Chem. Senses 23: 49-57, 1998
(y®
Scanning Electron Microscopy and Gramicidin Patch Clamp Recordings of
Microvillous Receptor Neurons Dissociated from the Rat Vomeronasal Organ
Didier Trotier, Kjell B. Doving1, Kirsten Ore1 and Camilla Shalchian-Tabrizi1
Laboratoire de Neurobiologie Sensorielle, Ecole Pratique des Hautes Etudes, 1 Avenue des
Olympiades, F-91 305 Massy, Franceand department of Biology, University of Oslo, Box 1051,
N-0316 Oslo, Norway
Correspondence to be sent to: Didier Trotier, Laboratoire de Neurobiologie Sensorielle, Ecole Pratique des Hautes Etudes, 1 Avenue des
Olympiades, F91 305 Massy, France, e-mail: [email protected]
Abstract
Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated
receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We
applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well
differentiated receptor neurons. The mean resting potential was -58 ± 14 mV (n = 39). The contribution of the sodium pump
current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 jiM
dihydro-ouabain. The input resistance was in the range of 1-6 Gfi and depolarizing current pulses of a few pA were sufficient
to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained
outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated
vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content.
Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor
cells were depolarized by diluted rat male urine.
Introduction
The precise characterization of the electrophysiological
properties of vomeronasal receptor neurons (VRN) is a
necessary step to understand how the vomeronasal organ
signals the presence of chemical stimuli to the accessory
olfactory bulb. Patch-clamp recordings from frog VRN have
been used to characterize voltage-gated conductances and
to demonstrate that these cells trigger action potentials in
response to depolarizing membrane currents in the pA
range (Trotier et al., 1993).
We have recently shown that the vomeronasal receptor
neurons of the frog are polarized at rest by the outward
current generated by the Na,K-ATPase sodium pump
(Trotier and D0ving, 1996a). In these cells the resting
membrane potential is set by the balance between the
sodium pump current and the inward rectifying current, /j,,
which is steadily activated by membrane hyperpolarization
(Trotier and Deving, 1996b). This uncommon way of
generating the membrane potential could be related to the
specific functional properties of the VRN or it could be
related to the fact that the recordings were made from an
amphibian. It was therefore of interest to investigate
whether the same mechanisms were present in mammalian
VRN.
Little is known about the properties of VRN in mammals.
© Oxford University Press
Liman and Corey (1996) described voltage-gated Na + , K+
and Ca2+ currents in isolated mouse VRN and
demonstrated that intracellular injection of cyclic AMP
(cAMP) failed to demonstrate the existence of the
cAMP-gated cationic channels observed in ciliated olfactory
receptor cells. In this sense rat VRN seem similar to frog
VRN (Trotier et al., 1994). More recently, Inamura et al.
(1997) recorded from VRN in slices of rat vomeronasal
epithelium using whole-cell recordings. They demonstrated
the presence of Na+, K+ and Ca2+ voltage-gated
conductances and found that the injection of 100 |^M
inositol 1,4,5-trisphosphate elicited an increase in
membrane conductance in about half of the cells.
In the present study we isolated VRN by dissociation of
rat vomeronasal organs. Using scanning electron microscopy, we observed that the dissociation procedure had a
limited effect on the morphology of many isolated VRN. We
recorded from these cells using the gramicidin-perforated
variant of the patch-clamp technique, at a temperature close
to the body temperature of the rat. The incorporation of
the antibiotic gramicidin ionophores into the patch of
membrane inserted in the lumen of the electrode allowed us
to record voltage-gated Na+ and K+ currents. Gramicidin
channels are permeable to Na + and K + ions but
50 D. Trotier et al.
impermeable to Cl" and Ca2+ ions and to small molecules
(Akaike and Harata, 1994; Kyrozis and Reichling, 1995;
Tajima et al., 1996). Therefore we believe that this technique
would be very useful to study the transduction process in
rodent vomeronasal receptor cells.
