic organization of the nuclei (for overview, see

Fig. 10). The signals of adjacent diodes were averaged such that

recordings from three diodes (MSO at 20· objective) to 32 diodes

(MNTB at 40· objective) were pooled. The size of the area covered by

the diodes in the relevant nuclei was always 8 438 lm 2 for the LSO,

4 219 lm 2 for the MSO, 7 031 lm 2 for the SPN, and 11 250 lm 2 for

the MNTB.

Optical signals are presented as the ratio DF ⁄ F (in percentage) of

the fluorescence change (DF) and the fluorescence baseline level (F).

The fluorescence baseline level for each diode was determined

immediately before the acquisition of the stimulus-induced optical

signals. From the fluorescence baseline level, the VR cannot be

inferred. However, we can exclude any negative effects of the VSD on

VR, because of normal VR values between )60 mV and )70 mV

measured with whole-cell patch-clamp recordings from SOC neurons

incubated in VSD solution.

RH795 shows a fluorescence decrease and a fluorescence increase

upon membrane potential depolarization and hyperpolarization,

respectively. To be in accordance with electrophysiological conventions,

the optical signals were inverted such that upward deflections

correspond to depolarizations and downward deflections correspond to

hyperpolarizations. For false colour presentations, a 16-colour scale

from red to purple was used to code the polarity of responses from

depolarizing to hyperpolarizing, with green colours representing the

baseline level (equal to zero-responses). Optical signals were quantified

by using the peak amplitude of DF. The D ⁄ H-shift was

determined on the basis of average DF ⁄ F-values and defined as the

age when depolarizing values changed to exclusively hyperpolarizing

values. The values from the last depolarizing age were statistically

compared with those from the hyperpolarizing period (Student’s

t-test). The first appearance of a significant difference between two

ages is depicted with asterisks in the diagrams (*, P < 0.05; **,

P < 0.01; ***, P < 0.001). Data are given as mean ± standard error of

mean. Values from fetuses aged between E18 and E20 were pooled.

Perforated-patch recordings

Slices were prepared as described above. To commence recordings,

they were transferred to a chamber continuously perfused (rate 3–

4mL⁄ min) with patch-clamp solution (composition equal to storage

solution). The recording chamber was mounted on an upright

microscope (Eclipse E600FN, Nikon, Tokyo, Japan) equipped with

differential interference contrast optics (Nikon objectives, 4· CFI

Achromat, 0.1 NA; 60· CFI Fluor W, 1.0 NA) and an infrared video

camera system (CCD camera, VX44, PCO; PC frame grabber card,

pciGrabber-4plus, PHYTEK, Mainz, Germany). Patch pipettes were

pulled from borosilicate glass capillaries (GB150–8P, Science Products,

Hofheim, Germany) with a vertical puller (PP-83, Narishige,

Tokyo, Japan). They had resistances of 3.5–5 MW when filled with

pipette solution [composition (mm): KCl, 140; EGTA, 5; MgCl2, 3;

Hepes, 5; pH 7.3 with KOH] and were connected via an Ag ⁄ AgCl

wire to an EPC-9 patch-clamp amplifier (HEKA elektronik, Lambrecht,

Germany). Patch pipettes were frontfilled with pipette solution

for approximately 60 s and then backfilled with pipette solution

supplemented with 2.5–5 lg ⁄ mL gramicidin D (Sigma-RBI,

Deisenhofen, Germany). To prove that recordings were obtained

under intact perforated-patch conditions, the Cl – concentration of the

pipette solution was reduced to 16 mm by equimolar substitution of

130 mm KCl with potassium gluconate in some experiments

performed on MNTB neurons (for details, see Results section).

Signals were digitized, low-pass filtered at 8.3 kHz, and sampled at

frequencies of 1–10 kHz.

Glycine responses were induced by pressure application of 1 mm

glycine via a patch pipette (tip diameter 4 lm) connected to a

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


Picospritzer (General Valve Corp., Fairfield, NJ, USA). Short puffs

(10 ms) of glycine were administered to the somata with 21–28 kPa

pressure at intervals of 10 s or 30 s. To determine E Gly, neurons were

stepped from a holding potential of )70 mV to various command

potentials (VC) between )120 mV and +30 mV by increments of

30 mV. To circumvent the problems of stimulus-related Cl – loading

and depletion (Ehrlich et al., 1999), the V C steps were applied from

)120 mV to +30 mV (‘up’ protocol) as well as from +30 mV to

)120 mV (‘down’ protocol) for each neuron. At each VC, glycineinduced

currents were elicited, and their peak amplitudes were

measured. Data obtained with ‘up’ and ‘down’ protocols were pooled

and processed with Origin 5.0 (Microcal Inc.) to obtain the best fitting

regression line and its x-intersect, which corresponds to E Gly. The time

of D ⁄ H-shift was defined by the average values for EGly and VR, i.e.

when EGly was persistently more negative than VR. Only cells with VR

more negative than )50 mV were analysed. Data are given as

mean ± standard error of mean.

Immunohistochemistry

Sprague–Dawley rat pups of either gender were used at age P0, P4,

and P8 (two animals obtained from different litters per age). Animals

were deeply anaesthetized with chloral hydrate (0.7 mg ⁄ g body

weight i.p.) and perfused transcardially with 0.01 m phosphatebuffered

saline (PBS, pH 7.4), followed by cold 4% (weight ⁄ volume;

w ⁄ v) paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The

brains were dissected and cryoprotected ⁄ postfixed in a 30% (w ⁄ v)

sucrose + 4% (w ⁄ v) paraformaldehyde solution for approximately

36 h at 4 °C. Coronal sections (30 lm) through the medullary

brainstem were cut with a freezing microtome and collected in PBS.

