Volume 16, Number 15,
Issue of August 1, 1996
pp. 4625-4637
Copyright ©1996 Society for Neuroscience
Electrophysiological Characterization of Chemosensory Neurons
from the Mouse Vomeronasal Organ
Emily R. Liman and
David P. Corey
Department of Neurology and Howard Hughes Medical Institute,
Harvard Medical School, and Massachusetts General Hospital, Boston,
Massachusetts 02114
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The mechanism of sensory transduction in chemosensory neurons of
the vomeronasal organ (VNO) is not known. Based on molecular data, it
is likely to be different from that mediating sensory transduction in
the main olfactory system. To begin to understand this system, we have
characterized the electrophysiological properties of dissociated mouse
VNO neurons with patch-clamp recording. Sensory neurons were
distinguished from nonsensory neurons by the presence of a dendrite, by
immunoreactivity for olfactory marker protein, and by the firing of
action potentials. The resting potential of VNO neurons was
approximately 
60 mV, and the average input resistance was 3 G
.
Current injections as small as 1-2 pA elicited steady trains of action
potentials that showed no sign of adaptation during a 2 sec stimulus
duration. The voltage-gated conductances in VNO neurons are distinct
from those in olfactory neurons. The Na+ current
is composed of two components; the major component was TTX-sensitive
(Ki = 3.6 nM). The
outward K+ current activates at 30 mV with
kinetics 10 times slower than for K+ currents in
olfactory neurons. The Ca2+ current is composed
of at least two components: an L-type current and a T-type current that
activates at 60 mV and is not found in olfactory neurons. We find no
evidence for cyclic nucleotide-gated channels in VNO neurons under a
variety of experimental conditions, including those that produced large
responses in mouse olfactory neurons, which is further evidence for a
novel transduction pathway.
Key words:
mouse;
olfactory;
vomeronasal;
sensory
transduction;
patch-clamp;
voltage-gated channel;
cyclic
nucleotide-gated channel
INTRODUCTION
Chemosensation in terrestrial vertebrates is
achieved by at least two distinct nasal organs: the olfactory
epithelium (OE) and the vomeronasal organ (VNO). The OE and VNO are
thought to detect different classes of chemicals and to mediate
different behavioral and neuroendocrine responses. In mice and other
rodents, the VNO has been shown to mediate (at least in part) the
response to pheromones, chemicals communicated between animals of the
same species that convey information primarily regarding reproductive
and social status (for review, see Keverne, 1983
; Halpern, 1987;
Wysocki and Meredith, 1987). In rodents and other mammals, the VNO
consists of two epithelial tubes encased in bone that lie at the base
of the rostral portion of the nasal cavity and send projections to the
accessory olfactory bulb (Fig. 1) (Cajal, 1911; Barber
and Raisman, 1974) (for review, see Keverne, 1983).
Fig. 1.
Schematic drawing of the mouse nasal cavity
showing the location of the VNO and the turbinates of the OE. The VNO
is shown encased in a bony shell. It communicates with the nasal cavity
through a duct, not drawn to scale. OE, olfactory
epithelium; VNO, vomeronasal organ; OB, olfactory
bulb; AOB, accessory olfactory bulb.
[View Larger Version of this Image (42K GIF file)]
Despite similarities in ontogeny (Garrosa et al., 1986) and morphology,
olfactory neurons and VNO neurons are likely to use distinct mechanisms
of sensory transduction. In olfactory neurons, one well studied pathway
for transduction involves seven-transmembrane odorant receptors, a
stimulatory heterotrimeric G-protein, adenylyl cyclase, and the
generation of cAMP (for review, see Reed, 1992; Ronnett and Snyder,
1992). cAMP opens a cationic heteromeric cyclic nucleotide-gated (CNG)
channel that depolarizes the membrane potential (Nakamura and Gold,
1987). Each of these components has been cloned, including a large
family of receptors, a G-protein (G
olf), an
adenylyl cyclase (ACIII), and two CNG channel subunits (rOCNC1, rOCNC2;
Jones and Reed, 1989; Bakalyar and Reed, 1990; Dhallan et al., 1990;
Ludwig et al., 1990; Buck and Axel, 1991; Bradley et al., 1994; Liman
and Buck, 1994). In situ hybridization, Northern blot, and
PCR analyses have shown that Golf, rOCNC1, and
ACIII are not expressed in the VNO (Dulac and Axel, 1995; Berghard et
al., 1996). rOCNC2 is expressed in the VNO (Berghard et al., 1996), but
it is also expressed in the OE, where it is a subunit of a channel with
rOCNC1 (Bradley et al., 1994; Liman and Buck, 1994). Because rOCNC2
does not form detectable channels when expressed alone in a
heterologous expression system, it is not clear whether it is
functional in the VNO. Recently, putative pheromone receptors have been
cloned from rat VNO, and they constitute a novel family of
seven-transmembrane receptors with no sequence similarity to the
olfactory receptors (Dulac and Axel, 1995).
To begin to understand transduction in the VNO, we have characterized
the electrical properties of freshly dissociated mouse VNO neurons.
Voltage- and ligand-gated conductances of olfactory neurons have been
characterized for a number of different vertebrate species, including
rat (Lynch and Barry, 1991; Trombley and Westbrook, 1991), mouse (Maue
and Dionne, 1987), salamander (Trotier, 1986; Firestein and Werblin,
1987), frog (Schild, 1989; Pun and Gesteland, 1991), and fish (Miyamoto
et al., 1992; Nevitt and Moody, 1992) and have established a role for
CNG channels in mediating sensory transduction in these cells (Nakamura
and Gold, 1987; Kurahashi, 1990; Firestein et al., 1991a; Dionne and
Dubin, 1994; Ache and Zhainazarov, 1995). A characterization of
electrical properties of VNO neurons has previously been undertaken
only in the frog and turtle (Trotier et al., 1993, 1996; Taniguchi et
al., 1995, 1996). In these experiments, no apparent differences were
found between VNO and olfactory neurons. In contrast, we find that ion
currents in mouse VNO neurons are distinct from those in olfactory
neurons. Furthermore, we find no evidence for functional CNG channels
in VNO neurons, which is additional evidence for different transduction
pathways.
