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Cell Calcium (2001) 29(3), 191–198


© 2001 Harcourt Publishers Ltd
doi: 10.1054/ceca.2000.0183, available online at http://www.idealibrary.com on

Intracellular calcium dynamics and


membrane conductance changes
evoked by Deiters’ cell purinoceptor
activation in the organ of Corti
L. Lagostena, F. Mammano
Biophysics Sector and INFM Unit, International School for Advanced Studies, via Beirut 2-4, 34014 Trieste, Italy

Summary. Deiters’ cells function as supporting cells for the sensory-motor outer hair cells of the mammalian cochlea
and are interconnected by gap junctions. Here the electrical and Ca2+ responses of Deiters’ cells evoked by purinergic
stimulation were investigated in the organ of Corti, the auditory sensory epithelium. Adenosine 5⬘-triphosphate (ATP,
50–100 ␮M) applied focally by pressure increased the intracellular free Ca2 concentration ([Ca2]i). At the same time
ATP evoked an early inward current that was followed by an outward component, reflecting a sustained Ca2-dependent
reduction of the pre-stimulus offset current. These responses were maintained when Ca2 was removed from the extra-
cellular medium (0 [Ca2]o), indicating a contribution to Ca2 signalling from P2Y metabotropic receptors. UV photolysis
of caged inositol 1,4,5-triphosphate (InsP3, 16 ␮M) produced Ca2 responses similar to those evoked by exogenous
ATP, accompanied by reduction of the offset current. In Deiters’ cells uncoupled by octanol (1 mM), ATP activated only
the early inward current, suggesting that functional gap junctions are required in the late phase of the current responses.
Following the delivery of UV flashes to pairs of Deiters’ cells loaded with caged InsP3, the electrical coupling ratio (CR),
monitored by double patch-clamp recordings, was strongly attenuated. These data support the idea that, by promoting
inflow of cations and by controlling gap-junction conductance in a Ca2-and InsP3-dependent way, ATP might serve a
protective role in the cochlea. © 2001 Harcourt Publishers Ltd

INTRODUCTION isolated from the cochlea have shown that both ionotropic
and metabotropic P2 receptors are present in the organ of
Extracellular ATP can affect cellular functions in a variety
Corti [4,5]. Recently, we have functionally localized P2X
of tissues [1]. In the cochlea, perfusion of ATP has been
and P2Y receptors at the apical pole of the outer hair cells,
shown to suppress both cochlear microphonic and endo-
providing evidence for an ATP-activated intracellular Ca2+-
cochlear potential [2]. Consistent with these findings,
release cascade linked to an InsP3-gated intracellular store
immunolabelling against P2 purinergic receptors has been
located at the base of the hair bundle [6].
observed in cochlear tissues obtained from adult guinea
Supporting cells in the organ of Corti possess gap junc-
pigs, suggesting that ATP might exert a humoral role [3].
tions [7] and are electrically and dye coupled [8], forming
Furthermore, electrophysiological and imaging investiga-
a syncitium that provides the basis for electrical and meta-
tions conducted on sensory and supporting cells acutely
bolic cell-to-cell communication. Gap junction communi-
cation in these cells is affected by the [Ca2+]i [9,10] which,
in the cochlea, is in turn altered by extracellular applica-
Received 9 August 2000
tion of nucleotides [4,6,11,12]. Here we report the Ca2+
Revised 10 November 2000
Accepted 14 November 2000
signals as well as the membrane conductance changes of
Published online 23 January 2001 Deiters’ cells evoked by purinergic stimulation in a prepa-
Correspondence to: Dr Fabio Mammano, International School for Advanced
ration of the isolated guinea pig cochlea that preserves
Studies, via Beirut 2-4, 34014 Trieste, Italy. Tel.: 39 040 3787 254; the structural integrity of the cellular matrix within the
Fax: 39 040 3787 243; E-mail: mammano@sissa.it organ of Corti.