The sections were kept free-floating in multiwell plates and washed

in PBS between the different incubation steps. First, sections were

preincubated for one hour in a PBS-based blocking solution containing

10% (v ⁄ v) normal goat serum, 3% (w ⁄ v) bovine serum albumin,

and 0.3% (v ⁄ v) Triton X-100. Thereafter, the primary antibody was

added to the solution (anti-KCC2, 1 : 300 v ⁄ v, Upstate, NY, USA).

After approximately 24 h incubation at 4 °C, the sections were bathed

for 2 h in the secondary antibody solution containing goat-anti-rabbit

IgG (Alexa Fluor 488, 1 : 1000 v ⁄ v, Molecular Probes) at room

temperature. They were finally mounted onto gelatin-coated slides and

embedded in Vectashield (Vector, Burlingame, CA, USA).

Labelled sections were analysed with a confocal laser scanning

microscope (Axioplan 2, LSM 510, Zeiss) equipped with Plan-Neofluar

objectives (10·, 0.3 NA; 40· oil, 1.3 NA, Zeiss). They were illuminated

with 488 nm light from an argon laser, passing through a set of

fluorescent filters (excitation 488 nm, emission 505–550 nm; Zeiss).

The confocal aperture was set between 66 lm and 71 lm. Optical

sections of 0.5-lm thickness were processed to obtain digital images at a

resolution of 2 048 · 2 048 pixels (Image Browser, v2.8, Zeiss).

Results

D ⁄ H-shift occurs at P4–5 in all LSO regions, yet displays

differences in the time course

In medial limb neurons of the gerbil LSO, yet not in lateral limb

neurons, Kotak et al. (1998) reported depolarized IPSC reversal

potentials at P3–5. In contrast, Ehrlich et al. (1999) found

depolarized EGly values throughout the rat LSO between P1–4. To

clarify the controversy regarding regional differences, we reinvestigated

the LSO with multiple-site VSD recordings after having

divided the nucleus into two medial, high-frequency regions and two

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722

D ⁄ H-shift in various SOC nuclei 2711

lateral, low-frequency regions, according to the tonotopic organization

(Fig. 1D).

VSD recordings (52 slices, P2–9) revealed strychnine-sensitive

responses at all ages. In false colour presentations (heat maps; Fig. 2),

their amplitude and polarity changed systematically with age from

warm colour (depolarizations) to cold colour (hyperpolarizations). At

P2 in each region, almost every diode signal appeared in warm colours

20 ms after stimulus onset, when the peak amplitudes occurred (Fig. 2,

Aa, cf. vertical dotted line in Fig. 2, Ab). Averaged strychninesensitive

responses from adjacent diodes displayed depolarizations in

all four regions (Fig. 2, Ab). By P5, the situation had changed in that

signal peaks in regions 1 and 2 appeared in cold and green colours,

whereas signal peaks in regions 3 and 4 appeared mostly in green

colours 15 ms after stimulus onset (Fig. 2, Ba). Accordingly, the

average responses in regions 1 and 2 corresponded to hyperpolarizations

and those in regions 3 and 4 to zero-responses (Fig. 2, Bb). At

P7, signal peaks in regions 1–3 appeared mostly in cold colours 8 ms

after stimulus onset (Fig. 2, Ca), and the average responses of these

regions corresponded to hyperpolarizations (Fig. 2, Cb). In region 4,

almost the entire spectrum from cold to warm colours was present at

P7, which is reflected in the average zero-response (Fig. 2, Cb).

Finally at P9, signal peaks 7 ms after stimulus onset displayed mainly

cold colours in all four LSO regions (Fig. 2, Da), corresponding to

hyperpolarizing average responses (Fig. 2, Db). Occasionally, the

blockade of inhibition by strychnine gave rise to action potentials,

which, due to the mathematical subtraction algorithm, appeared as

downward deflections in the strychnine-sensitive responses (asterisks

in Fig. 2, Db). These action potentials were excluded from further

analysis. The quantitative analysis showed that a D ⁄ H-shift occurred

in each LSO region at P4–5 (Fig. 3). In regions 1 and 2 (highfrequency

regions), hyperpolarizing responses had amplitudes that

were large already at P6 ()0.04 ± 0.01% and )0.03 ± 0.01%,

respectively; Fig. 3A and B). In contrast, amplitudes in regions 3

and 4 (low-frequency regions) were one order of magnitude smaller at

P6 ()0.006 ± 0.007% and )0.006 ± 0.006%, respectively; Fig. 3C

and D). Amplitudes of approximately the same size as seen at P6 in

the high-frequency regions were not present until P8–9 in the lowfrequency

regions (Fig. 3E), suggesting that the development of

efficient inhibition is delayed by 2–3 days in the latter regions.

D ⁄ H-shift occurs between P5 and P9 throughout the MSO

VSD recordings

Each MSO (62 slices, E18–20 until P9) was divided into a ventral

high-frequency region and a dorsal low-frequency region (insets in

Fig. 4). Between these two regions, there were no differences in the

strychnine-sensitive responses, as illustrated by the two depolarizing

responses at E18–20 and the two hyperpolarizing responses at P9

(traces in Fig. 4). In both regions, responses changed from being

exclusively depolarizing at E18–20 to exclusively hyperpolarizing at

P8–9. Altogether, the D ⁄ H-shift took place at P5–6 in both MSO

regions, yet hyperpolarizing amplitudes of considerable size did not

occur until P8 (diagrams in Fig. 4). This finding is corroborated by the

fact that the difference between depolarizing and hyperpolarizing

responses became significant between P5 and P8 in both regions

(Fig. 4, Ab and Bb; P ¼ 0.04 and 0.03, respectively).