Materials and methods
Cell preparation
Female rats of Wistar strain (2 months old) were
decapitated. The head was carefully cut along the middle
plane and the vomeronasal organs, situated on the ventral
aspect of each side of the nasal septum, were immediately
removed from their respective bone cavities. The sensory
epithelium, which is situated at the medial part of the organ,
was separated from the lateral non-sensory epithelium and
rinsed in calcium-free saline (CFS), comprising (mM) NaCl
(140), KC1 (4), MgCl2 (1) and HEPES (10), pH 7.4. It was
then transferred to CFS with 0.5 mM EGTA for 1 min and
placed for 40-50 min in CFS containing 0.8 mg/ml papain
(Boehringer Mannheim GmbH, Meylan, France). The
tissue was washed in CFS, placed in 80 ul of the same
solution and carefully pipetted through a fire-polished
pipette tip. An equal amount of Ringer's solution was
added and the cell suspension was placed in the recording
chamber. The Ringer's solution contained (mM): NaCl
(140), KC1 (4), CaCl2 (2), MgCl2 (1), glucose (10) and
HEPES (10), pH 7.4.
The recording chamber (~150 ul) was made by two
circular glass coverslips of 10 and 15 mm diameter kept
horizontal at a distance of 1.5 mm. Immediately after
dissociation, isolated cells were introduced into the chamber
and left undisturbed for some minutes until they adhered to
the coverslip. Then the perfusion (0.1 ml/min) of Ringer's
solution was started by means of a peristaltic pump (Mod.
Minipuls3; Gilson Medical Electronics, Villiers Le Bel,
France). A sample injector with a loop (Mod. 5020;
Rheodyne Inc., Cotati, CA) was used for introducing
another solution in the perfusion fluid. This system
permitted changes of the perfusion fluid with negligible
mechanical disturbances. Direct application of solutions
onto the cell was made from a patch pipette. The solutions
were driven with air pressure via a valve controlled from a
PC desk computer. The temperature of the solution in the
chamber was kept between 31 and 37°C by blowing hot air
into the chamber.
The recording chamber was placed under a Diastar
microscope equipped with a 30* objective with long
working distance (3.5 mm) and a Hoffman contrast
modulation (Modulation Optics, Greenvale, NY). The
microscope picture was presented on a TV screen with a
Sony DXC-107P video camera. The x, y and z coordinates
of the experimental chamber and the patch electrode
holder could be manipulated by a combination of handdriven micro-manipulators (Microcontrole, Paris, France)
and piezo-electric devices (Mod. P-840, Physik Instrument,
Waldbronn, Germany). The set-up was placed on a table
vibration damped by three air cushions (Physik Instrument).
Gramicidin-perforated patch-clamp recordings
Recordings were preferably made with the gramicidin
perforated whole-cell mode not only because recordings
obtained in this condition minimized modifications of the
cell cytoplasm but also because the membrane rupture with
suction, in conventional whole-cell mode, most often
destroyed the seal resistance. A stock solution of gramicidin
was made by dissolving 5 mg of gramicidin (Sigma) in 100
ul dimethyl sulfoxide (DMSO). The recording electrode
(7-14 MQ) was made of borosilicate glass capillaries
(GC150T-10; Clark Electromedical Instruments, Reading,
UK). After heat polishing the pipette was filled by dipping
the tip for ~20 s into a solution containing (mM): NaCl (10),
KC1 (130) and HEPES (10), adjusted to pH 7.4 with KOH.
Another solution, containing (mM): NaCl (130), KC1 (4)
and HEPES (10), adjusted to pH 7.4 with KOH, was used to
examine the ionic basis of the voltage-gated conductances.
The pipette was back-filled with the gramicidin solution
containing 2.5 ul of the stock solution of gramicidin added
to 1 ml of pipette solution which was vigorously stirred for
1 min with a whirl shaker. After obtaining a gigaohm seal
(< 6 GQ) the holding potential was set at -80 mV. The cell
capacitive current evoked by 5 mV voltage command pulses
were used to monitor the incorporation of gramicidin
channels into the patched membrane. In most cases the
access resistance to the cell interior reached a stable value
between 20 and 40 MQ within ~30 min.
Recordings were made with a patch clamp amplifier
(PC-501, Warner Instrument, Hamden, CT) and a probe
with 10 GQ feedback resistor. Membrane potentials and
currents were filtered (dc, 3 kHz) and stored on a computer.
