Download Ion Conductances in Supporting Cells Isolated From the Mouse

J Neurophysiol 89: 118 –127, 2003.
10.1152/jn.00545.2002.
Ion Conductances in Supporting Cells Isolated From the Mouse
Vomeronasal Organ
VALERIA GHIARONI,1* FRANCESCA FIENI,1* ROBERTO TIRINDELLI,2 PIERANGELO PIETRA,1
AND ALBERTINO BIGIANI1
1
Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, 41100 Modena;
and 2Istituto di Fisiologia Umana, Università di Parma, 43100 Parma, Italy
Submitted 11 July 2002; accepted in final form 19 September 2002
INTRODUCTION
The vomeronasal organ (VNO) is a chemosensory structure
involved in the detection of pheromones in many mammals
(Døving and Trotier 1998). VNO sensory epithelium contains
specialized neurons that are thought to transduce the chemical
information related to pheromones into action potentials to the
brain. Vomeronasal neurons are bipolar cells with an apical
dendrite that reaches the epithelium surface and an axon projecting to the accessory olfactory bulb. A wealth of information
is now available on the molecular and functional properties of
these neurons (reviewed in: Biasi et al. 2001; Keverne 1999;
*V. Ghiaroni and F. Fieni contributed equally to the work.
Address for reprint requests: A. Bigiani, Dipartimento di Scienze Biomediche, Sezione di Fisiologia, Università di Modena e Reggio Emilia, via Campi
287, 41100 Modena, Italy (E-mail: [email protected]).
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Tirindelli et al. 1998). VNO epithelium also contains supporting, glial cells (Carmanchahi et al. 1999; Garrosa and Coca
1991; Höfer et al. 2000; Naguro and Breipohl 1982; Vaccarezza et al. 1981). Like chemosensory neurons, these cells
are bipolar cells with an apical process reaching the epithelial
surface, where it branches in several tall microvilli, and a basal
process reaching the basal lamina. Unlike neurons, however,
data on the functional properties of supporting cells in the
VNO are not currently available.
In the olfactory epithelium, supporting cells possess a conspicuous resting K⫹ conductance that likely regulates the extracellular K⫹ (Masukawa et al. 1985; Trotier 1998; Trotier
and MacLeod 1986). It has been suggested that this control
might be important in setting the olfactory neurons at their
maximum sensitivity for odorant detection (Trotier and MacLeod 1986). In other sensory organs, glial cells show quite
complex membrane properties. In the retina, for instance, Müller cells are astrocyte-like cells expressing voltage-gated ion
channels, neurotransmitter receptors and various uptake carrier
systems (reviewed in: Newman and Reichenbach 1996). These
properties enable Müller cells to control the activity of retinal
neurons by regulating the extracellular concentration of neuroactive substances such as K⫹, GABA and glutamate. In
addition, it has been proposed that voltage-gated Na⫹ channels
in these cells could be activated by the functioning of adjacent
neurons: this way, glial cells could sense the activity of neighboring neurons (Chao et al. 1994). In short, supporting cells
seem to be key elements for controlling the transduction and
signaling in excitable tissues.
We have addressed the issue of the functional properties of
supporting cells in the VNO by studying with the patch-clamp
technique their membrane conductances. To this aim, we analyzed the ion currents in voltage-clamp conditions. We found
that vomeronasal supporting cells had unique electrophysiological properties, including a rather unusual (for glial cells)
ratio for resting ion permeabilities (PK:PNa:PCl ⫽ 1:0.23:1.4).
In addition, VNO supporting cells possessed voltage-gated K⫹
and Na⫹ currents with biophysical and pharmacological properties that differed from those of adjacent vomeronasal neurons. Our findings indicate that VNO supporting cells display
complex membrane properties and raise the possibility that
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0022-3077/03 $5.00 Copyright © 2003 The American Physiological Society
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Ghiaroni, Valeria, Francesca Fieni, Roberto Tirindelli, Pierangelo
Pietra, and Albertino Bigiani. Ion conductances in supporting cells
isolated from the mouse vomeronasal organ. J Neurophysiol 89:
118 –127, 2003. 10.1152/jn.00545.2002. The vomeronasal organ
(VNO) is a chemosensory structure involved in the detection of
pheromones in most mammals. The VNO sensory epithelium contains
both neurons and supporting cells. Data suggest that vomeronasal
neurons represent the pheromonal transduction sites, whereas scarce
information is available on the functional properties of supporting
cells. To begin to understand their role in VNO physiology, we have
characterized with patch-clamp recording techniques the electrophysiological properties of supporting cells isolated from the neuroepithelium of the mouse VNO. Supporting cells were distinguished from
neurons by their typical morphology and by the lack of immunoreactivity for G␥8 and OMP, two specific markers for vomeronasal
neurons. Unlike glial cells in other tissues, VNO supporting cells
exhibited a depolarized resting potential (about ⫺29 mV). A Goldman-Hodgkin-Katz analysis for resting ion permeabilities revealed
indeed an unique ratio of PK:PNa:PCl ⫽ 1:0.23:1.4. Supporting cells
also possessed voltage-dependent K⫹ and Na⫹ conductances that
differed significantly in their biophysical and pharmacological properties from those expressed by VNO neurons. Thus glial membranes
in the VNO can sustain significant fluxes of K⫹ and Na⫹, as well as
Cl⫺. This functional property might allow supporting cells to mop-up
and redistribute the excess of KCl and NaCl that often occurs in
certain pheromone-delivering fluids, like urine, and that could blunt
the sensitivity of VNO neurons to pheromones. Therefore vomeronasal supporting cells could affect chemosensory transduction in the
VNO by regulating the ionic strength of the pheromone-containing
medium.
ION CONDUCTANCES IN VOMERONASAL SUPPORTING CELLS
they might participate in the signal transduction and processing
in the VNO.
