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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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CECA-29.QXD 2/2/01 11:49 PM Page 192
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.
Effect of InsP3 on intracellular Ca2 and electrical
coupling
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
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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