MATERIALS AND METHODS
Dissociation of vomeronasal and olfactory neurons.
Six-week-old Cd1/nude mice were kept in an isolation facility and
killed by cervical dislocation or CO2 inhalation.
The dissociation protocol was a modification of that used by Maue and
Dionne (1987) for the dissociation of mouse olfactory neurons. The VNO
was removed by retracting the palate, breaking the front incisors, and
pulling out the intact VNO within its bony encasing. The epithelium was
dissected free of the bone, removed to divalent-free PBS supplemented
with penicillin-streptomycin solution (1:100 dilution; Gibco,
Gaithersburg, MD) and cut into small pieces. Crude collagenase (1 mg/ml
of type I; Sigma, St. Louis, MO) and trypsin (1 mg/ml; Sigma) were
added, and the tissue was incubated at 35°C for 30 min with
agitation. The small pieces of tissue were transferred through several
washes with divalent-free PBS to solution supplemented with DNase (40 mg/ml; Worthington Biochemical, Freehold, NJ). The tissue was gently
triturated with a fire-polished Pasteur pipette, and cells were either
immediately plated on a clean glass coverslip or stored at 4°C for up
to 5 hr and then plated. VNO neurons were also prepared without enzymes
by incubating the tissue in divalent-free PBS for 1-2 hr and then
scraping the surface of the epithelium with forceps, followed by gentle
trituration with a fire-polished Pasteur pipette. Mouse olfactory
neurons were prepared without enzymes for whole-cell recording and
either with or without enzymes for patch recording in an identical
manner to that used for VNO neurons.
Immunocytochemistry. The VNO was dissected free, fixed for
16 hr in 5% formalin, embedded in paraffin, sectioned (5 µm), and
cleared using standard protocols. A goat anti-rat olfactory marker
protein (OMP) antiserum was kindly provided by Frank Margolis and is
described in Farbman and Margolis (1980). OMP immunoreactivity was
detected with biotin-avidin-horseradish peroxidase using protocols
from Pixley (1992) and the manufacturer (Vectastain ABC Elite; Vector
Laboratories, Burlingame, CA). Blocking of nonspecific binding was with
5% rabbit serum; incubation with primary antibody (1:2000 dilution)
was for 1 hr at room temperature and incubation with biotinylated
secondary antibody (rabbit anti-goat; 1:500 dilution) was for half an
hour. Visualization with 3,3
-diaminobenzidine (DAB) substrate was as
recommended by the manufacturer (Vector).
Dissociated cells were prepared as described above, plated on clean
glass slides, and fixed for 5 min in PBS containing 4%
paraformaldehyde. Detection of OMP immunoreactivity was similar to that
described above except that nonspecific binding was blocked with 10%
fetal bovine serum and by including 0.1% Triton X-100 in all solutions
except those involved in the peroxidase reaction. Incubation with the
anti-OMP antibody (1:1000 dilution) and the biotinylated secondary
antibody (1:500 dilution) was for 1 hr at room temperature.
Electrophysiology. Voltage-clamp recordings were performed
with an Axopatch 200A (Axon Instruments, Foster City, CA) or Yale MarkV
patch-clamp amplifier. Currents were low-pass filtered at five times
the sampling frequency (typically 5 or 10 kHz) and digitized with the
LM900 Laboratory interface (Dagan, Minneapolis, MN). Patch pipettes
were constructed from borosilicate microcapillary glass (VWR
Scientific) and coated with SYLGARD (Dow Corning, Midland, MI). For
whole-cell recording from VNO neurons, pipettes had resistances after
fire polishing of 1-3 M. For excised patches and whole-cell
recording from olfactory neurons, pipettes were pulled to a smaller
diameter (5-10 M). Series resistance correction was used in most
experiments. In experiments used for quantitative analysis, the
uncorrected series resistance gave voltage errors of <5 mV. The bath
was connected to ground via a 1 M KCl agar
bridge. Junction potentials were measured to be <10 mV and were not
corrected.
Perforated patch recording was performed essentially as described (Horn
and Marty, 1988). Nystatin (Sigma) was dissolved in dimethylsulfoxide
(50 mg/ml) and was used at a final concentration of 0.25 mg/ml. Stock
and pipette solutions were discarded after 2 hr. Perforated patch mode,
with series resistance values <50 M, was achieved within 10-20
min. Experiments in which the capacitative current increased suddenly,
indicating a rupture of the patch membrane, were discarded. The patch
pipette solution (Nys1) contained Ba2+ to
stabilize seal formation.
Fast (<1 sec) and complete solution exchange was achieved by gravity
flow through an array of microcapillary tubes (1 µl; Drummond,
Broomall, PA) that was manually positioned. In most experiments, the
cell (or patch) was lifted from the bottom of the dish and positioned
in front of the array. In some experiments, cells were left adherent to
the bottom of the dish. Solution flow was directed downward by filing
the ends of the microcapillary tubes at the lower edge, providing
excellent solution exchange. Most traces are shown after leak
subtraction using the scaled response to hyperpolarizing steps.
Current-clamp recordings were performed with an Axopatch 200A
patch-clamp amplifier (Axon Instruments) in current-clamp mode. For
olfactory neurons, it was important that the clamp be in normal mode,
as the fast mode gave a ringing artifact. Curve fitting was performed
with least-squares optimization using the Origin program (Microcal
Software, Northampton, MA)
Recording solutions. The composition of solutions is shown
in Table 1. All biochemicals were
obtained from Sigma unless otherwise noted. Stock solutions of cAMP,
cGMP, 8-Br-cGMP, 8-CPT-cAMP (Calbiochem, La Jolla, CA), 
-conotoxin
GVIA (Alomone Laboratories, Jerusalem, Israel), and -Aga IVa
(provided by Pfizer, Groton, CT) were stored frozen and were diluted in
the intracellular or extracellular solution immediately before use.
Cytochrome C (1 mg/ml) was added to the extracellular solution
containing -Aga IVA. Stock solutions of nitrendipine and nifedipine
were made in EtOH and stored at 20°C. GTP, ATP, and phosphocreatine
were added to the pipette solution and stored as frozen aliquots.