The recorded membrane currents and potentials were
analyzed using the Biopatch software provided by Biologic
(France) or transferred to a SigmaPlot program (V3.06,
Jandel Co., San Rafael, CA) to analyze and present the data.
All values are represented as mean ± 1 SD.
Scanning electron microscopy
Isolated VRN adhering to glass coverslips were placed in
the fixative containing 4% formaldehyde, 0.3% picric acid
and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer,
pH 7.4. The preparations were critical point dried and
sputtered with a Pt/Ir mixture and observed in a scanning
electron microscope (Jeol JSM 6400). The same procedure
was used for scanning electron microscopy of the whole
vomeronasal organ. In that case anesthetized rats were
perfused briefly with 4% dextran in 0.1 M sodium
phosphate buffer, pH 7.4, before being perfused with the
fixative. The fixed vomeronasal organs were cut in
transverse sections to observe the shape and position of the
receptor neurons in situ.
Microvillous Receptor Neurons
51
Figure 1 Scanning electron microscopy of the rat vomeronasal organ. (A) Cross-section of the middle part of the vomeronasal organ
showing the lumen (L) and the sensory epithelium (E). A large blood vessel (V) is seen. Scale bar: 100 um. (B) Enlarged portion of the sensory
epithelium showing the packed microvillous receptor neurons. Scale bar: 20 (im. (C) Isolated receptor neuron showing a part of an axon, the
cell body, the dendrite and the terminal vesicle. Scale bar: 10 (im. (D) The terminal vesicle of the receptor cell shown in C bears numerous
microvilli. Scale bar: 1 urn.
Results
Morphological characteristics of rat vomeronasal neurons
in situ
Figure 1A shows a scanning electron microscopy picture of
a transverse section of the rat vomeronasal organ. The
lateral part of the organ, located at left in Figure 1A, is
mainly occupied by a large blood vessel (V) surrounded by a
vascularized erectile tissue. This erectile tissue is involved in
the pumping mechanism which draws liquid stimuli in and
out of the lumen of the organ (Hamlin, 1929; Meredith and
O'Connell, 1979; Meredith el al, 1980). In this example the
erectile tissue is turgescent and the crescent-shaped lumen
(L) is compressed. The sensory epithelium (E), which covers
the medial part of the lumen, has a thickness of 100-150
um. The lateral wall of the lumen opposite to the sensory
epithelium is exclusively covered by respiratory ciliated
cells (Vaccarezza et al, 1981). Observations at a higher
magnification, as illustrated in Figure IB, show that
vomeronasal receptor neurons present ovoid cell bodies 6-8
(im in diameter and 10-13 um long. Depending on the
position of the cell soma within the epithelium, the length
of the dendrite extending towards the surface of the
epithelium ranges from ~30 um to >100 um. The microvilli
on the receptor neurons form a carpet at the surface of the
sensory epithelium (Figure IB).
Morphological characteristics of isolated rat vomeronasal
neurons
Observations using scanning electron microscopy indicated
that many vomeronasal receptor neurons retained their
typical bipolar shape after isolation. A typical example is
presented in Figure 1C. The dendrite has a length of ~70 um
and a diameter of near 1 um. The tip of the dendrite
terminates by a small vesicle, ~7 um long and 3 um in
diameter, bearing numerous microvilli of ~2 um length
(Figure ID). The cell soma has a diameter of ~8 um and a
length of ~12 um. Frequently a thin cell protusion was seen
emerging from the basal region of the soma which, from its
size and location, was thought to be the initial part of the
axon. No significant difference appeared in the size of the
cell bodies in isolated (7-8 (im in diameter, 10—14 um in
length) and in situ rat VRN.
Recordings were obtained from neurons having the same
52
D. Trotier et al.
morphological features, as seen in Figure 2. The length of
the dendrite ranged from ~40 to 110 (am (mean = 69 ± 37
urn). The tip of the dendrite terminates by a small vesicle but
the Hoffman contrast modulation does not always allow a
precise visualization of the microvilli at the apex of the
dendrite. The diameter of the cell soma is 12 ± 2 urn and the
soma length 15 ± 2 urn (n = 42), distinctly larger than they
appeared in the scanning micrographs. This was probably
due to the shrinking that appeared by the fixation and
drying necessary for the scanning preparation. No alteration
of the cell morphology was observed during patch-clamp
recording.