Perforated-patch recordings

We performed complementary gramicidin perforated-patch recordings

from a total of 40 neurons aged P0–9, which were randomly selected

within the MSO. In a P3 neuron, glycine application resulted in inward


2712 S. Löhrke et al.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


currents at VC between )120 mV and )60 mV, and outward currents

at VC between )30 mV and +30 mV (Fig. 5A). In the cell illustrated in

Fig. 5A, E Gly amounted to )44 mV (Fig. 5B).

D ⁄ H-shift in various SOC nuclei 2713

Fig. 3. In the LSO, VSD recordings reveal differences in maturation of hyperpolarization between high-frequency region and low-frequency region. Each panel

shows strychnine-sensitive fluorescence changes obtained from individual slices (open symbols) and their average values (closed symbols) including standard error

of mean, with positive values representing depolarizing peak amplitudes and negative values representing hyperpolarizing peak amplitudes (number in brackets

equals the number of slices per age). Black and white bars depict the periods with exclusively depolarizing (de) and hyperpolarizing (hyper) glycine activity,

respectively. In all four regions, the D ⁄ H-shift occurred at P4–5. In regions 1 and 2 (A and B), the average amplitudes of hyperpolarizing responses at P6 were

considerably larger than those in regions 3 and 4 (C and D). In the latter two regions, amplitudes of similar size to those seen at P6 in regions 1 and 2 did not become

obvious until P8–9. (E) Summary of the time courses in D ⁄ H-shift depicted in diagrams A–D, demonstrating the difference between the high-frequency and the

low-frequency regions regarding the efficiency of inhibition (grey bar). Asterisks in A–D indicate the first appearance of statistical differences between the last day in

the depolarizing period and the corresponding day in the hyperpolarizing period. This way of data presentation and analysis is also used in Figs 4–6.

Based on EGly and VR values obtained from 40 MSO neurons, the

age-dependency of the polarity of glycine action was analysed

(Fig. 5C). E Gly and V R remained nearly constant between P0 and P8

Fig. 2. Shift from glycine-induced depolarization to hyperpolarization (D ⁄ H-shift) in the LSO revealed by VSD recordings. Electrical stimulation of the MNTB-

LSO pathway, in combination with strychnine application, revealed glycine responses. The strychnine-sensitive optical signals correspond to depolarizations or

hyperpolarizations if they are upward deflections or downward deflections, respectively. False colour depictions (panels a) show the outline of the LSOs and the four

regions, as well as the level of peak amplitude of strychnine-sensitive signals on a 16-colour scale from warm colours (de, depolarizations) to cold colours (hyper,

hyperpolarizations). Traces (panels b) correspond to average responses of adjacent diodes (A–C, 24 diodes, 40· objective; D, six diodes, 20· objective) from the four

regions illustrated in panels a. Arrows mark stimulus onset, vertical dotted lines mark peak amplitudes of responses from region 1, which corresponds to the time of

false colour depictions in panels a; Dt values represent the latency between stimulus onset and peak amplitude; horizontal dotted lines illustrate the baseline levels.

(A) At P2, all four regions showed mainly warm colours, i.e. depolarizations. (B) At P5, regions 1 and 2 showed cold and green colours, equivalent to

hyperpolarizations. Regions 3 and 4 displayed mainly green colours, equivalent to zero-responses. (C) At P7, regions 1–3 showed mainly cold colours,

corresponding to hyperpolarizations. In region 4, almost the entire colour spectrum was present, as shown by zero-responses (green colours), depolarizations (warm

colours), and hyperpolarizations (cold colours) side by side. As a consequence, the average response was zero (panel b). (D) At P9, all four regions showed

exclusively cold colours, corresponding to hyperpolarizations. Asterisks mark reversed action potentials that were excluded from further analysis (for details, see

text). Scale bars in D hold for panels A–D.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


2714 S. Löhrke et al.

Fig. 4. In the MSO, VSD recordings reveal the D ⁄ H-shift at P5–6. The MSO was divided into two halves, a ventral high-frequency region and a dorsal lowfrequency

region (inset in panels Aa and Ba). (Aa) Examples of a strychnine-sensitive depolarization and hyperpolarization from the high-frequency region at E18–

20 and P9, respectively. Arrows mark the time of electrical stimulation. (Ab) Data plotted from a total of 62 slices identify the D ⁄ H-shift at P5–6 in the highfrequency

region. Large average amplitudes of hyperpolarizing responses did not occur until P8. (Ba) Examples of strychnine-sensitive depolarizations and

hyperpolarizations from the low-frequency region at E18–20 and P9, respectively. (Bb) Like in the high-frequency region, the D ⁄ H-shift occurred at P5–6, with

hyperpolarizing amplitudes of considerable size from P8 in the low-frequency region.

(Fig. 5C), with EGly > VR. The D ⁄ H-shift occurred between P8 and

P9, demonstrated by EGly < VR at P9 and by the significant difference

of the E Gly values (Fig. 5C; P ¼ 3 · 10 )4 ). We further recorded

glycine responses in current-clamp mode at V R from the same

40 neurons analysed under voltage-clamp conditions. In 37 of them

(93%), membrane potential changes occurred according to the

predictions from the E Gly–V R relations, i.e. depolarizations if

EGly > VR, and hyperpolarizations if EGly < VR. At P0–8, 82% of

the neurons (27 of 33) showed depolarizations, whereas at P9 all four

neurons showed hyperpolarizations (Fig. 5D), thus providing further

evidence of the D ⁄ H-shift occurring at P8–9.