RESULTS
Preparation and identification of VNO sensory neurons
The VNO sensory epithelium contains three types of cells: sensory
neurons, supporting cells, and basal cells (Vaccarezza et al., 1981).
The sensory neurons are bipolar cells that send an axon to the
accessory olfactory bulb and a dendrite to the lumen of the epithelium.
The dendritic process terminates in a tuft of microvilli, where
transduction is likely to occur. The supporting cells form a layer
close to the lumenal surface of the epithelium, and they also have
microvilli, although they lack a dendritic process. Only sensory
neurons are immunoreactive for OMP (Farbman and Margolis, 1980). Figure
2A shows a section through the
epithelium labeled with an anti-OMP antibody. Labeling is seen of cells
in the deeper layers of the epithelium, the location of sensory
neurons, and not of cells in the most lumenal layer, the location of
supporting cells. The spotty labeling is similar to what has been shown
previously (Graziadei et al., 1980). Labeling is also present at the
lumen of the epithelium and likely corresponds to the labeling of
dendritic processes of VNO sensory neurons (Johnson et al., 1993).
Fig. 2.
Photomicrographs of VNO sensory epithelium and VNO
sensory neurons. A, A section through the VNO of the mouse
labeled with an antibody to OMP and visualized with peroxidase
staining. No staining was observed when the primary antibody was not
included (not shown). Note staining of the cell bodies of neurons
(n) and dendrites (d) but not of sustenticular
cells (sc). B, A dissociated cell with a long
dendritic process was labeled with anti-OMP antibody, confirming the
identity of these cells as sensory neurons. C-E,
Differential interference contrast photomicrographs of two dissociated
VNO neurons (C, D) and an olfactory neuron (E).
Scale bar is the same for all five panels (10 µm).
[View Larger Version of this Image (144K GIF file)]
To obtain dissociated VNO neurons, the VNO and its bony encasing were
removed from the nasal cavity and the epithelium was dissected free. At
this anterior position in the nasal cavity, the OE is dorsal to the VNO
and lies outside the bony encasing (Fig. 1), and thus no contamination
of VNO neurons by olfactory neurons is expected. For most experiments,
dissociated cells were prepared by incubating the epithelium in
divalent-free solution containing trypsin and collagenase and then
triturating gently. Dissociation using either of the two enzymes alone,
or no enzyme, did not produce as many healthy-looking cells. To
determine whether the enzymatic treatment altered the properties of the
ion channels, some experiments were done without the use of enzymes;
these experiments are explicitly stated as such. A population of cells
were identified as sensory neurons by morphological criteria. These
cells had an ovoid cell body with a mean diameter along the shorter
axis of 11.1 ± 1.4 (mean ± SD; n = 12) and a
dendritic process that varied in length and could be >40 µm. Some
short dendrites were probably the result of retraction during the
dissociation process. This identification was confirmed by labeling
with anti-OMP antibody (Fig. 2A,B) and by the detection of
action potentials in these cells (see next section). Two typical cells
are shown in Figure 2, C and D, as visualized
with differential interference contrast microscopy. Cells used for
electrophysiology all had a similar appearance, with a clearly
identifiable dendrite bearing microvilli. Mouse olfactory neurons were
also dissociated for comparative experiments, and a typical cell is
shown in Figure 2E at the same magnification. Note that VNO
neurons are several times larger than olfactory neurons.
Action potentials in response to current injections
The electrical properties of VNO neurons were examined by
whole-cell patch-clamp recording. The resting potential
(Vm) of VNO neurons was 58.3 ± 2.7 mV
(mean ± SEM unless otherwise noted; range 43 to 70 mV;
n = 9; Kin2 and
Tyrode's solutions), which was significantly more depolarized than the
resting potential of olfactory neurons measured under similar
conditions (81.8 ± 2.1 mV; n = 6). The input
resistance of 3.3 ± 1.0 G (n = 7) was similar to
that for olfactory neurons (2.9 ± 0.8 G; n = 8),
and the capacitance (8.8 ± 0.3 pF; n = 39) was several
times larger than that of olfactory neurons (1.8 ± 0.2 pF;
n = 7). The responses of a VNO neuron to current
injections of 4 to 4 pA are shown in Figure
3A. The response to hyperpolarizing current
injections gave a membrane time constant of 15.3 ± 2.2 msec
(n = 7). VNO neurons showed a remarkable sensitivity to
small current injections, firing repetitive action potentials with
injections of 1-2 pA. This sensitivity was also observed in
cell-attached patch-clamp recordings, in which we were routinely able
to elicit action potentials in response to depolarization of the
patch.
Fig. 3.
Different firing properties of VNO and olfactory
neurons. Current-clamp recordings from a VNO (A) and an
olfactory (B) neuron in response to current injections of
4 to 8 pA for 2 sec. Note the repetitive firing in the VNO neuron to
current injections of just 1 or 2 pA. The olfactory neurons required
larger current injections to elicit firing, and only fired one or a few
action potentials. For both cells, the internal solution was
Kin2 and the external solution was
Tyrode's. C, Firing frequency of VNO neurons as a function
of current injection for eight cells that fired repetitively (mean ± SEM). The line was fit by eye.
[View Larger Version of this Image (20K GIF file)]
VNO neurons fired repetitively with no sign of adaptation during a 2 sec step (Fig. 2A). This is in contrast to mouse olfactory
neurons, which under these conditions fire only one or a few action
potentials (Fig. 3B). The difference in response between
cell types was consistent across many cells. Of 20 VNO neurons, 14 fired sustained trains of action potentials, whereas only 1 of 8 olfactory neurons displayed such behavior. The firing rate as a
function of current injected is plotted in Figure 3C for
data from eight VNO cells that showed no adaptation to the current
injection. The firing frequency increased linearly for current
injections between 1 and 8 pA and saturated at currents above 20 pA, at
which point the membrane settled into oscillations. The maximal firing
frequency was ~16 Hz. Spontaneous action potentials were rarely
observed at rest; of 20 cells, only 1 spontaneously fired repetitive
action potentials. Similar properties were found for nonenzymatically
prepared cells; input resistance was 8.9 ± 2.9 G (n = 3), Vm was 61 ± 4 (n = 3), and repetitive firing could be elicited by injection of 1-6 pA of
current in 2 of 3 cells. To confirm these results, we performed
perforated patch recording from nonenzymatically prepared cells. No
differences were found (n = 4 cells): input resistance
was 3.1 ± 0.6 G, Vm was 60 ± 4 mV,
three of four cells fired repetitively with current injections of 1-2
pA, and spontaneous activity was observed in one cell.