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192 L Lagostena, F Mammano

MATERIALS AND METHODS excitation wavelengths were selected around the absorp-
tion maximum (494 nm) of Oregon green 488 BAPTA-1.
Cell preparation
Fluorescence emission was collected with an infinity-cor-
The preparation and methods of recording in the intact rected water-immersion objective (60, N.A. 0.90;
adult organ of Corti have been described previously LUMPlanFI, Olympus) and selected at 535 nm using a sec-
[13,14]. Adult guinea pigs (200–400 g) were anaesthetized ond filter set (XF23, Omega Optical). Fluorescence images
with chloroform and decapitated. The temporal bones were formed on a fast (15 MHz readout rate) CCD sensor
were placed in modified Leibowitz cell culture medium (IA-D1, DALSA, Ontario, Canada) that was cooled by a
(L-15) containing (in mM): NaCl, 137; KCl, 5.36; CaCl2, peltier device (Marlow Industries). The sensor’s output
1.25; MgCl2, 1.0; Na2HPO4, 1.0; KH2PO4, 0.44; MgSO4, was digitised at 12 bit/pixel by customised electronics to
0.81, at 4°C. For some experiments, Ca2+ ions were produce 128128 pixel images that were recorded in
excluded and the solution was supplemented by 2 mM real time to the RAM of a host PC. The typical inter-frame
EGTA (referred to as 0 [Ca2+]o conditions in the text). pH interval for these recordings was 16 ms. For each image
was adjusted to 7.35 with NaOH and osmolarity to 320 pixel, fluorescence signals were computed as ratios F/Fo
2 mOsm/L with D-glucose. = [F(t)F(0)]/F(0), where t is time, F(t) is fluorescence fol-
lowing a stimulus that causes calcium elevation within
Patch-clamp recordings and drug delivery the cell and F(0) is pre-stimulus fluorescence computed
by averaging 10–20 images. Both F(t) and F(0) were cor-
Conventional whole-cell patch-clamp recordings were
rected for mean background fluorescence computed
made under visual control after mounting the recording
from a 2020 pixel rectangle devoid of obvious cellular
chamber on a microscope stage. Three parallel rows of
structures. The fluorescence ratio magnitude was
Deiters’ cells, extending all along the cochlear duct, are
smoothed with a two-dimensional 33 median filter and
found in the organ of Corti. Cells in the third row are
encoded by 8 bit look-up tables to produce 256 pseudo-
encountered first while proceeding from the outer to the
colour indexed images.
inner side of the organ along a radial direction. To gain
access to third row Deiters’ cells at the apex of the guinea
pig cochlea (fourth turn), a cut was made through the over- UV flash photolysis of caged compounds
lying layer of Hensen’s cells with a suction pipette, after Photolysis of intracellular caged compounds was pro-
first locally applying collagenase type I (2.5 mg/ml) under duced by the arc of a Xenon flashlamp ( JML-C2; Hi-Tech
pressure from a pipette. Cells were maintained at room Sci. Ltd). Flashes passing through a UVII bandpass filter
temperature (24–26°C). List EPC-7 patch-clamp amplifiers (wavelengths approximately 300–400 nm) were launched
(Heka) were used to drive pipettes that had been pulled on into a light guide coupled to the epifluorescence port of
a vertical puller (PP-83, Narishige) from 2.0 mm o.d. borosil- the microscope with a 45° dichroic mirror. The output of
icate glass (Clark Electromedical). Current and voltage were the light guide was focussed onto the organ of Corti
sampled at rates between 6 and 50 kHz using a standard through the objective. The cell-impermeant form of D-
laboratory interface (1401Plus, Cambridge Electronic myo-inositol 1,4,5-triphosphate, P4(5)-(1-(2-nitrophenyl)-
Design) controlled by customized software. Pipettes were ethyl) ester (caged InsP3, 16 ␮M; Molecular Probes) was
filled with an intracellular solution containing (in mM): loaded into selected cells through the patch pipette. The
KCl, 150; MgCl2, 2.0; Na2HPO4, 8.0; NaH2PO4, 1.0; EGTA, flash duration was estimated as 1 ms.
0.5 mM; adjusted to pH 7.3 with KOH and brought to Results are expressed as mean SE.
320 mOsm/L with D-glucose. For fluorescence imaging,
the intracellular solution was supplemented with the cell-
RESULTS
impermeant form of the Ca2+-selective fluorescent dye
Oregon Green 488 BAPTA-1 (100 ␮M; Molecular Probes). ATP-activated currents of Deiters’ cells uncoupled by
The pipette resistance was typically 5 Megohms (M⍀) when octanol
measured in the bath. No correction was applied to the
Deiters’ cells were electrically uncoupled by supplement-
data for liquid-junction potentials (estimated not to exceed
ing the extracellular medium with octanol (1 mM) that
 4 mV). ATP was pressure applied through a patch pipette
blocks gap junctions in this preparation [14]. In these
using a gated PicoPump (PV800, World Precision
conditions, brief (50 ms) focal applications of ATP
Instruments).
(100 ␮M), repeated at 5 min intervals, evoked transient