METHODS
Dissociation of vomeronasal supporting cells
lular solution. The access resistance of the patch pipette tip was
estimated by dividing the amplitude of the voltage steps by the peak
of the capacitive transients (from which stray capacitance had been
subtracted). Values typically ranged from about 10 to 15 M⍀. Leakage and capacitive currents were not subtracted from currents under
voltage clamp, and all voltages have been corrected for liquid junction
potentials (Neher 1992).
Input resistance of vomeronasal cells was measured as the slope of
the linear current-voltage (I-V) relationship around ⫺80 mV. Cell
membrane capacitance was measured by integrating the capacitative
current transient during application of a 10-mV voltage step from a
holding potential of about ⫺80 mV (Bigiani and Roper 1993).
Analysis of electrophysiological data
Results are presented as means ⫾ SE. Data were analyzed using a
Student’s t-test. Significance level was taken as P ⬍ 0.05.
The Goldman-Hodgkin-Katz equation (GHK) (Hille 2001) was
used to evaluate the relative membrane permeabilities to K⫹, Na⫹,
and Cl⫺. Values of zero-current potential (V0) were measured at
different concentration of the relevant ion and corrected for the shunt
to ground by the seal resistance (Bigiani et al. 1996; Lynch and Barry
1991). This allowed us to estimate the cell’s resting potential (Vr) at
different concentrations of the relevant ion.
Correction of V0 for the shunt to ground by seal resistance was
obtained by applying the following equation (for details, see Bigiani
et al. 1996; Lynch and Barry 1991)
V r ⫽ 关V0 ⫺ 共Gseal/Gin兲 䡠 Eseal兴/共Gm/Gin兲
where Gseal is the seal conductance evaluated from the seal resistance,
Gin is the input conductance evaluated from the input resistance, Gm
is the membrane conductance evaluated from Gin and Gseal, and Eseal
is the potential across the pipette-membrane seal resistance, which is
assumed to be nonselective and to represent only the liquid junction
potential between the pipette and the bath solution.
Concentration-inhibition curves for the effect of the ion channel
blockers, tetraethylammonium (TEA) or tetrodotoxin (TTX), on voltage-gated ion currents were obtained by adding increasing concentrations of the blocker into the bath solution and by measuring the
corresponding change in ion current magnitude. The data were fitted
to the logistic equation
%I ⫽ 100兵1 ⫺ 1/关1 ⫹ 共C/IC50兲n兴其
I/Imax ⫽ 1/兵1 ⫹ exp关共V ⫺ V0.5兲/k兴其
J Neurophysiol • VOL
(2)
where %I is the percent fraction of the ion current blocked by TEA or
TTX, C is the blocker concentration, IC50 is the blocker concentration
that produces 50% inhibition of the voltage-gated current, and n is the
Hill coefficient.
Steady-state inactivation curve for sodium currents were obtained
by fitting the data with a Boltzmann equation
Whole cell recording
Membrane currents of single VNO cells were studied at room
temperature by whole cell patch-clamp (Hamill et al. 1981), using an
Axopatch 1D amplifier (Axon Instruments, Union City, CA). Signals
were recorded and analyzed using a Pentium computer equipped with
Digidata 1320 data acquisition system and pClamp8 software (Axon
Instruments). pClamp8 was used to generate voltage-clamp commands and to record the resulting data. Signals were prefiltered at 5
kHz and digitized at 50- or 100-␮s intervals.
Patch pipettes were made from borosilicate glass capillaries (Garner Glass, Claremont, CA) on a two-stage vertical puller (model PB-7,
Narishige, Tokyo, Japan). The standard pipette solution contained (in
mM) 120 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 11 EGTA, 2 ATP, and
0.4 GTP, pH 7.3 with KOH. In some experiments, KCl was replaced
by an equal concentration of CsCl, K gluconate, or Cs gluconate.
Pipette resistances typically were 5– 8 M⍀ when filled with intracel-
(1)
(3)
where I/Imax is current elicited during a test pulse and normalized to
the maximal current, V is the voltage at which the membrane was held
for 300 ms before the test pulse, V0.5 is the membrane potential at
which the current is 50% inactivated, and k the slope.
Immunohistochemical procedures
For immunohistochemistry, glass attached VNO cells were gently
perfused with 4% paraformaldehyde, washed in phosphate-buffered
saline solution (PBS), and air dried. Cells were blocked in 1% albumin, 0.3% Triton X-100 in PBS for 20 min, and incubated with
anti-G␥8 antibody (1:400) (Tirindelli and Ryba 1996) or olfactory
marker protein (OMP) antiserum (1.10000, kindly provided by Dr. F.
Margolis, University of Maryland, Baltimore) overnight at 4°C. Specific immunoreactivity was detected by the biotin-avidin-horseradish
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Adult male C57BL/6J and CD-1 mice were used in this study.
Vomeronasal neurons and supporting cells were isolated with a standard enzymatic-mechanical procedure (e.g., Liman and Corey 1996;
Maue and Dionne 1987). Briefly, mice were deeply anesthetized by
CO2, followed by dislocation of cervical vertebrae. The VNO was
removed within its bony encasing and then carefully dissected free of
the bone. The sensory epithelium, which is situated at the medial part
of the organ, was separated from the lateral nonsensory epithelium,
rinsed in divalent-free Tyrode solution (in mM: 140 NaCl, 5 KCl, 10
HEPES, 10 glucose, 10 Na pyruvate, and 2 EGTA, pH 7.4 with
NaOH), and cut into several small pieces. Collagenase (1 mg/ml of
type A; Roche, Mannheim, Germany) and trypsin (1 mg/ml; Sigma,
St. Louis, MO) were added, and the tissue was incubated at room
temperature (22–25°C) for 60 –70 min with agitation (for details, see
Maue and Dionne 1987). After centrifugation, the surnatant was
replaced with regular Tyrode solution (in mM: 140 NaCl, 5 KCl, 2
CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 10 Na pyruvate, pH 7.4
with NaOH) supplemented with DNase (0.1 mg/ml; type I, Boehringer Mannheim, Germany) and incubated for 15 min with agitation.