To understand the firing properties, we compared the voltage-gated
conductances in VNO and olfactory neurons. Figure 4
shows representative current families for two VNO neurons (one prepared
enzymatically and the other prepared nonenzymatically) and two
olfactory neurons, and shows the corresponding I-V plots
measured at the peak of the inward current or the end of the sustained
outward current. The inward current corresponds primarily to the
Na+ current (see next section), and differences
between the inward currents in olfactory and VNO neurons are not
apparent at this resolution. In contrast, clear differences are
apparent between the outward currents, carried by
K+, in the two cell types; the outward current in
the olfactory neurons activates faster and is larger than that of VNO
neurons. These findings prompted a more thorough investigation of
voltage-gated conductances in VNO neurons.
Fig. 4.
Differences in voltage-gated currents between VNO
and olfactory neurons. A, Whole-cell currents in two VNO
neurons, dissociated either enzymatically (top set of
traces) or mechanically (bottom set of traces). Voltage
steps were in 10 mV increments from 70 to +60 mV from a prepulse
potential of 120 mV. I-V curves were plotted from the
family of currents at the top of the panel for the peak
inward current (filled circles) or the sustained
current at the end of the test pulse (open circles).
B, Whole-cell currents in two olfactory neurons were
recorded under identical conditions. I-V curves for the
top set of traces are shown. Note that the outward current
in the olfactory neuron activated more rapidly. All recordings were
with Kin2 in the pipette and
Tyrode's in the bath.
[View Larger Version of this Image (31K GIF file)]
Na+ current
Na+ currents were recorded from
nonenzymatically treated cells under conditions in which
K+ currents were blocked. A family of
Na+ currents in response to step depolarizations
is shown in Figure 5A. The current is carried
almost entirely by Na+ as it was abolished by
replacing external Na+ with
NMDG+ (data not shown). The peak current
I-V curve (Fig. 5B) shows that this current
activates at 50 mV, reaching a maximum at 0 mV. The average magnitude
of the Na+ current at 0 mV was 1272 ± 129 pA
(n = 7).
Fig. 5.
Na+ currents in VNO neurons
A, A family of Na+ currents in
response to voltage steps between 60 and +30 mV from a prepulse
potential of 120 mV recorded with Csin1 in
the pipette and Tyrode's in the bath. B, I-V
curves for the peak inward current from the data shown in A
(closed circles) or in the presence of 10 nM TTX (open circles). C,
Dose-response curve for TTX block of Na+
currents. Na+ currents were evoked by a step to
10 mV from a prepulse potential of 120 (mean ± SEM from 6 cells). Dotted line is the fit to single-site binding
isotherm (Ki = 6.4 nM). A better fit (solid line) is
obtained by assuming that 10% of the current is insensitive to TTX;
the remaining current is fit with a Ki of
3.6 nM. D, On-rates
(filled circles) and off-rates (open
circles) of TTX block. Solid lines are apparent linear
fits from which kon and
koff were calculated. Data were obtained as
shown in the inset for voltage step from 120 to 10 mV;
block of the current and recovery are fitted with single exponential
functions. E, Steady-state inactivation curve; a prepulse
for 300 msec to the voltages shown was followed by a test pulse to 10
mV, and the current elicited was normalized to the maximal current.
Data shown are from a single cell in control solution
(filled circles) or with 30 nM TTX (filled squares) or
from five cells (mean ± SEM; open circles). Lines are
fits to a single Boltzmann equation (dotted and dashed
lines) or the sum of two Boltzmann equations (solid
line). F, Recovery from inactivation at 100 mV
in control solutions (closed circles) or with 10 nM TTX (open circles). Using a
paired-pulse paradigm, the current elicited by the second pulse was
measured and normalized to the first preceding test pulse and plotted
against the interpulse interval. The smooth curves are fits
to a single exponential equation of the form
I/Imax = 1 e(t0
t)/
with values of (
, t0) for control and
TTX experiments of (8.0, 0.28) and (20.2, 0.54), respectively. All data
shown are from nonenzymatically prepared cells.
[View Larger Version of this Image (23K GIF file)]
The Na+ current was sensitive to TTX; 10 nM blocked more than half the current as seen in
the I-V curve of Figure 5B. However, high
concentrations of TTX did not completely abolish the current,
suggesting that a component of the current might be TTX-insensitive.
Figure 5C shows dose-response data obtained from six cells.
The data were poorly fitted by a single binding curve
(Ki of 6.4 nM) and
were better fitted by assuming that some channels were insensitive to
TTX; in this case, the best fit is obtained with a value of
Ki = 3.6 nM for most
of the channels, and a TTX-insensitive component that is 10% of the
total. The data were not sufficient to determine whether the second
component might be sensitive to high concentrations of TTX. To confirm
these results, we measured the on-rate and off-rate for TTX block at
several concentrations (Fig. 5D). A linear fit to the
on-rate gives a value of kon of 2.9 × 106 s
1
M1 (calculated from the inverse of
the slope) and koff of 10 × 103 s1 (the
y-intercept). Separate measurements of
koff at several concentrations, shown in
Figure 5D, gave a similar value. The calculated
Ki
(koff/kon) of
3.4 nM is remarkably similar to the value
obtained from the fit to the dose-response data and supports the
assumption of a TTX-insensitive component of the current.