Together, our data from VSD recordings and perforated-patch

recordings revealed a D ⁄ H-shift at P5–9 in the MSO. Unlike the LSO,

regional differences were not found in the MSO.

D ⁄ H-shift occurs around birth throughout the SPN

VSD recordings

Each SPN (46 slices, E18–20 until P6) was divided into a medial

high-frequency region and a lateral low-frequency region (insets in

Fig. 6A and B). At a given age, average strychnine-sensitive responses

from the two regions were very similar (Fig. 6, Aa and Ba). Regarding

the age-dependency of the responses, there was also no difference

between the medial and the lateral SPN region (Fig. 6, Ab and Bb).

Between P0 and P6, hyperpolarizing responses were predominant,

except for some zero-responses (in six of 37 slices in the medial

region, and in seven of 40 slices in the lateral region). Depolarizing

responses were not found between P0 and P6, yet they were most

often seen at E18–20 (in five of six slices in both regions). The D ⁄ Hshift

occurred between E18 and P0 in both regions (Fig. 6, Ab and Bb;

P ¼ 0.01 and 0.004, respectively).

Perforated-patch recordings

Recordings from 40 neurons, randomly selected within the SPN,

revealed that the average EGly was more negative than VR at P1 and

thereafter, indicating that the D ⁄ H-shift occurred between E18 and P1

(Fig. 6, Cb). Current-clamp recordings at VR, obtained from 36 neurons,

showed glycine-induced depolarizations and hyperpolarizations

in 33 of the cells (92%), matching with the EGly–VR relations. From

P1 on, hyperpolarizations occurred in 84% of the neurons (21 of 25)

and depolarizations in 16% (four of 25; Fig. 6C). The results from

current-clamp recordings are consistent with the conclusion that the

D ⁄ H-shift occurs around birth.

Together, the results from VSD recordings and perforated-patch

recordings show that the D ⁄ H-shift occurs around birth (between E18

and P1) in the SPN. This is approximately one week earlier than in the

LSO and MSO. No regional differences were found in the SPN.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


Fig. 5. In the MSO, perforated-patch recordings reveal the D ⁄ H-shift at P8–9. (A) Glycine-induced current responses obtained from a voltage-clamped P3 neuron

at command potentials (VC) between +30 mV and )120 mV. Arrow marks the start of glycine application for all traces. (B) Current–voltage relations obtained from

the neuron in A, stepped from )120 mV to +30 mV (‘up’ protocol; closed symbols) as well as from +30 mV to )120 mV (‘down’ protocol; open symbols). EGly

corresponds to the potential at the x-intersect of the regression line and amounted to )44 mV. (C) EGly-age relation and VR-age relation obtained from 40 MSO

neurons. Open symbols represent individual EGly values, and closed symbols with error bars represent average EGly values with standard error of mean. Number in

brackets equals the number of cells per age. The broken line corresponds to the regression line obtained from the V R value of all cells. For the sake of clarity,

individual V R values are not shown. The D ⁄ H-shift occurred at P8–9. (D) A glycine-induced depolarization from the same P3 neuron as in panels A and B, as

opposed to a glycine-induced hyperpolarization from a P9 neuron (E Gly ¼ )95 mV), both current-clamped at V R.

No D ⁄ H-shift throughout the MNTB between P0 and P10

VSD recordings

Each MNTB (40 slices, P3–10) was divided into three regions along

the mediolateral axis, according to the tonotopic organization from

high-frequency to low-frequency (insets in Fig. 7). In the medial highfrequency

region, average strychnine-sensitive responses were predominantly

hyperpolarizing (at 7 of 8 days) and declined in amplitude

between P3 and P10 (closed symbols in Fig. 7, Ab). In the central

region, amplitudes of average strychnine-sensitive responses jittered

around the baseline during the time period analysed (closed symbols

in Fig. 7, Bb). Finally, in the lateral region, average strychninesensitive

responses were mainly depolarizing (at 5 of 8 days; closed

symbols in Fig. 7, Cb). Representative examples of response traces

obtained from the three regions at P3 and P10 are depicted in Fig. 7,

Aa, Ba and Ca. With the exception of the small hyperpolarization in

the medial region at P3 (Fig. 7, Aa) and the small depolarization in the

lateral region at P3 (Fig. 7, Ca), zero-responses occurred. Regarding

all individual strychnine-sensitive responses obtained between P3 and

P10 (120 recordings from 40 slices), depolarizations, hyperpolarizations,

and zero-responses occurred in each region (Fig. 7, Ab, Bband Cb). Irrespective of the location, 49% zero-responses, 27% hyperpolarizations,

and 24% depolarizations were found (59 of 120, 32 of 120,

and 29 of 120, respectively). Furthermore, the dynamic range of

responses, i.e. the span between maximum depolarization and

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722

D ⁄ H-shift in various SOC nuclei 2715

maximum hyperpolarization, decreased between P3 and P10 in each

region. No D ⁄ H-shift was found in any of the three MNTB regions.

Perforated-patch recordings

In perforated-patch recordings, EGly was determined from 25 neurons

(P0–9) that were randomly located within the MNTB and visually

identified as principal cells (large oval-shaped cell body, eccentric

nucleus). Except for three cases, all neurons showed EGly > VR, implying a depolarizing glycine action (Fig. 8A). As shown by the fact

that all mean EGly values were more positive than the VR regression

line (Fig. 8A), no obvious age-dependency of EGly was observed

between P0 and P9, implying that no D ⁄ H-shift takes place in the

MNTB during this period. This was confirmed by current-clamp

recordings (24 cells), which showed glycine-induced depolarizations

in all 21 neurons with EGly > VR, independent of their age (Fig. 8B).