Membrane properties at rest
Figure 2 Photomicrograph of a microvillous sensory neuron from the rat
vomeronasal organ using diffraction interference contrast. This picture is
representative of the shape of isolated rat vomeronasal neuron used for
patch-clamp recording. Note the long coiled dendrite —100 urn long and
the terminal knob. A brush of short and thin microvilli could be observed on
the knob. A thin axon is seen protruding from the basal pole of the cell
body. Scale bar: 20 u.m.
The mean membrane potential, measured as the zero
current voltage after incorporation of gramicidin into the
patched membrane, ranged from -36 to -85 mV (mean =
-58 ± 14 mV, n = 38). In six cells having a resting potential
negative to -80 mV, a particular activity was observed: the
membrane potential spontaneously jumped from the resting
level to the firing voltage threshold and then back to the
resting level. An example is presented in Figure 3A. The
origin of these transitions between the two levels of
polarization, which has been observed in frog VRN (Trotier
et al, 1994), remains to be determined. In other recordings
the membrane potential fluctuated only slightly around the
mean value (Figures 4B, 5 and 8). The input resistance,
measured by injection of current pulses of a few pA, was in
the range of 1-6 GQ. Action potentials were generally
triggered by injection of depolarizing current pulses of <5
pA. In the example shown Figure 3B, the injection of 1 pA
triggered a single action potential after a latency of 220 ms.
Increasing the current intensity to 3 and 5 pA reduced the
latency to 90 and 55 ms, inducing two and three action
B
A
40-.
40-.
E
0-
-40
1
-20 -
-60
-80 .
E
0.
-20
i
2
o
3
20-
ntia
20-
bra
E
5
•40-
-60 -80
-100
10
Time (s)
15
100
200
300
400
Time (ms)
Figure 3 (A) Spontaneous impulse activity of a rat VRN. The membrane potential spontaneously jumped from the resting level near-95 mV
to the firing voltage threshold and then back to the resting level. Every membrane depolarization triggered overshooting action potentials. (B)
High sensitivity to small depolarizing currents. The injection of a 1 pA depolarizing current step, during the time indicated by the lower trace,
triggered a single action potential. Increasing the current intensity to 3 and 5 pA reduced the latency of the first action potential and increased
the impulse frequency. Resting potential: -64 mV. Different cells in A and B.
Microvillous Receptor Neurons
53
B
A
60
4
n
I -
1 2
^T
|
20 •
-20
o
I -2
-40
cc
-60
t
i ""°
10
15
20
25
30
35
S -ioo -
10
16
20
30
28
Time (s)
Time (s)
Figure 4 Reduction of the sodium pump current by lowering the external K+ concentration depolarized rat VRN. (A) The membrane was
held at -80 mV and a Ringer's solution containing 4 \xM K+ was applied from a stimulating pipette during the time indicated by the lower bar.
The reduction of the external K+ concentration evoked a reduction of ~4 pA of the outward sodium pump current. The current transients seen
at 20 and 27 s were not always observed and corresponded probably to spontaneous openings of membrane channels. (B) In current-clamp
conditions the application of a Ringer's solution containing 4 \iM K+ (indicated by the bar) reduced the sodium pump current and depolarized
the cell membrane. When the depolarization reached —65 mV repetitive action potentials were elicited.
potentials respectively. The action potentials presented a
large positive overshoot and the spike height commonly
reached 80 mV. The spike duration at -30 mV was ~10 ms
and each action potential was followed by an afterhyperpolarization.