The kinetic and steady-state properties of the TTX-sensitive and
TTX-insensitive components of the Na+ current
were compared by recording currents in the presence or absence of TTX
(>10 nM). No difference was observed in
activation properties. Both components of current activated at 50 to
40 mV (Fig. 5B, and data not shown). The time to
half-maximal current, a measure of activation kinetics, was 0.62 ± 0.14 msec (n = 5) in the absence of TTX and was not
significantly different in the presence of TTX at 10 nM (0.61 ± 0.13; n = 4), 100 nM (0.3 msec), or 300 nM
(0.5 msec). In contrast, inactivation properties of the two currents
were markedly different. Steady-state inactivation curves measured in
the presence and absence of TTX are shown in Figure 5E. In
the presence of 30 nM TTX, inactivation of the
residual component was shifted by ~30 mV in the hyperpolarizing
direction. A Boltzmann equation fitted to the data gives values for the
midpoint V1/2 = 94 mV and slope
k = 12.8 mV where I/Imax = 1/[1 + exp((V1/2 Vm)/k)]. If TTX-sensitive and
-insensitive currents differ in steady-state inactivation, this should
be evident in the shape of the combined inactivation curve in the
absence of TTX. Indeed, the inactivation curve in the absence of TTX is
not well fitted by a single Boltzmann equation (Fig. 4E);
instead, it is well fitted by the sum of two Boltzmann equations, one
of which has the shifted V1/2 obtained in
the presence of TTX. The fit shown gives a value of
K1/2 for the TTX-sensitive component of
66 mV and slope of 5.9 mV. To confirm the difference in
inactivation properties between the two currents, we measured the time
course of recovery from inactivation using a paired-pulse paradigm.
Figure 5F shows that at 100 mV, recovery from inactivation
of the combined current could be fitted with a single exponential with
a time constant of 6.7 msec (mean = 9.1 ± 0.9 msec;
n = 4). In the presence of 10 nM
TTX, recovery of the current was slower and was fitted with a time
constant of 20 msec. Similar results were obtained in the presence of
30 nM TTX (
= 30 msec).
These results are most readily explained by a difference in
inactivation properties of the TTX-sensitive and -insensitive
components; for the TTX-insensitive component, the inactivation state
of the channel is stabilized relative to other states of the channel,
perhaps by a higher affinity binding of the inactivating particle. An
alternative explanation is that TTX alters the apparent inactivation
properties of the TTX-sensitive channel via a state-dependent binding
mechanism (Cohen et al., 1981; Bean et al., 1983). Although this can
account for a hyperpolarizing shift in the inactivation curve obtained
at concentrations at which the sensitive component is not entirely
blocked, the poor fit by a single Boltzmann equation of the
inactivation curve in the absence of TTX argues against this
mechanism.
Na+ currents were also recorded from
enzymatically treated cells, and similar values were obtained for
gating properties, including the voltage at which currents were
activated, the midpoint of inactivation, and the time constant for
recovery from inactivation. In these cells, however, TTX sensitivity
appeared to be diminished, and data from these experiments was,
therefore, not included in the analysis.
A slowly activating K+ current
Outward K+ currents in response to step
depolarizations of short and long duration are shown in Figure
6, A and B; these were typical of
the currents seen in most VNO neurons, either enzymatically or
nonenzymatically prepared. Measurements of the reversal potential of
tail currents in 5 mM K+
and 20 mM K+ confirmed that
the outward conductance is K+-selective (data not
shown). The I-V curve in Figure 6C for the
family of traces in Figure 6A shows that the current begins
activating at potentials above 30 mV. This is also seen in an
activation curve plotted from isochronal tail current measurements at
120 mV after voltage steps to varying potentials (Fig.
6D). The activation curve was relatively shallow and was fit
with a Boltzmann equation with V1/2 = 11.5 mV and K = 16 mV. During a prolonged
depolarization lasting 50 sec, the K+ current
displayed only slow inactivation; the time course of inactivation could
be fitted with the sum of two exponential functions with time constants
of 2.4 sec (67%) and 49 sec (33%; Fig. 6B). This is
consistent with either the presence of at least two different channel
species that inactivate at different rates or with a single channel
type that has multiple open or inactivated states.
Fig. 6.
Outward K+ current.
A, A family of K+ currents in response
to step depolarizations between 30 and +80 mV from a prepulse
potential of 120 mV. B, Time course of inactivation of the
K+ current from a nonenzymatically prepared cell
(+60 mV). The decay of the current was fit (solid line) with
the sum of two exponential functions with time constants of 2.4 sec
(67%) and 49 sec. C, I-V curve for currents in
A measured at 100 msec. D, Activation curve,
measured from the amplitude of the tail current at 120 mV after a 100 msec depolarizing step to the potentials indicated (filled
circles). The data were fitted by a Boltzmann equation with
V1/2 = 9.2 mV and K = 18 mV. E, Time to half activation measured from enzymatically
prepared VNO neurons (solid circles; n = 7),
nonenzymatically prepared VNO neurons (solid squares;
n = 5), and olfactory neurons (open circles;
n = 5). Note that the y-axis is a
logarithmic scale. F, I-V curves, measured as in
B in the presence of 2 mM
Ca2+ (control, open circles) in
nominally Ca2+-free solution (with
Mg2+ replacing Ca2+,
closed circles) and in the presence of 10 mM TEA (diamonds). Solutions for most
recordings were Kin4 and
Kout2 + TTX (10 µM).
[View Larger Version of this Image (21K GIF file)]
The activation of K+ currents was relatively slow
at all potentials. This was quantified by measuring the time to
half-maximal activation as a function of voltage (Fig. 6E).
At +40 mV, the current reached half activation in 12.0 ± 1.3 msec
(n = 7) in enzymatically prepared cells and was not
significantly different in nonenzymatically prepared cells (8.5 ± 1.0 msec; n = 5). The VNO K+ current
activated an order of magnitude slower than the outward current in
mouse olfactory neurons, which reaches half activation in 1.3 ± 0.1 msec (n = 5; Fig. 6E). Our value is similar
to that previously reported for rat olfactory neurons (1.5 msec; Lynch
and Barry, 1991). Because the time it takes for an action potential to
go from 0 to +40 mV in VNO cells is only 1-3 msec, only a fraction of
the K+ current would be activated during an
action potential.