inward currents (Fig. 1, left ) whose amplitude was insen-
Ca2 fluorescence imaging
sitive to changes in the position of the puff pipette along
Fluorescence imaging of intracellular Ca2+ was performed the cell body (Fig. 1, right ). Results similar to those
as described previously [15]. Briefly, a narrow range of displayed in Figure 1 were obtained in 4/4 cells tested.

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Purinergic responses of cochlear Deiters’ cells 193

is that only those receptors that had recovered from


desensitization contributed to the subsequent response.
However, the ATP-evoked current did not desensitize
within the first 3 s of the first ATP application (Fig. 2A,
trace 1). This permitted us to study the voltage-depen-
dence of the ATP-evoked currents by applying voltage
ramps from 118 mV to +50 mV before and during appli-
cation of ATP (50 ␮M, 3 s, Fig. 2B). The I-V relationships
constructed in this way reversed near 0 mV and displayed
inward rectification (Fig. 2C). Results similar to those
shown in panel A were obtained in 3/3 cells whereas
those in panels B, C were reproduced in 7/7 cells.

Effect of ATP on membrane conductance and



intracellular Ca2 of Deiters’ cells coupled to the
syncitium

Application of ATP (100 ␮M, 50 ms) in octanol-free


medium evoked whole-cell current responses (Fig. 3A,
bottom) that consisted of the early and transient inward
phase due to activation of P2X ionotropic receptors
followed by a delayed and sustained component whose
Fig. 1 Invariance of the ATP-evoked current to changes of puff direction was outward relative to the pre-stimulus baseline
pipette position. The same Deiters’ cell, bathed in extracellular fluid current. For simplicity, although inappropriately, this sec-
containing the uncoupling agent octanol (1mM), was repeatedly ond component will be temporarily referred to as an out-
stimulated by brief (50 ms) applications of ATP (100 ␮M, 5 min
interval) from a puff pipette whose location was varied over a range ward current. In 10/10 octanol-treated cells, one of which
of positions (numbered 1 to 6 on the cell image at right). Similar is shown in Figure 3A (top), the outward current was
current responses were elicited at all positions. Traces have been never observed, suggesting that gap junctions in the
offset for clarity. Scale bar, 10 ␮M.
open state were required for its generation. Washing out
octanol restored the outward current and increased the
To investigate the type of purinoceptors present on offset current due to an increased syncitium conduc-
Deiters’ cells, pyridoxalphosphate-6-azophenyl-2⬘,4⬘- tance. Repeated applications of ATP in octanol-free
disulfonic acid (PPADS, 30 ␮M), a selective antagonist to medium produced a progressive reduction of the offset
P2 purinoceptors [16], was added to the superfusate. In current associated with development of the outward cur-
two cells, responses to 30 ␮M ATP were tested and found rent (Fig. 3B). A contribution from ionic conductances to
before introduction of PPADS. Within 5 min, both current this current is unlikely, as outward K currents, the only
and Ca2+ responses to ATP were completely suppressed ionic currents found in Deiters’ cells, have been shown to
(data not shown). Cells did not recover for up to 40 min. depend solely on voltage (which was kept constant in
These results are consistent with P2X2 receptor immuno- these experiments) for their activation [17]. Therefore,
fluorescence imaging which indicates expression of ATP- the simplest explanation for the results shown in Figure 3
gated ion channels on the surface of Deiters’ cells, is that the ATP-dependent outward current merely