The remaining three neurons, whose EGly < VR, showed glycineinduced

hyperpolarizations.

Although gramicidin perforated-patch recordings are usually recognized

for measurements at undisturbed native [Cl – ]i (Kyrozis &

Reichling, 1995), we wanted to exclude that the positive EGly values

of MNTB neurons, which we observed and, which represented a high

[Cl – ] i, were due to unintentional whole-cell recordings, i.e. to dialysis

of the neurons with the pipette solution ([Cl – ]pip). To do so, we

changed from our standard pipette solution containing 146 mm Cl –


2716 S. Löhrke et al.

Fig. 6. In the SPN, the D ⁄ H-shift occurs around birth. For VSD recordings, the SPN was divided into two halves, a medial high-frequency region and a lateral lowfrequency

region (inset in panels Aa and Ba). Perforated-patch recordings were obtained from randomly selected neurons throughout the SPN. (Aa) Examples of a

strychnine-sensitive depolarization and hyperpolarization from the high-frequency region at E18–20 and P6, respectively. (Ab) In the high-frequency region the shift

occurred between E18 and P0 (43 slices). (Ba) Examples of a strychnine-sensitive depolarization and hyperpolarization from the low-frequency region at E18–20

and P6, respectively. (Bb) In the low-frequency region, the shift occurred also between E18 and P0 (46 slices). (Ca) Glycine-induced depolarization from an E18–20

neuron (EGly ¼ )53 mV) and hyperpolarization from a P7 neuron (EGly ¼ )100 mV), both current-clamped at VR. (Cb): Perforated-patch data revealed the D ⁄ H

shift at P0–1 (40 neurons).

(high [Cl – ]pip) to a pipette solution with only 16 mm Cl – (low [Cl – ]pip)

and recorded from 12 MNTB neurons aged P3–9. The average EGly

obtained with low [Cl – ] pip amounted to )21 ± 8 mV and was not

significantly different from that obtained with high [Cl – ] pip

()16 ± 7 mV, n ¼ 13, P ¼ 0.6). In addition, most MNTB neurons

(10 of 12) recorded with low [Cl – ]pip displayed EGly values that were

more positive ()32 mV to +29 mV) than those predicted in the case of

whole-cell recordings ()55 mV; Fig. 8C). This demonstrates that our

sample of MNTB neurons were indeed analysed in perforated-patch

recordings and that these neurons had a high native [Cl – ] i. It also

demonstrates that gramicidin ionophores are impermeable to Cl – .

In addition to the visual identification of principal MNTB neurons,

we validated their nature in some cases by means of electrophysiological

characterization. To do so, we assessed the pattern of action

potential firing in 11 of the 25 neurons. Seventy-three percent (eight of

11) of the neurons responded with a single action potential at the start

of depolarizing current steps (Fig. 8D), which is typical of principal

MNTB neurons (Wu & Kelly, 1991). These eight neurons, aged P2–6,

had mean VR and EGly values of )62 ± 2 mV and )17 ± 10 mV,

respectively, and showed glycine-induced depolarizations in seven of

eight cases. The remaining three cells showed repetitive action

potentials in response to depolarizing current injections (Fig. 8D),

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


Fig. 7. In the MNTB, VSD recordings show no D ⁄ H-shift between P3 and P10. The MNTB was divided into three regions along the mediolateral, high-to-low

frequency axis (inset in panels Aa, Ba, and Ca). (Aa) Recordings from the medial, high-frequency region, showing a small hyperpolarization and no response at P3

and P10, respectively. (Ab) In the medial region, average strychnine-sensitive responses were mostly of hyperpolarizing nature and their amplitude declined with age

(closed symbols and solid regression line; 40 slices). (Ba) Recordings from the central region, showing zero-responses both at P3 and P10. (Bb) In the central region,

zero-responses occurred between P3 and P10 on average (closed symbols and solid regression line). (Ca) Recordings from the lateral, low-frequency region, showing

a small depolarization and a zero-response at P3 and P10, respectively. (Cb) In the lateral region, average responses were mostly of depolarizing nature between P3

and P10 (closed symbols and solid regression line).

characteristic of nonprincipal neurons (Banks & Smith, 1992) or of

presynaptic calyces of Held (Forsythe, 1994). These results provide

further evidence that the majority of recordings in the MNTB were

indeed obtained from principal neurons.

Together, VSD recordings and perforated-patch recordings demonstrated

no D ⁄ H-shift between P0 and P10 in the MNTB. On the

single-cell level, mostly depolarizing glycine responses occurred,

whereas recordings from cell ensembles showed depolarizing and

hyperpolarizing glycine responses to almost the same amount.

KCC2 immunoreactivity in the SOC is present already at P0

In order to assess whether the occurrence of the D ⁄ H-shift correlates

with the developmental expression profile of the Cl – extrusion protein

KCC2, we performed immunohistochemistry in the SOC. At P0, the

LSO, the MSO, and the SPN already showed strong KCC2

immunoreactivity, whereas labelling in the MNTB was weak

(Fig. 9). Until P8, immunoreactivity appeared to decrease slightly in

the LSO, the MSO, and the SPN, whereas the signal in the MNTB

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722

D ⁄ H-shift in various SOC nuclei 2717

remained unchanged (Fig. 9A). In all nuclei, immunoreactivity was

quite strong in the neuropil, yet weak in the somata (Fig. 9B). A

change in the staining pattern was observed between P0 and P8, when

an increase in perisomatic and peridendritic labelling occurred

(Fig. 9B). Perisomatic and peridendritic labelling became obvious at

different ages in the four nuclei, i.e. already at P0 in the SPN, at P4 in

the LSO and the MSO, but not until P8 in the MNTB. Most

interestingly, plasma membrane-associated KCC2 immunoreactivity

was found already several days before the respective D ⁄ H-shift in all

four SOC nuclei (Fig. 9B, vertical broken bars; Fig. 10). Together, the

KCC2 immunohistochemical data, in combination with the electrophysiological

results, demonstrate that the mere presence of KCC2

protein is not sufficient for hyperpolarizing glycine action in the

various SOC nuclei.