Little if any of the macroscopic K+ current
could be attributed to Ca2+-activated
K+ channels. This was assessed either by removing
Ca2+ in the external bath (and replacing it with
Mg2+) or by adding 100 mM
Cd2+ or 2 mM
Co2+ to the bath. The currents in Figure
6F are representative of the majority of cells; the
I-V curve does not show the ``N'' shape characteristic of
Ca2+ activated K+ currents,
and removal of Ca2+ actually enhanced the current
(most likely because of surface-charge effects). In this cell, the
absence of a Ca2+-activated
K+ current could not be attributed to rundown of
Ca2+ channels, because a robust
Ca2+ current could be elicited in the presence of
20 Ba2+ at the end of the experiment. Of 14 cells
tested, 13 showed little evidence of a
Ca2+-activated K+ current.
Removal of Ca2+ caused a 0.7 ± 5% increase in
the current (n = 5; +30 mV). Cd2+
at 100 mM, a concentration that was effective at
completely abolishing the Ca2+ current (next
section), had little effect on the K+ currents,
with only 7.2 ± 3.5% block (n = 7; +30 mV). This
small amount of block is most likely attributable to nonspecific
effects, as it was similar at all potentials. On the other hand, the
K+ current was sensitive to block by 10 mM tetraethylammonium (78 ± 2% block,
n = 7; +30 mV) and 5 mM
4-aminopyridine (40 ± 8% block; n = 2; +30 mV).
An inward-rectifier potassium current
We determined the magnitude of the inward-rectifier
K+ current with hyperpolarizing ramps in the
presence of elevated extracellular K+. In these
experiments, equimolar Na+ was replaced by
K+ in the external solution, and
Na+ channels were blocked with 5 mM TTX. Voltage ramps in the presence and absence
of 20 mM K+ showed that a
K+ conductance was present at voltages more
negative than 50 mV (Fig. 7A). The
difference current is shown in the bottom panel. The current was
roughly linear over the voltage range of 120 to 50 mV, and the mean
slope conductance for four cells was 0.75 ± 0.19 nS (n = 4). The extrapolated reversal of 44.4 ± 2.5 mV (n = 4) is closed to the Nernst potential of 50 mV predicted for a
K+-selective conductance.
Fig. 7.
An inward-rectifier K+
current. A, Response to voltage ramps (0.88 V/sec) in the
absence of external K+ and with 20 mM K+ replacing
Na+ in the bath solution. The difference curve
shows that the conductance carried by K+
activates at close to the reversal potential for
K+ (approximately 50 mV) and is linear between
80 and 50 mV. B, Voltage steps in the presence and
absence of 20 mM external
K+. The inward K+
conductance activates with depolarizing voltage as seen by the tail
currents at repolarization to 120 mV. Plotted below is a steady-state
I-V curve measured at the end of a 100 msec test pulse.
Note that the current saturates below 120 mV. External solution was
Naout1 or Kout4, and
internal solution was Kin3.
[View Larger Version of this Image (20K GIF file)]
The inward K+ current, examined with step
hyperpolarizations, showed evidence of deactivation at negative
potentials (Fig. 7B). During a hyperpolarizing step from
120 to 140 mV, the current relaxed to a smaller value, whereas
depolarizing steps led to activation of the current as seen in the
increase in the currents at repolarization to 120 mV. This is also
apparent in the I-V curve measured at the end of the test
pulse (Fig. 7B), which shows a saturation in the size of the
currents below 120 mV. The small magnitude of the inward-rectifier
current (21.6 ± 4.9 pA at 78 mV in 20 mM
K+; n = 5) is consistent with the
high input resistance of these cells and might be sufficient to
maintain the resting potential. [Since submission of this manuscript,
a report has appeared showing that the resting potential in frog VNO
neurons is set by the balance between a hyperpolarizing sodium pump and
a slowly activating, depolarizing cation conductance,
Ih; (Trotier and Doving, 1996). In
perforated patch recording from nonenzymatically prepared mouse VNO
neurons, we have also observed a current that, based on kinetics, is
likely to be Ih.]
Two components of Ca2+ current
Ca2+ currents recorded with 20 Ba2+ in the external solution are shown in Figure
8A. At this slow time scale, two
components of current are apparent. A transient component activated at
depolarization to 60 mV and reached a peak at 20 mV. Inactivation
of this current proceeded to near completion with a time constant 38 ± 4 msec (measured at 10 mV; n = 9). A second,
sustained component activated at 10 mV and reached maximal
conductance at +20 mV (Fig. 8A, bottom
panel). I-V relations shown in Figure 8B
were measured from the traces in Figure 8A either
at the peak of the current or late in the voltage step (at the times
indicated by the filled circle and square in Fig.
8A). The bimodal I-V relationship for
the late current (squares) is indicative of two components
of current; at negative potentials, the current is carried by the
transient component (that is not fully inactivated), whereas at
positive potentials it is carried by the sustained component. The
I-V relationship of the peak current at negative potentials
(below 10 mV) reflects the activation of the transient component
alone, whereas at positive potentials it reflects the activation of
both components. Both components were blocked by the addition of
Cd2+ (100 µM), which
abolished 92.6 ± 2.2% (n = 4) of the current
(measured at +10 mV). In three nonenzymatically prepared cells, similar
currents were observed, and the time constant for decay of the
transient component at 10 mV (32 ± 6 msec; n = 3)
was similar to the value obtained from enzymatically prepared cells
(see above).
Fig. 8.
Two components of Ca2+
current. A, A family of Ca2+ currents
recorded with 20 Ba2+ as the charge carrier.
Currents in response to depolarizations from a prepulse potential of
100 mV to 60 to 20 mV in 10 mV steps are shown in the top
panel. Currents in response to depolarizations to 10 to +70 mV
in 20 mV steps from same prepulse potential are shown in the
bottom panel. Two components of current are apparent in
these traces, a transient current that is activated at low voltages and
a sustained current that is activated at high voltages. B,
I-V curves from the family of currents in A
showing the peak current (filled circles) and the
sustained current (filled squares) measured at the
times indicated in A by the same symbols. The open
diamonds are the current calculated by multiplying the peak
current by the fractional inactivation at that potential (from
C). C, Steady-state inactivation curve
measured as the peak current elicited by a step depolarization to 10
mV after a 500 msec prepulse to varying potentials. The solid
line is a fit to the Boltzmann equation with
V1/2 = 54 mV and K = 8
mV. D, The amplitude of the sustained component of
Ca2+ current plotted against the transient
component (at 10 mV) for individual cells. No correlation was
apparent. E, Block of transient and sustained components by
100 µM Ni2+,
10 µM Nitrendipine (DHP), and 200 nM w-aga-IVa. The transient and
sustained components were measured at the peak and at the end of the
pulse for 100 msec voltage steps from 80 to 0 mV. The peak current
should be the sum of both transient and sustained components, whereas
the current at the end of the pulse will mainly represent the sustained
component. For all measurements of Ca2+ currents,
the internal solution was Csin3 or
Csin4, and external solution was
Baout.