Finally, small tissue samples were gently triturated with a fire-polished pipette (tip diameter about 100 ␮m) and immediately plated on
the bottom of a chamber that consisted of a standard glass slide onto
which a silicon ring 1–2 mm thick and 15 mm ID was pressed. The
glass slide was precoated with Cell-Tak (approximately 3 ␮g/cm2; BD
Biosciences, Bedford, MA) to improve adherence of isolated VNO
cells to the bottom of the chamber. As a control, we checked that
enzymatic treatment with collagenase and trypsin did not affect the
membrane properties of VNO supporting cells. To this aim, minced
tissue was incubated with agitation in divalent-free Tyrode for 70 min
at room temperature and gently triturated with a fire-polished pipette.
Although the cell yield was low, this procedure allowed us to established that membrane properties of VNO supporting cells were not
affected by enzymatic treatment. VNO neurons were also prepared as
for supporting cells and used for comparing membrane properties.
The recording chamber was placed on the stage of an inverted
Olympus microscope (model IX70, Olympus, Tokyo, Japan), and
isolated VNO cells were viewed with Nomarski optics. During the
experiments, VNO cells were continuously perfused with Tyrode by
means of a gravity-driven system. Drugs were dissolved in modified
Tyrode solution to maintain osmolarity. All chemicals were from
Sigma, except tetrodotoxin (Alomone Laboratories, Jerusalem, Israel).
119
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V. GHIARONI, F. FIENI, R. TIRINDELLI, P. PIETRA, AND A. BIGIANI
peroxidase-diaminobenzidine method (ABC kit) as recommended by
the supplier (Vector, Burlingame, CA).
RESULTS
Identification of supporting cells and neurons isolated from
the VNO
Passive membrane properties
The electrical properties of VNO supporting cells were
examined by whole cell patch-clamp recordings. We did not
observe any significant differences between the membrane
properties of supporting cells dissociated using enzymes, or no
enzyme. Therefore data were pooled together.
In a first series of experiments, we determined the passive
membrane properties of supporting cells. In the whole cell
patch-clamp configuration, a parameter often used to estimate
the cell’s resting potential is the zero-current potential (V0). V0
is the voltage at which no current is required in voltage clamp.
With KCl pipette solution, V0 was ⫺29 ⫾ 1 mV (n ⫽ 39),
which was significantly more depolarized than V0 of sensory
neurons measured under similar conditions (⫺53 ⫾ 3 mV; n ⫽
19). The input resistance (Rin) of supporting cells was 1.7 ⫾
0.2 G⍀ (n ⫽ 14), whereas in sensory neurons, Rin was almost
four times larger (6.4 ⫾ 1.3 G⍀; n ⫽ 37). Since seal resistance
did not change significantly when we patched onto supporting
cells or on sensory neurons, findings on Rin suggested that
supporting cells were endowed with a substantial “resting”
conductance. Finally, in supporting cells the membrane capacitance (Cm: 9.2 ⫾ 0.6 pF; n ⫽ 22) was significantly larger than
that of sensory neurons (5.1 ⫾ 0.4 pF; n ⫽ 39). Normalization
of Rin to Cm revealed that the low membrane resistance of
supporting cells was due only in part to their larger membrane
surface area (Rin/Cm: 0.19 G⍀/pF in supporting cells; 1.26
G⍀/pG in sensory neurons).
Resting membrane permeabilities
FIG. 1. Differential interference contrast photomicrographs of a sensory
neuron (A) and a supporting cell (B) isolated from the mouse vomeronasal
organ (VNO). Cells were immunostained for G␥8, a specific marker for
vomeronasal neurons. Strong staining can be detected in the neuron up to the
dendritic knob, whereas the supporting cell was not stained (the darkness in the
soma of the supporting cell is shadow of the interference contrast optics). b,
basal process; d, dendrite; k, dendritic knob. Scale bar, 10 ␮m.
J Neurophysiol • VOL
Glial cells of several tissues are characterized by a large
resting potassium conductance (reviewed in Syková et al.
1998). Substitution of K⫹ with Cs⫹ (a potassium channel
blocker; Rudy 1988) in the pipette solution markedly affected
V0 in supporting cells (⫺6 ⫾ 3 mV; n ⫽ 8). However, a
depolarized V0 in control conditions (about ⫺29 mV on average) suggested that the membrane of supporting cells was
permeable also to other ions. For example, substitution of
extracellular Na⫹ ions with N-methyl-D-glucamine (NMDG)
caused a pronounced hyperpolarization (V0 ⫽ ⫺45 mV ⫾1.5;
n ⫽ 11), suggesting the presence of Na⫹ pathways in the cell
membrane. Interestingly, 30 –100 ␮M amiloride, which blocks
the passive, epithelial Na⫹ channels in many cell types (Garty
and Palmer 1997), had no effect on vomeronasal supporting
cells (n ⫽ 7).
To estimate the relative membrane permeabilities to the
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The VNO sensory epithelium contains three types of cells:
sensory neurons, supporting cells, and basal cells (Carmanchahi et al. 1999; Garrosa and Coca 1991; Vaccarezza et al.
1981). Both sensory neurons and supporting cells possess
cytoplasmatic processes, whereas basal cells are ovoid elements. Sensory neurons have a round cell body with an apical
dendrite reaching the epithelium surface and an axon projecting to the accessory olfactory bulb. Supporting cells have
oblong cell bodies with narrow prolongation that lay between
the dendrites of the sensory neurons (Carmanchahi et al. 1999;
Höfer et al. 2000; Vaccarezza et al. 1981). We used these
morphological features to identify VNO neurons and supporting cells after dissociation. Isolated sensory cells were round,
ovoid in shape and possessed a clear dendritic process that
ended in a knoblike structure from which microvilli protruded
(Fig. 1A). Consistently with previous reports (e.g., Liman and
Corey 1996; Moss et al. 1998), the axon was usually not
present in isolated neurons, likely lost during the dissociation
procedure. On the contrary, isolated supporting cells were
rectangularly shaped and possessed narrow distal prolongations (Fig. 1B). Supporting cells were further distinguished
from sensory neurons by the lack of immunoreactivity for G
protein ␥-subunit (G␥8), a specific marker for vomeronasal
neurons (Tirindelli and Ryba 1996) (Fig. 1). In addition, only
sensory neurons were immunoreactive for OMP (data not
shown), consistently with previous results (Liman and Corey
1996).