Contribution of the sodium pump current to set the
resting potential
We previously demonstrated that the reduction of the
sodium pump activity of frog VRN, either by lowering the
external K+ concentration or by applying dihydro-ouabain,
largely decreased the resting membrane potential (Trotier
and D0ving, 1996a). In the present study we made similar
observations on most rat VRN. For example, lowering the
K+ concentration in the bath from 4 mM to 4 uM reversibly
reduced the outward sodium pump current (Figure 4A) in
8/11 VRN. In current-clamp conditions the low K+-evoked
decrease of the sodium pump activity induced the
depolarization of 11/13 tested cells (Figure 4B). Moreover,
the application of Ringer containing 50-100 uM dihydro-ouabain, a specific sodium pump inhibitor, evoked
membrane depolarization of 7/12 cells (Figure 5).
Voltage-dependent currents
In gramicidin patch-perforated recordings depolarizing
voltage pulses from -80 mV activated a fast transient inward
current followed by a sustained outward current (Figure
6A,C). Current-voltage relationships (Figure 6B) indicated
that the initial transient inward current appeared near
-50 mV, reached a maximum (0.4-1 nA) near -40 mV
and then gradually decreased to reverse near 50 mV. This
-100
20
40
60
80
100
120
Time (s)
Figure 5 Application of dihydro-ouabain depolarized rat VRN. Dihydroouabain (100 uM) was applied during the time indicated by the lower bar.
The membrane depolarized from the resting level near -75 mV to near -60
mV and a burst of action potentials was elicited. Action potentials were
truncated at -20 mV.
inward current mostly resulted from the activation of
voltage-dependent sodium channels because most of the
current was blocked by 1 |iM tetrodotoxin in the bath and
because replacing K+ by Na+ in the recording pipette shifted
its reversal potential to near 0 mV (not shown). The
outward potassium current appeared at test potentials
beyond -30 mV and increased gradually with the level of
depolarization. It was almost completely blocked by
replacing K+ by Na+ in the recording pipette (Figure 6D). In
3/27 cells the outward current reached its maximal
amplitude within ~50 ms (Figure 6A) whereas in the
remaining 24 cells the current activated much more rapidly
54 D. Trotierefa/.
B
100 ms
1000
600
-500
•1000
100 ms
750
600
100 ms
I "
~
0 -I
\r
0
-250
-200 -I
-500
Figure 6 Voltage-dependent currents. In (A) and (C) the membrane of two different cells was depolarized by voltage pulses from -70 to 80
mV with 10 mV increments. Holding potential: -80 mV. (B) I-V curve of the recording presented in A. The outward current amplitude (open
circles) was measured at the end of the voltage pulse. The inward current (closed circles) was measured at the peak. (D) Block of the outward
current by internal Na + ions. In A, C and D the membrane was depolarized during the time indicated by the lower traces.
Gramidicine-perforated patch clamp recording. In A and C the pipette contained mainly 140 mM KG and 10 mM NaCI, whereas in D the
pipette contained mainly 140 NaCI and 4 mM KCI. The transient small inward current seen at the end of the voltage step in D was not
systematically observed.
Effect of stimulation with rat urine
Urine is a major vector of pheromones in rodents and
specific effects are elicited by exposing individuals to the
urine of the same or the opposite sex (for review see
Halpern, 1987; Wysocki and Meredith, 1987). It was of
interest to see whether the isolated microvillous receptor
cells in patch-clamp studies could respond to urine. In
natural conditions it seems that there is little dilution of the
2 , -60
0-
1
Current
and an initial transient component was observed (arrow in
Figure 6C). These differences in the kinetic of activation of
the outward current suggest a diversity of voltage-gated
potassium channels.
In a few cells we noticed the presence of an inward current
activated by long-lasting hyperpolarizing voltage pulses
more negative than about -80 mV. This current, very similar
to the current /(, analyzed in detail in frog VRN (Trotier and
D0ving, 1996b), slowly increased with time during the
steady hyperpolarization (Figure 7). The current amplitude
increased, and its time course of activation decreased, with
increasing hyperpolarizations. It was reversibly blocked by
the specific blocker Cs+ added (5 mM) to the bath (not
shown).
-60
M1
-2-4-6-
-100
||U
II
ill
-8-
!*•
-10.