[View Larger Version of this Image (21K GIF file)]
To characterize the transient component further, we measured the
voltage dependence of inactivation (Fig. 8C). A fit to the
Boltzmann equation gives values of V1/2 = 50.0 ± 1.2 mV (n = 3) and K = 8.5 ± 0.1 mV (n = 3). Multiplying the inactivation curve
by the peak current gives the values plotted in Figure 8B
(open diamonds), which are nearly identical to those of the
late current at these potentials, confirming that this part of the late
current is the same as the noninactivated portion of the transient
component. On average, the magnitude of the peak transient current was
169 ± 13 pA at 10 mV (n = 25), and the magnitude of
the sustained component at the same potential was 134 ± 24 (n = 25; 10 mV). The two components, plotted against
each other for individual cells (Fig. 8D), were seen to vary
independently, and distinct populations of cells expressing one
component or the other were not observed. The sustained component
displayed more variability and was also more labile.
The pharmacology of both components was examined (Fig. 8E).
The transient component was insensitive to -conotoxin (3 µM; 2.7 ± 7.8% increase; n = 3) and -aga-IVA (200 nM; 1.3 ± 2.3%
increase; n = 3), but was blocked by
Ni2+ (100 µM; 74 ± 1%
decrease; n = 5). The sustained component was partially
blocked by dihydropyridines (37 ± 3% block by 10 µM nitrendipine, n = 5; 62 ± 11% block by 1 µM nifedipine,
n = 3), partially blocked by -conotoxin GVIA (3 µM; 21 ± 3.6% decrease, n = 3), and was relatively insensitive to -aga IV (200 nM; 7 ± 4% decrease, n = 3).
The kinetics and pharmacology of the transient component are consistent
with its identification as T-type current, whereas the sustained
component appears to be composed mainly of L-type, and perhaps some
N-type current.
CNG channels
We used a variety of approaches to try to detect a CNG conductance
in VNO neurons. Based on the properties of vertebrate and invertebrate
olfactory, retinal, and gustatory CNG channels (Michel and Ache, 1992;
Gomez and Nasi, 1995; Kolesnikov and Margolskee, 1995; Yau and Chen,
1995), we assumed that CNG channels, if present in the VNO, would be
localized on microvilli, be permeant to either
Na+ or K+ or both, and be
blocked by divalent cations. The most straightforward approach was to
excise patches under divalent-free conditions and to expose the
intracellular surface to cyclic nucleotide. Because the extremely small
size of the microvilli precluded patch-clamp recording, we instead
obtained excised patches from the dendritic knob. In olfactory neurons,
the corresponding region has a relatively high density of CNG channels
(Frings et al., 1992).
In 17 patches excised from the dendritic knob of enzymatically prepared
VNO neurons and in 6 patches excised from nonenzymatically prepared
cells, we detected no response to saturating concentrations of cAMP or
cGMP (0.5-1.0 mM), as measured with ramp
depolarizations from 80 to +80 mV. In many of these patches, square
openings of single voltage-activated K+ or
Na+ channels were seen, indicating that the
cytoplasmic surface of the patch was not obstructed. In all
experiments, the patch pipette contained a low-divalent
Na+ solution (Naout3),
and the bath contained divalent-free Na+ solution
(6 experiments; Napatch) or
K+ solution (17 experiments;
Kpatch).
In olfactory neurons, in contrast, we obtained responses in three out
of four patches from the dendritic knob of enzymatically prepared cells
and four out of five patches from nonenzymatically prepared cells (Fig.
9B). The response to saturating
concentrations of cAMP or 8-CPT-cAMP (0.5-1 mM)
averaged 12 ± 5 (n = 3) and 20 pA ± 12 pA
(n = 4) in patches from enyzmatically and
nonenzymatically prepared cells, respectively (measured at 70 mV;
Naout3 and Napatch
solutions). Responses of similar magnitude were obtained with cGMP or
8-Br-cGMP (0.5-1 mM).
Fig. 9.
Response of VNO and olfactory neurons to cyclic
nucleotides. A, Holding current for a VNO neuron exposed to
0.5 mM 8-Br-cGMP in whole-cell recording at 80
mV. Responses to ramp depolarizations (0.22 V/sec) are shown below for
time points before and during 8-Br-cGMP exposure, as indicated by the
arrows in the time course. Recordings were performed with
Csin3 solution in the pipette and low
divalent (Naout2) external solution. No change
in the current was observed, even though a change of only 1 pA would
have been detected. B, Current at 80 mV in an excised
patch from the dendritic knob of an olfactory neuron exposed to the
same solution of 0.5 mM 8-Br-cGMP. A large
current was evoked that declined rapidly after removal of 8-Br-cGMP.
Shown below are responses to ramp depolarizations (1.3 V/sec) before and at the peak of the response (indicated by the
arrows). Solutions were Csin3 in the
pipette and low divalent (Naout2) in the
bath.
[View Larger Version of this Image (24K GIF file)]
The failure to observe a response to cyclic nucleotides in excised
patches from VNO neurons might be attributed to a low density of
channels, consistent with the extremely high sensitivity of VNO cells
to current injections. To improve our ability to detect a CNG
conductance, we used whole-cell recording. In one series of
experiments, cells were dialyzed with cAMP or 8-Br-cGMP, a treatment
that elicits several hundred picoamperes of current in olfactory
neurons. Solution exchange between the patch pipette and the cell
occurred within seconds of patch rupture, as monitored by loading the
pipette with high Na+ solution
(Nain) and observing the change in the reversal
potential of the Na+ current. The diffusion of
cyclic nucleotide at high concentrations is expected to be only 2-3
times slower than that of Na+ (Pusch and Neher,
1988). Thus, at the time when dialysis with Na+
is 95% complete, the cyclic nucleotide will be at >50% of its final
concentration. With 0.5 mM cAMP or cGMP in the
pipette, this gives a concentration of 0.25 mM in
the cell, which is a saturating concentration for most CNG channels.