In this study, we present data obtained from 159 unambiguously identified supporting cells isolated from the mouse
vomeronasal organ. Just for the purpose of comparison, we
also report data obtained from 87 sensory neurons. Further
information on membrane properties of these sensory neurons
isolated from the mouse VNO can be found in a published
report (Liman and Corey 1996).
ION CONDUCTANCES IN VOMERONASAL SUPPORTING CELLS
⫹
⫹
relative permeabilities to Cl and to Na , in the absence of
K⫹. The data in Fig. 2B clearly show that in resting conditions,
glial membranes were six times more permeable to chloride
ions than to sodium ions. Moreover, these data show that
vomeronasal supporting cells had a significant resting Na⫹
permeability, consistently with the effect of NMDG on V0.
Both ion channels and electrogenic pumps can affect V0
measurements used to evaluate ion permeabilities. In glial cells
of other tissues, the occurrence of electrogenic Na⫹,K⫹ATPase has been documented (e.g., Walz 1989). However,
with our ionic conditions it was unlikely that Na⫹,K⫹-ATPase
could play any role in setting up V0 in vomeronasal supporting
cells. Intracellular Na⫹ concentration in the millimolar range is
required for pump activity (e.g., Glitsch 2001). On the contrary, our standard pipette (intracellular) solution was nominally Na⫹-free (see METHODS). Indeed, application of ouabain (a
known Na⫹,K⫹-ATPase blocker at concentrations in the micromolar range) did not significantly affect V0 (control: ⫺30 ⫾
6 mV; with 50 ␮M ouabain: ⫺31 ⫾ 7 mV; n ⫽ 4). However,
it is possible that Na⫹ influx through opened channels could
cause a local increase in the intracellular Na⫹ concentration
near the pump (the so-called “fuzzy space”: Lederer et al.
1990). This condition could be enhanced when using a highNa⫹ extracellular solution, while recording with a pipette
solution in which K⫹, a competitive inhibitor of Na⫹ at intracellular Na⫹-binding sites of the pump (e.g., Glitsch 2001), is
replaced by Cs⫹ (Fig. 2B, rightmost point). However, even in
these conditions, application of 50 ␮M ouabain had no effect
on V0 (control: ⫺15 ⫾ 5 mV; with ouabain: ⫺15 ⫾ 4 mV;
n ⫽ 4).
In conclusion, the resting membrane of VNO supporting
cells was permeable to the main ions occurring in the experimental solutions, namely K⫹, Na⫹, and Cl⫺, with a relative
permeability ratio of PK:PNa:PCl ⫽ 1:0.23:1.4. Since the contribution of the Na⫹,K⫹-ATPase to resting potential was negligible, ion permeabilities were likely due to the presence of
opened (passive) ion channels.
Voltage-gated K⫹ currents
⫹
FIG. 2. Effect of variations in extracellular K
or Na⫹ concentration
([K⫹]o and [Na⫹]o, respectively) on the value of resting membrane potential
(Vr) in vomeronasal supporting cells. Resting membrane potential was calculated by correcting V0 for the shunt to ground by seal resistance, and for liquid
junction potentials (see METHODS). Points represents means ⫾ SE (bars) from
7 to 11 measurements. A: effect of variation in [K⫹]o on Vr in the presence of
only K⫹ and Cl⫺ as main ions. Intracellular solution: K gluconate; bath
solution: Na⫹-free Tyrode [Na⫹ replaced by N-methyl-d-glucamine
(NMDG)]. Data points were fitted using the Goldman-Hodgkin-Katz equation
in the hypothesis that the membrane was permeable only to K⫹ and to Cl⫺.
The best fit of the data points was obtained for a permeability ratio PCl/PK of
1.4. B: effect of variation in [Na⫹]o on Vr in the presence of only Na⫹ and Cl⫺
as main ions. Intracellular solution: Cs gluconate; bath solution: K⫹-free
Tyrode. Data points were fitted using the Goldman-Hodgkin-Katz equation in
the hypothesis that the membrane was permeable only to Na⫹ and to Cl⫺. The
best fit of the data points was obtained for a permeability ratio PCl/PNa of 6.1.
J Neurophysiol • VOL
Glial cells in several tissues display complex membrane
properties due to voltage-gated ion channels, including K⫹
channels and Na⫹ channels (reviewed in Sontheimer 1994;
Verkhratsky and Steinhäuser 2000). The presence of voltagegated ion channels in VNO supporting cells was investigated
by recording membrane currents elicited by voltage steps from
a holding potential of about ⫺80 mV. With normal Tyrode
solution in the bath and the pipette solution containing 120
KCl, depolarizing voltage pulses elicited the current time
courses shown in Fig. 3A. After an early capacitive transient,
current exhibited a pronounced outward trace. These outward
currents were observed in all supporting cells we tested under
the above ionic conditions, although their magnitude varied
from cell to cell. Outward currents were recorded also from
sensory neurons. However, in these cells they were preceded
by a fast, transient inward current (Fig. 3B) mediated by
sodium ions (see also Liman and Corey 1996).
The outward current in both supporting cells and sensory
neurons was carried by potassium ions, as indicated by their
sensitivity to TEA (Fig. 4, A and B) and by their complete
block when CsCl was used instead of KCl in the pipette
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main ions occurring in our experimental solutions, namely K ,
Na⫹, and Cl⫺, V0 was measured at different extracellular
concentrations of a given ion (K⫹ or Na⫹) and in bi-ionic
conditions (i.e., only 2 main permeant ions present in the
experimental solutions: either K⫹ and Cl⫺, or Na⫹ and Cl⫺).