Discussion

Three main results were obtained in the present study. (i) Within the

four major SOC nuclei (LSO, MNTB, MSO, SPN), D ⁄ H-shifts are


2718 S. Löhrke et al.

Fig. 8. In the MNTB, perforated-patch recordings show no D ⁄ H-shift between P0 and P9. (A) Principal neurons (n ¼ 25), randomly selected in the MNTB,

showed almost no age-dependent change in EGly and VR (broken regression line) between P0 and P9. Most neurons (22 of 25) had an EGly that was more positive

than VR, indicating depolarizing glycine action. (B) Glycine-induced depolarizations from a P3 and a P8 neuron, both current-clamped at VR. (C) Same individual

EGly values as shown in panel A, but plotted with the information about the used pipette chloride concentration ([Cl – ]pip). Open symbols correspond to recordings

obtained with a high [Cl – ]pip (146 mm), closed symbols to those with a low [Cl – ]pip (16 mm). Horizontal lines mark EGly values that would be expected in case of

whole-cell recordings with high or low [Cl – ] pip. Most neurons (10 of 12) recorded with the low [Cl – ] pip showed E Gly more positive than )55 mV (lower horizontal

line), demonstrating intact perforated-patch recordings and high native intracellular chloride concentrations. (D) Most tested neurons (eight of 11) generated a single

action potential in response to a 200 pA-current injection at V R, which is characteristic for principal MNTB neurons. The remaining three showed a train of action

potentials, which is characteristic for non-principal MNTB neurons and the presynaptic calyces of Held.

staggered in time and occur over a period of almost two weeks. (ii)

Throughout the LSO, the D ⁄ H-shift occurs at P4–5. However, the size

of hyperpolarizing amplitudes increases in delayed time course in the

low-frequency regions compared to the high-frequency regions by

approximately two days. (iii) In the LSO, MSO, and MNTB,

immunoreactivity of the Cl – outward transporter KCC2 is present

already at birth and therefore at a time when glycine action is still

depolarizing, indicating that the mere presence of the KCC2 protein

does not correlate with a low [Cl – ]i.

The D ⁄ H-shift, a common phenomenon in the mammalian CNS

In many, if not all, mammalian brain regions, the inhibitory transmitters

glycine and GABA induce depolarizations early in development. It is

generally accepted that these depolarizations are important for synapse

stabilization (reviewed in Ben-Ari, 2002). Sorting the D ⁄ H-shift from

different brain regions shows a chronological order along the caudorostral

axis, namely from the spinal chord perinatally (Wu et al., 1992;

Hübner et al., 2001), over the brainstem after P3 (Singer et al., 1998), up

to the cerebellum (Brickley et al., 1996), the hippocampus (Ben-Ari

et al., 1989), and the cerebral cortex (Owens et al., 1996) between P7 and

P11. Previously, we discussed that the D ⁄ H-shift in the rat LSO

(between P5–8; Ehrlich et al., 1999) fits perfectly into this chronological

order along the caudorostral axis. The data presented here change our

view in so far as even within a local complex of different nuclei, the time

of D ⁄ H-shift varies for almost two weeks (from E18 to P1 in the SPN

until P10–12 in the MNTB, Fig. 10). Therefore, we now conclude that a

strict chronological order of the D ⁄ H-shift along the caudorostral axis

does not exist.

The reason for the differences in D ⁄ H-shift timing within the SOC

is unclear. Nonetheless, the development from depolarizing to

hyperpolarizing glycine action is completed by the onset of hearing

(around P12) in each of the four SOC nuclei analysed. This

demonstrates the importance of inhibition for sound localization.

The LSO is involved in sound localization by detecting interaural level

differences based on the integration of ipsilateral excitation and

contralateral inhibition (reviewed in Irvine, 1992). The MSO, which is

also involved in sound localization, processes interaural time differences.

The excitatory inputs converging from both ears in the MSO are

modulated by inhibition (Brand et al., 2002). So far, the knowledge

about the role of inhibition in the SPN and MNTB is insufficient. In

the MNTB, the strength of hyperpolarizing glycine action increases

even after hearing onset (Awatramani et al., 2004a), indicating that

some developmental steps of the glycinergic input may be experiencedependent.

Such an experience-dependent mechanism has indeed been

reported recently for the gerbil MSO (Kapfer et al., 2002).

We reinvestigated the LSO with multiple-site VSD recordings in

order to clarify the controversial results obtained by Kotak et al.

(1998), who observed EIPSC > VR only in the medial, high-frequency

limb of P3–5 gerbils, and Ehrlich et al. (1999), who described

EGly > VR throughout the LSO of P1–4 rats. Our results, demonstrating

depolarizing glycine action throughout the LSO until P4, are in

accordance with the latter study. Kotak et al. (1998) reasoned that

negative E Cl appear earlier in the lateral, low-frequency limb of gerbils

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D ⁄ H-shift in various SOC nuclei 2719

Fig. 9. KCC2 immunoreactivity in the SOC nuclei is present already at P0. Confocal laser scanning images of KCC2-labelled SOC sections at P0, P4, and P8.