I
-12-140
1
2
3
4
5
Time (s)
Figure 7 Activation of the inward rectifying current /h by membrane
hyperpolarization. Increasing the membrane potential from -60 to -100 mV
during 4 s evoked a current jump through the leak resistance followed by
the slow activation of /h.
urine during behavioral sampling, i.e. licking. The urine is
mixed with glandular secretions during its transport
towards the opening of the organ and its active pumping
into the lumen, but we estimate that this dilution would not
be larger than ~l:10. In the example shown in Figure 8 we
Microvillous Receptor Neurons
c-
- 3 0 -i
E
r* -40
ss
5
-50
»
^0-
A
-70
S
-80
'I*'V
10
20
30
40
50
60
Time (s)
Figure 8 Stimulation of a female rat VRN with male urine diluted 1:20 in
Ringer's solution. The urine was applied through the perfusion system and
was present around the cell during the time indicated by the bar. In response
to the stimulation the cell depolarized and repetitive action potentials were
elicited. Action potentials were truncated at -30 mV.
demonstrate that a receptor neuron isolated from a female
rat responded to male urine diluted 1:20 in Ringer solution.
The urine was introduced into the bath through a loop in
the perfusion system to avoid mechanical stimulation of the
recorded cell. The stimulation induced a long-lasting
depolarization which triggered a burst of action potentials.
Of eight cells tested with male urine, four responded with
a series of action potentials similar to the one shown in
Figure 8.
Discussion
The present study is, to our knowledge, the first microscopic
and electrophysiological characterization of isolated rat
vomeronasal receptor neurons. We made an effort to obtain
isolated cells with little modification of their morphology
observed in situ. We consider this point to be very important
as it has been shown in many other cell preparations that
the modification of cell morphology is accompanied by
activation of membrane currents, such as swelling-induced
chloride currents (e.g. Lascola and Kraig, 1996; Meng and
Weinman, 1996; Zhang and Lieberman, 1996). Isolated
VRN were placed into the recording chamber just after
mechanical disruption of the organ following the enzymatic
treatment with papain. In this condition they rapidly
adhered to the glass coverslip and the fixation of both the
soma and the dendrite was essential to maintain their
bipolar morphology for a few hours. Another favorable
factor in this context seems to be the treatment with papain
because mouse VRN isolated with a mixture of collagenase
and trypsin seldom presented dendrites >40 um in length
(Liman and Corey, 1996). We have made similar
observations using similar techniques with frog olfactory
receptor cells (unpublished data).
Light and scanning microscopy observations indicated
that many rat VRN kept their morphology after dissociation, including the presence of the vesicle at the apex of
55
the dendrite. We did not notice any signs of deterioration
during the time of the experiment. Recordings were
obtained from receptor cells having a long dendrite (>40um
in length and ~1 urn in diameter) terminating with a vesicle.
We could not observe microvilli under Hoffman contrast
but microvilli were shown clearly by scanning electron
microscopy. Since the scanning electron microscopy revealed
the presence of microvilli at the terminal vesicle, it is highly
probable that they were present also in the dissociated and
isolated cells used for the patch-clamp studies. Most
recorded neurons presented, at the basal pole of the soma, a
thin process which was considered as the initial part of the
axon. More than 100 cells with these morphological
characteristics were usually available in the recording
chamber.
We could record from the VRN with little modification
of the intracellular content. This is utmost importance
for the study of the transduction process because there
are indications, in rat (Inamura et al, 1997) and turtle
(Taniguchi et al, 1995, 1996) VRN, that the transduction
process is linked to the activation of second messenger
pathways. Therefore we discarded the direct whole-cell
recording by rupture of the patched membrane and also the
nystatine perforated patch-clamp method as nystatine
channels are permeable to Na and K ions but also to Cl ions
(Abe et al, 1994). We have demonstrated that gramicidine
channels are efficiently incorporated into the patched
membrane at a rate apparently similar to other cells
(Ebihara et al, 1995; Kyrozis and Reichling, 1995;
Zhainazarov and Ache, 1995; Tajima et al, 1996). The
residual access resistance (20-40 MQ) was low enough
to record membrane currents although it would have
certainly introduced a distortion, brought about by the
series resistance errors (Marty and Neher, 1995), in the
current-voltage curves of voltage-dependent currents
presented in Figure 6B.