The osmolarity and the ionic strength of the extracellular
solutions were maintained by substituting sodium or potassium
with NMDG⫹, a nonpermeable ions. Although V0 is assumed
to be an estimation of the cell’s resting potential, it requires
correction for the shunt to ground by the seal resistance and
also for liquid junction potential changes during solution exchange (see Eq. 1). This correction allowed us to obtain a better
estimation of the resting potential (Vr) in supporting cells in
different ionic conditions. First we evaluated the relative permeabilities to K⫹ and to Cl⫺ in the absence of Na⫹. As
revealed by the data shown in Fig. 2A, in resting conditions, the
membrane of supporting cells was highly permeable to chloride ions, in addition to potassium ions. Next, we evaluated the
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V. GHIARONI, F. FIENI, R. TIRINDELLI, P. PIETRA, AND A. BIGIANI
nonlinear in symmetric K⫹ (Fig. 5). In addition, the I-V plot
exhibited an inward deflection for voltages between ⫺40 and 0
mV (arrow in Fig. 5, right), reflecting the influx of K⫹ ions
through voltage-gated channels, which were closed for more
negative potential. Thus outward potassium currents were
likely mediated by the activation of voltage-gated K⫹ channels
in supporting cells.
When external Na⫹ was substituted by K⫹ to obtain symmetric conditions for K⫹, the I-V plot was shifted rightward
(Fig. 5, right). In these conditions, K⫹ is the main cation both
in the extracellular and intracellular solution. Thus when the
voltage-gated channels open up (at about ⫺40 and ⫺30 mV)
solution (data not shown). As indicated by the concentrationinhibition curves shown in Fig. 4C, the sensitivity of potassium
current (IK) to TEA was larger in supporting cells than in
neurons. Also 4-aminopyridine (4-AP, another K channel
blocker, especially for inactivating channels) (Rudy 1988) affected IK. Interestingly, IK in supporting cells was less sensitive
to 4-AP than to TEA, whereas neuronal IK was sensitive to
both channel blockers (Fig. 4D). Consistent with the weak
effect of 4-AP on iK in supporting cells was the observation
that IK amplitude (at a reference potential of ⫹46 mV) was not
significantly affected by the holding potential (Vh): 777 ⫾ 37
pA with Vh ⫽ ⫺84 mV; 809 ⫾ 29 pA with Vh ⫽ 64 mV;
786 ⫾ 44 pA with Vh ⫽ ⫺44 mV; 760 ⫾ 37 pA with Vh ⫽
⫺24 mV (n ⫽ 3). Thus IK in supporting cells did not show any
significant inactivation. As reported by Liman and Corey
(1996), IK in sensory neurons displays slow inactivation.
Outward rectification in potassium currents could be due to
the activation of voltage-gated channels or to leak channels that
rectify because of nonsymmetric potassium concentration gradient (Goldstein et al. 2001). Thus we perfused the cells with
a high-potassium Tyrode solution in which Na⫹ was substituted by K⫹ (symmetric K⫹). In these conditions, currents
through leak channels (like the KCNK channels of the 2-Pdomain potassium channel family; Goldstein et al. 2001)
should produce a linear current-voltage (I-V) relationship.
However, we found that I-V plots in supporting cells remained
J Neurophysiol • VOL
⫹
FIG. 4. Outward K currents in cells isolated from the VNO neuroepithelium. Membrane currents were elicited by a series of depolarizing pulses
between ⫺74 and ⫹106 mV, in 10-mV increments, from a holding potential
of ⫺84 mV. Pipette solution: KCl. A: in supporting cells, outward currents
recorded in regular Tyrode (control) were totally abolished by 10 mM TEA. B:
in sensory neurons, on the contrary, 10 mM TEA blocked only partially the
outward currents. C: dose-response curves for the effect of TEA on supporting
cells (䡺) and sensory neurons (E). Percentage inhibition of the total K⫹ current
was evaluated for the current elicited by a depolarizing voltage step to ⫹96
mV from a holding potential of ⫺84 mV. Points represent mean ⫾ SE (bars)
of 4 –10 measurements. Sigmoidal curves are the best fit of the data obtained
with the logistic equation (see METHODS) with an IC50 of 0.35 mM and a Hill
coefficient of 1 in supporting cells and with an IC50 of 3.39 mM and a Hill
coefficient of 0.61 in sensory neurons. D: comparison of the effect of 2 mM
TEA and 2 mM 4-AP on the outward potassium current in supporting cells and
sensory neurons. Percentage inhibition of the total K⫹ current was evaluated
for the current elicited by a depolarizing voltage step to ⫹96 mV from a
holding potential of ⫺84 mV. The histograms represent mean values ⫾ SE
(bars) from 6 – 8 measurements. Note that in supporting cells, K⫹ currents were
weakly sensitive to 4-AP (⬍10% reduction), whereas they were strongly
affected by TEA (90% reduction).
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FIG. 3. Membrane currents in cells isolated from the vomeronasal sensory
epithelium. Cells were held at ⫺84 mV and stepped in 10-mV increments from
⫺74 to ⫹106 mV. Pipette solution: KCl. Bath solution: Tyrode’s. A: supporting cells were characterized by outward currents only. Note the large noise that
was typically associated with outward currents in these cells. As shown by the
current-voltage (I-V) relationship on the right, outward currents typically
activated at approximately ⫺30 mV. B: sensory neurons were characterized by
both inward currents (shaded dot) carried by Na⫹, and outward currents (empty
dot). As revealed by the corresponding I-V plots (right), inward currents
activated at about ⫺50 mV, and outward current at about ⫺30 mV. In all I-V
plots, outward currents were measured at the end of 30-ms voltage pulses.
Each point represents the means ⫾ SE (bars) from 27 supporting cells, and 20
sensory neurons. Im, membrane current; Vm, membrane potential.