(A) At all three ages, immunoreactivity was stronger in the LSO, MSO, and SPN than in the MNTB. In the LSO, MSO, and SPN, immunoreactivity appeared to

decrease from P0 to P8, whereas in the MNTB, immunoreactivity remained unchanged. (B) High-power images from the preparations shown in A. At P0, intense

labelling occurred in the neuropil (n) of the LSO and MSO, whereas very weak labelling appeared in the somata (s). Until P8, the immunoreactivity pattern changed

in both nuclei, i.e. perisomatic (arrows) and peridendritic labelling (arrowheads) became clearly visible. In the SPN, perisomatic and peridendritic labelling was

clearly visible already at P0, becoming more distinctive by P8. In the MNTB, clear perisomatic immunoreactivity became apparent only at P8. Vertical broken bars

indicate the time of D ⁄ H-shift obtained from VSD and perforated-patch recordings. Scale bars, 100 lm (A) and 10 lm (B); dorsal up; lateral to the right.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


2720 S. Löhrke et al.

Fig. 10. Overview of the time of D ⁄ H-shift in LSO, MSO, SPN, and MNTB.

The days of D ⁄ H-shift, as revealed by VSD recordings, are provided within the

nuclei, those obtained with perforated-patch recordings are marked with a cellpipette

symbol. In the MSO and the two low-frequency regions of the LSO, a

delayed development of inhibition occurred. The asterix indicates data from

Ehrlich et al. (1999).

than in the medial limb, namely before P3–5. In contrast, our results

show that the opposite is true in the rat LSO (Fig. 3). Interestingly,

differences in the development in auditory nuclei between highfrequency

and low-frequency regions, in that the latter reach maturity

later, are a common phenomenon (summarized and discussed in

Friauf, 1992). Therefore, it is surprising that the observations by Kotak

and coworkers represent an exception to this phenomenon.

KCC2 and the D ⁄ H-shift

Gene-silencing experiments have demonstrated that the K-Cl cotransporter

KCC2 renders glycine hyperpolarizing in LSO neurons (Balakrishnan

et al., 2003). Electron microscopy has demonstrated the

presence of KCC2 protein in the plasma membrane of LSO neurons

already during the depolarizing phase of glycine, and the perisomatic

distribution of KCC2 does not change substantially until P60 (P. Blaesse,

unpublished observations). This implies that KCC2 activity is regulated

by post-translational modifications and ⁄ or protein–protein interactions.

Here, we show a continuous presence of KCC2 immunoreactivity also in

the MSO, the SPN, and the MNTB between P0 and P8. Thus, in all four

SOC nuclei, the mere emergence of KCC2 appears to be unrelated to the

D ⁄ H-shift. It should be noted though that we performed laser scanning

microscopy in the present study, which cannot unequivocally localize

the proteins to the plasma membrane. Earliest perisomatic and

peridendritc staining in the various nuclei was detected at different ages

(SPN at P0; MSO and LSO at P4; MNTB at P8). Regarding the

peridendritic staining, the differences may reflect the developmental

neuropil reorganization (e.g. a reduction in the number of dendrites due

to pruning; Rietzel & Friauf, 1998). Together with the electrophysiological

results, KCC2 immunohistochemistry shows that the glycinergic

inhibition in the SPN develops earlier than in the other SOC nuclei.

Finally, it is very likely that KCC2 renders glycine hyperpolarizing in the

MSO, the SPN, and the MNTB, as it does in the LSO. Final proof may be

obtained by means of recordings from KCC2 knockout animals.

Optical imaging with VSDs, an electrophysiological approach

In the present study, we performed VSD recordings as well as

gramicidin perforated-patch recordings in a complementary way to

address several questions related to neuronal chloride homeostasis. In

general, the results obtained with the two methods were similar: Each

method demonstrated an early D ⁄ H-shift in the SPN, a later

occurrence in the MSO and the LSO, and no shift in the MNTB

until P10 (Fig. 10). However, when comparing the results of the two

methods in detail, several differences became apparent. For example,

in the MSO at P8, VSD recordings showed exclusively hyperpolarizing

responses (Fig. 4), whereas perforated-patch recordings revealed

that EGly > VR in most cases (Fig. 5). In the SPN at E18–20, VSD

responses were on average depolarizing (Fig. 6, Ab and Bb), whereas

the average EGly was more negative than the average VR (Fig. 6, Cb).

Finally, in the MNTB, amplitudes of VSD responses were very small

throughout the age period tested (Fig. 7), whereas depolarizations

were strong and EGly > VR in perforated-patch recordings (Fig. 8A

and B). Possible reasons for the different results obtained with the two

methods are: (i) different numbers and ⁄ or types of glycine receptors

(synaptic vs. extra-synaptic) are activated by the two different

stimulation methods (electrical stimulation of MNTB inputs vs.

glycine puff-application on somata). Additionally, locally different

chloride regulations must be assumed (Jarolimek et al., 1999). (ii) In

VSD experiments, various numbers of cells contribute to responses

obtained even with a single photodiode (e.g. the area covered with a

20· objective is 37.5 lm · 37.5 lm; for details, see Srinivasan et al.,

2004). Thus, VSD recordings mostly represent average responses of

several adjacent neurons (ensemble activity), in contrast to patchclamp

recordings, which are obtained from single neurons. (iii)

Regarding the MNTB, it is possible that our electrical stimulation in

the region of the centre line failed to activate the glycinergic MNTB

input. Indeed, the VSD responses from the MNTB were very small on

average, and the percentage of zero-response was high (49%; Fig. 7).