extending from the cell body to the phalangeal process of reflects a reduction of the offset current due to increased
cells in the third (outermost) row [3]. Cells in the remain- resistance of the syncitium formed by Deiters’ cells.
ing rows show more confined immunolabelling patterns To study the effect of ATP on intracellular Ca2,
[3] suggesting that their responses to ATP might differ selected Deiters’ cells in the syncitium were loaded with
from those reported here, particularly for application of the cell-impermeant form of the fluorescent Ca2+ indica-
ATP confined to the endolymphatic pole. However, due tor Oregon Green 488 BAPTA-1 (100 ␮M). Application
to the fragility of the Deiters’ cell matrix in situ, we were of ATP (100 ␮M, 2–3 s) evoked the expected sequence of
unable to test this hypothesis directly. currents responses (Fig. 4A, top) accompanied by a rise of
When ATP was applied for several seconds at 4 min the Ca2+ fluorescence signal that spread throughout the
intervals, a progressive reduction of the ATP-evoked cur- cell body (Fig. 4A, bottom). As shown in the frame
rent was noted. Receptor desensitization increased with sequence of Figure 4B, when ATP was delivered to a
increasing duration of the application. What determined selected portion of the cell (in this case, the phalangeal
the radical difference in the responses 1, 2 and 3 (Fig. 2A) process) a concentration gradient formed initially within

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194 L Lagostena, F Mammano

Fig. 2 Desensitization and voltage dependence of the ATP


response. (A) Whole cell currents evoked by application of ATP (bar
1: 2 s; bar 2: 10 s; bar 3: 20 s) to the same Deiters’ cell repeated at
4 min intervals; dashed line: zero-current reference level; holding
potential Vh60 mV. (B) Top: voltage waveforms applied from a
holding potential Vh60 mV. Voltage ramps ranging from
118 mV to 50 mV were delivered before and during the
application of ATP (50 ␮ M, horizontal bar ). Bottom: whole cell
current response. (C) Current-voltage (I-V) relationships derived
from data portions shown as solid lines in B (bottom) by plotting
current (I) vs membrane potential (Vm) before (Control, dash-dot
line) and during pressure application of ATP (dotted line). Difference
trace (solid line) is the ATP-sensitive fraction of the I-V. Potentials
were corrected for the error due to access resistance (8.7 M⍀).
Records in A-C were obtained with 1mM octanol in the superfusate.

Fig. 3 Effect of repeated brief applications of ATP. (A) Top: whole


cell current evoked by ATP (100 ␮M, 50 ms, arrow) in the presence
of 1mM octanol. Bottom: response of the same cell 15 min after
replacing the octanol-containing medium with standard extracellular
solution (wash-out ). Dotted line indicates pre-stimulus offset
current. Holding potential, Vh20 mV. (B) Successive ATP-
applications (100 ␮M, 50 ms, arrows) repeated at 4 min intervals in
octanol free-medium. Holding potential, Vh40 mV. Records in
A, B are from two different cells.

clearly present (Fig. 5, top) whereas the [Ca2+]i (bottom)


increased uniformly throughout the cell body (inset
frames), indicating release from intracellular stores. The
the cytoplasm. The gradient then relaxed, producing a onset of the outward current coincided with the [Ca2+]i
uniform elevation of [Ca2+]i within 10.2 [/] 2.4 s approaching its maximum, reflecting a Ca2+-dependent
(n = 7) from the onset of the ATP application. In 0 [Ca2+]o, input conductance decrease mediated by gap junction
the two distinct phases of the whole cell current were still closure. Similar results were found in 4/4 cells.