ION CONDUCTANCES IN VOMERONASAL SUPPORTING CELLS
123
5. Outward K⫹ currents in supporting cells are mediated by voltagegated channels. Current-voltage (I-V) relationship for membrane currents were
evaluated under conditions of high internal and low external K⫹ (control;
pipette solution: 155 mM K⫹; bath solution: 5 mM K⫹), and under symmetric
K⫹ (pipette solution: 155 mM K⫹; bath solution: 155 mM K⫹). A linear I-V
plot would be expected under symmetric K⫹ in the case of leak channels. Note
that under symmetric K⫹, an inward current (arrow) activates at a membrane
voltage of about ⫺40 mV (deflection below the line of leakage current). This
is consistent with K⫹ channels being opened by membrane depolarization and
with K⫹ ions flowing into the cell (EK ⫽ 0 mV in symmetric conditions). Each
point represents the means ⫾ SE (bars) from 10 supporting cells. Straight lines
represent the extrapolated leakage (passive) current obtained by fitting the
current values at membrane voltages more negative than ⫺45 mV. Im, membrane current; Vm, membrane voltage.
FIG.
the potassium current predominates and sets the behavior for
the I-V plot. In particular, the membrane current reverses at 0
mV, which is the equilibrium potential of K⫹ (EK) in symmetric conditions. In addition, the I-V plot was shifted downward
for negative membrane potentials (Fig. 5, right), consistently
with the presence of resting potassium channels. On the other
hand, the increased in the current amplitude for very positive
voltages was unusual, since an increase of [K⫹]o lowers the
driving force for outward K⫹ flux and would be expected to
decrease rather than increase outward currents. Such a dependence on [K⫹]o have been reported for outward rectifier K⫹
channels in other tissues (Fink et al. 1996; Pardo et al. 1992;
Scamps and Carmeliet 1989), and could be due an action of
extracellular K⫹ on the channel protein.
Ca2⫹-dependent K⫹ currents mediated by maxi-K channels
(BK channels) have been described in glial cells of other
tissues (reviewed in Verkhratsky and Steinhäuser 2000). Application of 20 nM charybdotoxin (CTX), a blocker of BK
channels (Knaus et al. 1994), did not affect the outward potassium currents in supporting cells (n ⫽ 6; data not shown).
Consistent with the absence of BK channels was also the
finding that 1 mM Cd2⫹, an inorganic blocker of Ca2⫹ channels (Bean 1992), was unable to reduce outward potassium
currents (n ⫽ 6; data not shown). As demonstrated by an early
study (Liman and Corey 1996), also vomeronasal neurons do
not possess Ca2⫹-activated K⫹ currents.
Voltage-gated Na⫹ currents
Voltage-gated, TTX-sensitive sodium currents (INa) were
elicited regularly in vomeronasal neurons by depolarizing the
J Neurophysiol • VOL
FIG. 6. Voltage-gated inward currents in supporting cells isolated from the
mouse VNO. A: membrane currents (Im) were elicited by a series of depolarizing voltage pulses in 10-mV increments from different holding potential (Vh).
When holding potential was set to value more negative than ⫺84 mV, inward
currents appeared in the records (arrows). Note that outward current (upward
deflections in the records) were always present. B: current-voltage (I-V) relationships for the inward currents shown in A. These currents activate at about
⫺50 to ⫺60 mV and peaked at about ⫺40 mV. Note that the current amplitude
increased by hyperpolarizing the holding potential, suggesting that a large
fraction of the channels was removed from inactivation. C: unlike inward
currents, outward potassium currents were scarcely affected by hyperpolarized
holding potentials.
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membrane from a holding potential of about ⫺80 mV (Fig.
3B). In these conditions, on the contrary, supporting cells
displayed only outward potassium currents (Fig. 3A). However, when the holding potential was set to more negative
values, early, transient inward currents appeared also in the
current records from supporting cells (Fig. 6A). Membrane
hyperpolarization affected markedly the amplitude of inward
currents (Fig. 6B) but not that one of outward potassium
currents (Fig. 6C). The transient inward current disappeared
when NMDG replaced Na⫹ in the bath (Fig. 7) and was
blocked reversibly by 0.5 ␮M TTX (data not shown): in short,
it was a Na⫹ current (INa). Activation threshold for INa in
supporting cells was between ⫺60 and ⫺50 mV, which is
similar to the value for neuronal INa (Figs. 3B and 6B).
To compare the amplitude of INa in supporting cells with that
one of INa in sensory neurons, INa was elicited by a two-pulse
stimulation as follows: from a holding potential of ⫺84 mV,
124
V. GHIARONI, F. FIENI, R. TIRINDELLI, P. PIETRA, AND A. BIGIANI
DISCUSSION
One of the current challenging issues in olfaction research is
the evaluation of the operation of vomeronasal neuroepithelium as detector and transducer of pheromonal signals. The
goal of our study was to contribute to the understanding of the
functional properties of such a chemosensory structure. We
have examined the electrophysiological properties of supporting cells isolated from the mouse VNO. The main finding is
that these cells possess quite unique membrane properties that,
to our knowledge, have not been described for other types of
glial cells.
⫹
FIG. 7. Effect of Na -free solution on the inward current in supporting
cells isolated from the mouse VNO. Na⫹ was replaced by NMDG in the
Tyrode’s solution. Membrane current were elicited by a series of depolarizing
pulses between ⫺94 and ⫹106 mV, in 10-mV increments, from a holding
potential of ⫺104 mV. Inward current recorded in regular Tyrode (control)
were totally abolished by Na⫹-free solution (middle). The effect was reversible, as indicated by the recovery of the currents during washout (wash). Note
that the outward potassium currents was unaffected by the application of
Na⫹-free solution.
J Neurophysiol • VOL
FIG. 8. Voltage dependence of the steady-state inactivation of voltagegated Na⫹ currents in supporting cells and sensory neurons isolated from the
mouse VNO. A standard 2-pulse voltage protocol (top) was used for this
analysis. Current magnitudes of the test pulse (⫺34 mV) were normalized to
its maximal value and plotted against the prepulse potential (bottom). Each
point represents the mean ⫾ SE of 15–19 measurements. Data were fitted to a
Boltzmann equation. For supporting cells, the half-maximal voltage (V0.5) was
⫺101 mV and the slope (k) was 8.9 mV. For sensory neurons, V0.5 was ⫺77
mV and k was 10.6 mV. Pipette solution: CsCl. Bath solution: standard
Tyrode’s. Duration of prepulse potentials: 300 ms.