(iv) In our perforated-patch experiments, the time of D ⁄ H-shift was

defined by EGly and VR. Recently, Tyzio et al. (2003) showed that with

this experimental approach, V R may be determined incorrectly when

measured in small cells. For VSD recordings, such errors are unlikely

to occur, indicating that the D ⁄ H-shift determination by VSD

recordings is more reliable than with perforated-patch recordings.

We also addressed the question of region-specific changes (along

the frequency axis) in the various nuclei. Regarding the latter question,

VSD recordings are favoured over perforated-patch recordings

because of the unique possibility to simultaneously obtain concurrent

recordings from large slice areas (e.g. entire nuclei).

In the MNTB, results from perforated-patch recordings as well as

VSD recordings showed no D ⁄ H-shift until P10. In most perforatedpatch

recordings (21 of 24 cells), glycine induced depolarizations,

whereas depolarizing and hyperpolarizing responses were found to

almost the same extent with VSD recordings (27% hyperpolarizations;

24% depolarizations, and 49% zero-responses). Our perforated-patch

recordings were obtained mainly from somata of MNTB principal

neurons (cf. Fig. 8). Our data are in agreement with those obtained by

Awatramani et al. (2004b), who found relatively positive GABA

reversal potentials (EGABA > VR) in P5–7 principal neurons, indicating

a high [Cl – ]i at this age. Only at the age of P13–15, did they find

E Gly < V R in principal neurons. These data, together with those of the

present study, demonstrate that the D ⁄ H-shift occurs between P10 and

P12 in principal MNTB neurons.

Our VSD recordings from the LSO identified an age-dependent

decrease of the latency between stimulus onset and peak amplitude

(Fig. 2). This acceleration of synaptic transmission is in agreement

with the reduction in latency of contralaterally elicited PSPs, measured

in LSO neurons between E18 and P17 with sharp microelectrodes

(Kandler & Friauf, 1995a). In the MSO (Fig. 4) and the SPN (Fig. 6),

peak amplitudes also occurred at shorter latencies with increasing age.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 2708–2722


It is likely that at least some of these kinetic changes are due to agerelated

changes in passive membrane properties (Sanes, 1993; Kandler

& Friauf, 1995b), the development of a glycine uptake mechanism

(Friauf et al., 1999), and the exchange of ‘immature’ against ‘mature’

glycine receptor subunits (Friauf et al., 1997).

In conclusion, the general conformity of VSD results with

electrophysiological results demonstrates that optical imaging with

VSDs is a powerful electrophysiological method and that VSD

recordings are well suitable to investigate the maturation of neuronal

inhibition and age-related changes in [Cl – ] i under native conditions.

The D ⁄ H-shift and general developmental gradients

As the D ⁄ H-shift in the various SOC nuclei occurred over a period

lasting almost two weeks (at E18–P1 in SPN; at P4–5 in LSO; at P5–9 in

MSO; after P10 in MNTB), one may wonder whether this time

difference is reflected by other developmental gradients, such as cell

birth or synapse formation. Regarding cell birth, the peak of neurogenesis

occurs between E12 and E16 with differences among the various

nuclei (Altman & Bayer, 1980). MSO neurons are born first (peak at

E12), followed by SPN neurons (E13), LSO neurons (E14–16), and

finally by MNTB neurons (E15–16). Thus, the period of neurogenesis

lasts much less than the period of the D ⁄ H-shifts, and moreover, there is

no match between the sequences. Concerning synapse formation, the

available data also contradict the idea that the successive D ⁄ H-shifts are

due to a developmental gradient in general. First, efferent fibers from the

cochlear nucleus form collaterals almost synchronously between E18–

20 in the rat LSO, MSO, and MNTB (Kandler & Friauf, 1993). Second,

in all three nuclei, glycinergic and glutamatergic synaptic transmission

is present by E18 (Kandler & Friauf, 1995a). Third, immunoreactivity

to the a1 glycine receptor subunit becomes present at P0 in LSO and

SPN, at P4 in MNTB, and at P8 in MSO (Friauf et al., 1997), thus

demonstrating a different gradient from those mentioned above. Finally

glycine transporter 2 immunoreactivity appears around birth in all

auditory brainstem nuclei, thus demonstrating no gradient (Friauf et al.,

1999). Taken together, it appears that the different ages at which the

D ⁄ H-shift represent a developmental gradient that is unique in the

maturation of auditory brainstem structures because the period lasts

almost two weeks, much longer than other periods known so far.

Acknowledgements

We thank Sascha Ehrhardt for excellent technical assistance, and Drs Christian

Lohr and Joachim Deitmer for support with the confocal microscope. Financial

support was provided by the Deutsche Forschungsgemeinschaft (Lo 718 ⁄ 1–3

and Fr 772 ⁄ 8–2).

Abbreviations

[Cl – ]i, intracellular chloride concentration; [Cl – ]pip, pipette chloride concentration;

D ⁄ H-shift, shift of glycine ⁄ GABA action from depolarization to

hyperpolarization; E, embryonic day; E GABA, GABA reversal potential; E Gly,

glycine reversal potential; F, fluorescence baseline level; DF, fluorescence

change; i.p., intraperitoneally; KCC2, K-Cl cotransporter; LSO, lateral superior

olive; MNTB, medial nucleus of the trapezoid body; MSO, medial superior

olive; NA, numerical aperture; P, postnatal day; SOC, superior olivary

complex; SPN, superior paraolivary nucleus; VC, command potentials; VR,

resting membrane potential; VSD, voltage-sensitive dye.

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