In the present study we observed that isolated rat VRN
were much more polarized (-58 ± 14 mV) than rat VRN
recorded in slices (-45 ± 2.5 mV; Inamura et al, 1997). This
indicates that rat VRN were not depolarized by the isolation
procedure and remained in a good physiological condition.
Resting potentials of isolated mouse VRN were in the same
range (-58 ± 3 mV; Liman and Corey, 1996).
These recorded resting potential values could, however,
underestimate the real resting membrane potential. In
whole-cell recordings the pipette-membrane seal resistance
(^seai) a cts as a shunt pathway and must be much higher
than the cell input resistance to give an accurate measurement of the resting membrane potential. We have observed
the depolarizing effect of a low RX!L\ in frog VRN because
the improvement of R^a\ from 5-10 GQ (Trotier et al, 1993)
to >40 GQ (Trotier and Doving, 1996a,b) increased the
estimation of the resting membrane potential from -61 to
-88 mV. In the present recordings a good seal was rather
difficult to obtain and Rseal rarely exceeded 5 GO. Although
56
D. Trotier et al.
there is no indication about the quality of the seal in other
studies, it is probable that Rsea\ values were in the same range
(Liman and Corey, 1996; Inamura et al., 1997).
Given the technical difficulty of getting a high Rxai value,
the estimation of the cell input resistance from the recorded
input resistance should be made with caution.
The input resistances we measured on isolated rat VRN
(1-6 Gfi) are similar to those measured from rat VRN in
slices (1.5 GQ; Inamura et al., 1997) and isolated mouse
VRN (3.3 Gn, Liman and Corey, 1996), i.e. in the same
range as the R^^ values. There is a possibility that the cell
input resistance is much higher than those estimations. For
example, cell-attached estimations of the cell input
resistance of rat olfactory receptor cells indicate values in
the order of 40 Gn (Lynch and Barry, 1989) whereas in
whole-cell recordings the input resistance is only ~3 GQ.
(Liman and Corey, 1996).
The present study shows that rat VRN were depolarized
by decreasing the external K+ concentration and by
application of dihydro-ouabain, two treatments that
decrease the sodium pump activity. Therefore we may
conclude that the sodium pump current helps set the resting
membrane potential. In frog VRN the membrane is largely
polarized by the sodium pump current (Trotier and Doving,
1996a,b) and resting membrane potentials more negative
than the equilibrium potential for K+ ions are commonly
recorded. This effect of the pump current was easily
demonstrated because the seal resistance was high and thus
had a limited shunting effect on the cell input resistance. In
the present recordings on isolated rat VRN the exact
contribution of the pump current to the steady membrane
polarization is much more difficult to assess because of the
rather limited R^] values.
Isolated rat VRN were excitable and generated full-sized
action potentials either spontaneously or in response to
depolarizing current pulses of a few pA. We did not
characterize all the properties of the activated voltagedependent currents, but the major currents are the fast
inward sodium current blocked by TTX and the outward
potassium currents blocked by internal sodium ions. These
currents appear to be at least qualitatively similar to those
already observed in frog VRN (Trotier et al., 1993), mouse
VRN (Liman and Corey, 1996) and rat VRN (Inamura et
al., 1997).
Though preliminary, the recordings show that exposure
of isolated rat VRN to urine induced a depolarization that
triggered a series of action potentials lasting for the entire
stimulation period. Since our recordings were made with the
diluted urine in the perfusion fluid there was no mechanical
stimulation during the presentation. Thus the observed
responses were likely due to chemical(s) present in the urine.
Only half of the cells tested responded to the exposure of
urine, suggesting a specificity of the responses. However, our
present recordings are too preliminary to interpret these
responses as specific responses of vomeronasal receptor cells
to urinary pheromones.
In conclusion, microvillous receptor cells could be
isolated in good conditions from the rat vomeronasal organ.
Patch-clamp recordings were obtained with incorporation
of gramicidine channels into the patched membrane and
indicated that cells were polarized and excitable. We hope
that this technique will help us to understand the transduction process activated by pheromonal molecules.
Acknowledgements
The present study was supported by grants from the European
Science Foundation (#207, Strasbourg, France) and the University
of Oslo, Norway.
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Accepted October 7, 1997