Resting membrane permeabilities in vomeronasal
supporting cells
One of the characteristic features of glial cells in several
tissues is their membrane selectivity for K⫹, and a low (in
some cases, negligible) permeability to Na⫹ and Cl⫺ (Bigiani
2001; Kettenmann et al. 1983; Newman 1985, 1989; Ransom
and Sontheimer 1995; Sugihara and Furukawa 1996; Walz and
Schule 1982; Walz et al. 1984). Results from the ion substitution experiments described here demonstrate that vomeronasal
supporting cells have membranes that are permeable not only
to K⫹, but also to Na⫹ and Cl⫺. An estimate based on fitting
the data with the GHK equation suggests that the ratio of K⫹
to Na⫹ permeabilities (PK/PNa) is about 4, and the ratio of K⫹
to Cl⫺ permeabilities (PK/PCl) is about 0.6. These permeability
ratios seem quite peculiar to VNO supporting cells (Table 1).
Thus there is something fundamentally different about the
membrane construction of supporting cells in the VNO.
Role of glial resting ion permeabilities in VNO physiology
The VNO is a chemosensory structure located at the base of
the nasal cavity and shaped as a blind-end sac (Døving and
Trotier 1998; Halpern 1987). VNO receives stimuli from the
nasal cavity through an active vascular pumping mechanism
controlled by the autonomic nervous system (Meredith 1994;
Meredith et al. 1980). This way, mucus can flow in and out
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cell membrane was hyperpolarized to ⫺124 mV for 300 ms
and then depolarized to ⫺34 mV to activate INa. In these
conditions, the maximal amplitude of INa was significantly
lower in supporting cells (⫺292 ⫾ 114 pA; n ⫽ 20) than in
sensory neurons (⫺ 868 ⫾ 158 pA; n ⫽ 18).
The absence of INa in supporting cells held at ⫺80 mV
indicated that the inactivation properties of these currents differed from those of the channels expressed by sensory neurons.
To study the voltage dependence of the steady-state inactivation, we used a typical two-pulse voltage protocol (prepulse
and test pulse) that allowed the evaluation of the noninactivated fraction of the sodium current as a function of a prepulse
membrane potential. Steady-state inactivation curves show that
in supporting cells inactivation was shifted by about 24 mV in
the hyperpolarizing direction (Fig. 8, bottom). Also in astrocytes, V0.5 is about 25 mV more negative than in neurons
(Barres et al. 1989).
Finally, the sensitivity of INa to TTX was less pronounced in
VNO supporting cells than in neurons (Fig. 9, bottom). For
VNO neuronal Na⫹ channels, a high sensitivity to TTX has
been reported also by Liman and Corey (1996).
As a whole, these data indicated that supporting cells of the
VNO expressed voltage-gated Na⫹ channels with specific biophysical and pharmacological properties.
ION CONDUCTANCES IN VOMERONASAL SUPPORTING CELLS
125
Compared with neuronal microvilli, those of supporting cells
reach further into the lumen of the VNO. Thus it is tempting to
speculate that at this level excess of ions could be absorbed and
then transferred into the blood vessels via glial basal processes
coursing through the neuroepithelium. By modifying the ionic
strength of the pheromone-containing medium, supporting
cells may also influence directly the biophysical properties of
pheromonal proteins, such as MUPs. Thereby, VNO supporting cells can affect pheromone detection by neurons. Further
studies on the membrane localization of ion permeabilities in
glial membranes (apical, basal, lateral) are required to fully
elucidate their role in the management of inorganic ions in the
VNO.
Voltage-gated K⫹ and Na⫹ conductances in vomeronasal
supporting cells
from the VNO (Meredith 1998). Volatile pheromones (Novotny et al. 1985) can reach the VNO after solubilization in the
mucous as usual odorants. On the contrary, nonvolatile pheromone substances, such as the proteins of the major urinary
protein complex (MUP) (Hurst et al. 2001; Mucignat-Caretta et
al. 1995) can reach the vomeronasal neuroepithelium by direct
influx of urine into the VNO lumen with licking (Wysocki et
al. 1980). Given the relatively high concentration of K⫹, Na⫹,
and Cl⫺ in rodent urine (150 mM K⫹ and 200 mM Na⫹, with
Cl⫺ as main counter-anion) (Wüthrich 2000), it is reasonable
to conceive that these ions might interfere with the pheromone
detection by sensory neurons by altering their membrane excitability at the apical ends. It is therefore tempting to speculate
that high resting membrane permeabilities to K⫹, Na⫹, and
Cl⫺ in supporting cells may play a role in mopping-up the
excess of inorganic ions that enter in the VNO lumen with
certain pheromone-containing fluids, like urine. Supporting
cells are bipolar elements with an apical process reaching the
epithelium surface, and a basal process reaching the basal
lamina that separates the neuroepithelium from the blood vessels (Carmanchahi et al. 1999; Garrosa and Coca 1991; Höfer
et al. 2000; Naguro and Breipohl 1982; Vaccarezza et al.
1981). Apical processes of supporting cells bear tall and wide
microvilli that protrude inside the VNO lumen and seem to
cover up the short microvilli of sensory neurons (Höfer et al.
2000; Naguro and Breipohl 1982; Vaccarezza et al. 1981).
J Neurophysiol • VOL
TABLE 1. Relative ion permeabilities in supporting (glial) cells
from different tissues and in resting conditions
Supporting Cells
PK
PNa
PCl
Reference
Vomeronasal glial cells
Retinal glial (Müller) cells
Astrocytes
Olfactory glial cells
Taste glial cells
1
1
1
1
1
0.23
⬍0.01
*
0.025
*
1.4
*
*
*
0.017
this study
Newman 1985, 1987
Walz et al. 1984
Trotier 1998
Bigiani 2001
* Not present or negligible.