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Purinergic responses of cochlear Deiters’ cells 195

Fig. 5 Responses to ATP in 0 [Ca2]o. Top: whole cell current


elicited by focal application of ATP (100 ␮M, 50 ms; arrow). Early
inward phase is followed by delayed outward phase (relative to pre-
stimulus offset current, dashed line). Bottom: per cent Ca2
fluorescence change averaged over the whole cell body. Holding
potential Vh = 20 mV. Inset frames (a–c): fluorescence-ratio
(F/Fo) images encoded according to the colour-scale bar below
frame montage and captured at the times marked by upward
triangles below Ca2 trace. Scale bar, 10 ␮M.


Effect of InsP3 on intracellular Ca2 and electrical
coupling

To establish direct evidence for a second-messenger sig-


nalling pathway in Deiters’ cells, we loaded the cell cyto-
plasm with caged InsP3 (16 ␮ M) through the patch
pipette. Representative responses evoked by the photoly-
sis of this membrane impermeant compound by a 1-ms
UV flash (wavelength 300–400 nm) are shown in Figure 6.
Fig. 4 Simultaneous recoding of whole cell currents and Ca2
responses. (A) Top: whole cell current evoked by the application of The InsP3-evoked current (top) was monophasic, resem-
ATP (100 ␮M, bar). Note large and transient inward current followed bling the sustained reduction of the pre-stimulus offset
by an outward phase (relative to pre-stimulus offset current, dashed current produced by ATP in octanol-free medium (com-
line). Bottom: Ca2+-fluorescence per cent variations measured
concurrently from the number- and colour-coded regions of pare Fig. 3 and Fig. 5, top). Following flash delivery, Ca2+
interests (ROIs) superimposed on the cell fluorescence image fluorescence increased rapidly throughout the cytoplasm
(inset; scale bar, 10 ␮m). (B) Nine selected fluorescence-ratio (Fig. 6A, bottom), equilibrating within 9.71.2 s (n3).
(F/Fo) images from the same sequence used to generate the
traces in (A), encoded according to the colour-scale bar below The frame montage in Fig. 6B highlights the distributed
frame montage. Yellow trace at the bottom of each frame nature of the fluorescence change evoked by InsP3.
reproduces timing of ATP application. Relative timing of frame Consistent with anatomical reports that a network
capture is marked by a vertical bar below this trace. Each image is
also tagged by the absolute frame number (in bracket) and by the of endoplasmic reticulum (ER) canaliculi (termed canalic-
time of acquisition relative to the onset of ATP delivery (in ms). ular reticulum) is present in Deiters’ cells [18], these
results suggest that Ca2+ might be released diffusely in

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196 L Lagostena, F Mammano

the cytoplasm from InsP3-gated intracellular stores asso- coupling between Deiters’ cells in the organ of Corti, pairs
ciated to the ER. of cells were patch-clamped and maintained under cur-
It is known that gap-junction communication is affected rent clamp near their zero-current potential (Fig. 7A). The
by the [Ca2+]i [19]. To assess and quantify electrical voltage responses evoked by injecting current into one
cell (conventionally defined as cell 1) were recorded
simultaneously from both cells and the average of 10 to
20 such responses was computed off line. In response to
depolarizing current-step stimuli (Fig. 7B, top), the voltage
of cell 1 was found to rise to a steady-state value, increas-
ing by V1, while the voltage of cell 2 reached a lower
steady state value, increasing by V2 with similar time
course (middle). From these parameters, the cell coupling
ratio was computed as CRV2/V1. We monitored CR
before (middle) and after (bottom) UV flash photolysis of
intracellular caged InsP3 (16 ␮M). CR was found to
decrease after flash delivery, indicating an InsP3- and Ca2+-
dependent modulation of gap junction permeability.
Similar results were obtained from two other cell pairs.
Taken together these data indicate that the permeability
of gap junctions connecting adjacent Deiters’ cells is
modulated by [Ca2+]i changes due to release of Ca2+ from
InsP3-gated intracellular stores.