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⫹
FIG. 9. TTX sensitivity of the voltage-gated Na
currents recorded in
supporting cells and sensory neurons isolated from the mouse VNO. Na⫹
current was elicited by a test pulse to ⫺34 mV after hyperpolarizing the
membrane to ⫺124 mV for 300 ms (top). Percentage inhibition of the maximal
Na⫹ current was evaluated for a series of TTX concentrations and then plotted
to obtain the dose-response curves shown (bottom). Points represent means ⫾
SE (bars) of 5–11 measurements. Sigmoidal curves are the best fit of the data
obtained with the logistic equation (see METHODS) with an IC50 of 0.48 ␮M and
a Hill coefficient of 0.91 in supporting cells (䡺) and with an IC50 of 0.015 ␮M
and a Hill coefficient of 0.54 in sensory neurons (E).
Like glial cells in other tissues (reviewed in Sontheimer
1994; Verkhratsky and Steinhäuser 2000), supporting cells
isolated from the mouse VNO possessed both voltage-gated
K⫹ and Na⫹ channels. Interestingly, all supporting cells we
patched (n ⫽ 159) displayed both K⫹ and Na⫹ currents,
although their magnitude varied among cells. In addition, we
could not reveal any significant variability in the properties of
these currents among supporting cells, which in this respect
made up a homogeneous cell population. On the contrary,
variability in the expression of ion currents has been shown for
other types of glial cells. For example, in patch-clamp recordings from hippocampal slices, only 10% of astrocytes expressed Na⫹ currents (Sontheimer and Waxman 1993).
Although voltage-gated K⫹ currents in vomeronasal supporting cells were similar to the delayed-rectifier type (KDR)
described in other glial cells (reviewed in Sontheimer 1994),
they presented some unique features. In astrocytes and
Schwann cells, KDR channels are moderately sensitive to TEA,
with blocking concentration ranging from 5 to 50 mM, and
IC50 ranging from about 6 to 11 mM (Sontheimer 1994). On
the contrary, KDR currents in vomeronasal supporting cells
were highly sensitive to TEA, as indicated by an IC50 of 0.35
mM. Moreover, KDR channels in astrocytes are equally sensitive to both TEA and 4-AP (Sontheimer 1994), whereas in
vomeronasal supporting cells they display a lower sensitivity to
4-AP than TEA.
Another unique feature of KDR currents in vomeronasal
supporting cells was their unusual noise (see Fig. 3A). In cone
photoreceptors, large fluctuations in the current records have
been attributed to the activation of Ca2⫹-dependent K⫹ currents by Ca2⫹ ions entering through voltage-gated channels
(Barnes and Hille 1989). However, in vomeronasal supporting
cells we found no evidence of such Ca2⫹-dependent currents.
126
V. GHIARONI, F. FIENI, R. TIRINDELLI, P. PIETRA, AND A. BIGIANI
Outward currents through two-P-domain K⫹ channels may
exhibit conspicuous noise (e.g., Lesage et al. 2000; Patel et al.
1998). However, our experiments with symmetric K⫹ indicated that outward currents in vomeronasal supporting cells
were mediated by voltage-gated channels. Molecular studies,
such as single-cell RT-PCR (e.g., Rabe et al. 1999) for identifying the potassium channel genes, will be helpful in the
identification of the K⫹ channels expressed by vomeronasal
supporting cells.
Unlike K⫹ channels, Na⫹ channels in vomeronasal supporting cells were electrophysiologically (steady-state inactivation
properties) and pharmacologically (low sensitivity to TTX)
similar to the glial type Na⫹ channels expressed in protoplasmic astrocytes (Sontheimer and Waxman 1992). They differed,
however, from Schwann cell Na⫹ channels, which are highly
sensitive to TTX (IC50 ⫽ 2 nM) (Howe and Ritchie 1990).
As in the case of glial cells in other tissues, the functional
importance of voltage-gated K⫹ and Na⫹ channels in vomeronasal supporting cells remains to be established. Spatial buffering, the uptake and redistribution of excess K⫹ from the
extracellular space, may involved both resting K⫹ channels
and voltage-activated K⫹ channels (reviewed in Sontheimer
1994). Like in astrocytes, Na⫹ currents in vomeronasal supporting cells were usually small in amplitude. As a consequence, current density for INa (max amplitude/Cm) was about
32 pA/pF, well below the mean value of 170 pA/pF for INa in
sensory neurons, which are electrically excitable (Liman and
Corey 1996). Furthermore, Na⫹ channels were more than 95%
inactivated at a membrane potential of about ⫺75 mV (Fig. 8).
Vr of supporting cells could be at best ⫺72 mV in the presence
of a Na⫹-free extracellular solution and with a low Cl⫺ intracellular solution (see Fig. 2A). Thus as in other glial cells, the
mismatch of resting potential and inactivation properties would
prevent electrogenesis in vomeronasal supporting cells. It is
possible that TTX-blockable Na⫹ channels in glial cells serve
roles that do not require voltage-dependent activation. In astrocytes, these channels, even at rest, allow a small percentage
of Na⫹ ions to leak into cells (Sontheimer et al. 1994). It has
been proposed that glial Na⫹ channels may represent a return
pathway for the function of the glial Na⫹/K⫹-ATPase by
permitting Na⫹ ions to enter the cell to maintain intracellular
Na⫹ at concentrations necessary for the activity of the pump
(Sontheimer et al. 1994). Thus it is likely that also in VNO
voltage-gated ion channels in supporting cells may play a key
role in the control of chemical composition of intercellular
fluid, which, in turn, can affect the excitability of sensory
neurons.
We thank Dr. Paolo Pelosi (Università di Pisa) for helpful comments. We
thank G. Nespoli for excellent technical assistance.
This work was supported by the Italian Board of Education and University
(Ministero dell’Istruzione dell’Università e della Ricerca, Cofin 2001 to A.
Bigiani and R. Tirindelli).
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