DISCUSSION

The organ of Corti is a polarized epithelium whose upper


surface separates endolymph, an unusual extracellular
fluid rich in K, from perilymph, which communicates
with cerebrospinal fluid. Cochlear supporting cells,
Deiters’ and Hensen’s cells, are an integral part of the
epithelium and have been proposed to take an active part
in protection against trauma during exposure to high-
intensity sound [20]. Both cell types receive direct inner-
vation [21,22], suggesting that they may participate also
in a neural feed-back control loop.
In our experiments we found functional evidence for a
uniform distribution of P2X receptors (Fig. 1) in Deiter’s
cells, consistent with immunohistochemical studies con-
ducted in whole-mount preparations and in radial sec-
tions of the whole cochlea [3,23]. This suggests that ATP
may exert its action on Deiters’ cells through two parallel
and distinct pathways. One pathway, humoral in nature,

Fig. 6 UV photolysis of intracellular caged InsP3. (A) Top: whole


cell current; note reduction of the offset current (dashed line) after
flash (curly arrow); holding potential Vh20 mV. Inset:
fluorescence image of the patched Deiters’ cell, loaded with caged
InsP3 (16 ␮M) through the pipette, with 4 superimposed colour- and
number-coded ROIs; scale bar, 10 ␮m. Bottom: per cent Ca2+
fluorescence changes measured from the corresponding ROIs in
inset. (B) nine selected fluorescence images encoded according to
the colour-scale bar below frame montage. Yellow trace at the
bottom of each frame reproduces timing of UV flash. Relative timing
of frame capture is marked by a vertical bar below this trace. Each
image is also tagged by the absolute frame number (in bracket) and
by the time of acquisition relative to flash delivery (in ms).

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Purinergic responses of cochlear Deiters’ cells 197

through release from intracellular stores. This, in turn, may


cause suppression of intercellular communication during
those same conditions that are responsible for the eleva-
tion of ATP concentration in cochlear fluids. Activation of
this complex purinergic signaling mechanism in response
CR
to stressors, such as noise and ischemia, may thus con-
tribute to a protective scheme that is ultimately responsible
for uncoupling the ‘cochlear amplifier’ [3,20]. Although the
exact subtype of receptor involved remains to be estab-
lished, activation of the P2Y metabotropic pathway can
potentially extend the time course of this protective func-
tion for the entire duration of the noxious stimuli as P2Y1
CR
and P2Y2 receptors do not readily desensitize [1].

ACKNOWLEDGEMENTS
Fig. 7 Effect of intracellular InsP3 on electrical coupling. (A) Video Supported by: grant Progetto di Ricerca Avanzata CADY,
image showing a patch-clamped pair of adjacent Deiters’ cells in
row 3 of the organ of Corti. Outer hair cells are clearly visible above
Istituto Nazionale di Fisica della Materia; grant
Deiters’ cell cups. Scale bar, 20 ␮M. (B) voltage responses (V1 N.9906194385, Ministero dell’Università e della Ricerca
and V2) evoked by injecting current into one of the two Deiters’ Scientifica to FM. We thank David A. Williams and
cells (I1480 pA, 43 ms, upper trace) to measure coupling ratios
(CR) before (control, middle traces) and after flash photolysis of
Jonathan F. Ashmore for critical comments and helpful
intracellular caged InsP3 (16 ␮m, bottom traces). suggestions.

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