Because dialysis with Na+ was faster than we
could measure (<5 sec), we slowed the onset of dialysis in some
experiments by loading the tip of the pipette with
Cs+-containing solution. In these experiments, we
found no evidence for an increased conductance during the time course
of the dialysis. Measuring the current at 120 mV at a time well after
dialysis was complete (130-800 sec), we found an increase of only 2 ± 10 pA with 0.5 mM 8-Br-cGMP (n = 3) and 0.5 ± 1.2 pA with 0.5 mM cAMP
(n = 3). The negligible size of the induced current
under conditions of high concentrations of cyclic nucleotide indicates
that CNG channels are unlikely to be present or to mediate
transduction.
In a second series of experiments, we measured the response of
whole-cell currents to bath perfusion of membrane-permeant analogs of
cAMP or cGMP. To enhance the likelihood of seeing a response, we used
two different conditions: a high K+ (20 mM) external solution to reveal a
K+-selective conductance or a low divalent
external solution to unblock channels. In neither condition did we see
any significant response. With 20 mM
K+ and 125 mM
Na+ in the bath (Kout4),
an inward-rectifier current was evident at 78 mV, but the magnitude
of the current, 20 ± 4 pA (n = 4), was unaffected by
constant perfusion for >1 min with 1 mM
8-Br-cGMP (20 ± 5 pA; n = 4). In the presence of low
divalent external solution, the holding current at 78 mV was 14 ± 4 pA (n = 4), and no significant increase was seen
after exposure to 0.5 mM 8-Br-cGMP (15 pA ± 4, n = 4). Figure 9A shows the results of
one experiment performed under these conditions. No change in the
whole-cell current was observed in response to application of
8-Br-cGMP, although an increase in the current of only 1 pA would have
been detectable. As a control, an excised patch from an olfactory
neuron was exposed to the same solution of 8-Br-cGMP; it gave a
response of 10-20 pA (Fig. 9B). Finally, to test for the
unlikely possibility that a CNG channel was not detected because of
``rundown'' from internal perfusion of the cell or patch, we recorded
responses in perforated-patch mode. In current-clamp mode, in which
1-2 pA was sufficient to elicit action potentials, no activity was
elicited by bath application for several minutes of 8-CPT-cAMP (0.5 or
1 mM; n = 2) or 8-Br-cGMP (1 mM; n = 2).
DISCUSSION
High sensitivity and temporal integration in single
sensory neurons
Mouse VNO neurons, like mouse olfactory neurons (Maue and Dionne,
1987; Lynch and Barry, 1989), fired action potentials to current
injections of just 1-2 pA. This remarkable sensitivity is attributable
in part to the high input resistance of the cell membrane of olfactory
and VNO neurons. Our measurement of ~3 G, on average,
underestimates the real membrane resistance (Schild, 1989). A better
estimate is the highest input resistances that we measured, 8 G
(from enzymatically prepared cells) and 16 G (from nonenzymatically
prepared cells), which are similar to values reported for olfactory
neurons (Trotier, 1986; Firestein and Werblin, 1987; Lynch and Barry,
1989, 1991). A current injection of 0.5-1 pA thus could cause a
depolarization of 8-16 mV, bringing Vm to
50 to 40 mV, which is just the voltage range over which the
Na+ current is steeply activated. The sensitivity
of VNO neurons may be enhanced by a resting potential (approximately
60 mV) that is close to the firing threshold, and by the contribution
of the transient Ca2+ current that is activated
close to the resting potential.
We found that mouse VNO neurons fire tonically to maintained
current injections of as little as 1 pA, without sign of adaptation.
Under similar conditions of whole-cell recording, rodent and amphibian
olfactory neurons fire only a single or short burst of action
potentials, and repetitive firing can only be elicited with a pulsitile
current injection protocol (Trombley and Westbrook, 1991). With
perforated patch recording, sustained firing of olfactory neurons has
been observed, but only at higher current levels (5-8 pA, which is
near saturation for VNO neurons) (Leinders-Zufall et al., 1995). The
sustained firing of VNO neurons would allow temporal integration,
enhancing sensitivity in the detection of small signals
perhaps even
the binding of single pheromone/odorant molecules. This higher
sensitivity may be unnecessary for the olfactory system, which may
achieve additional sensitivity by the large numbers of sensory neurons
and the enormous degree of convergence in the olfactory bulb (Kauer,
1986; Ressler et al., 1994; Vassar et al., 1994). Instead, olfactory
neurons may be optimized to detect changes in the environment, showing
adaptation to sensory input at several different levels.
It is not known whether sensory transduction results in an increase or
a decrease in the activity of VNO neurons. In reptiles, stimuli
detected by the VNO generally cause an increase in activity in the
accessory olfactory bulb (Tucker, 1971; Jiang et al., 1990); in
mammals, similar experiments have been more difficult. Our data are
consistent with either possibility. Most VNO neurons did not fire
spontaneously, suggesting that information might be encoded by an
increase in firing frequency. However, our results also suggest that
VNO neurons could fire tonically in vivo if a small inward
current was activated at rest, and sensory transduction might, then,
reduce firing frequency.
Contribution of voltage-activated currents to electrical
response properties
The Na+ current in mouse VNO neurons
consists of a major component that is TTX-sensitive and a minor
component that is TTX-insensitive. These currents are similar in
inactivation properties and pharmacology to two
Na+ currents, TTX-sensitive and TTX-insensitive,
described in rat olfactory neurons. However, in rat olfactory neurons
both currents have not been shown to coexist in the same cell; the
TTX-insensitive current is seen in freshly dissociated rat olfactory
neurons (Rajendra et al., 1992), whereas the TTX-sensitive current is
seen in cultured rat olfactory neurons (Trombley and Westbrook, 1991).
In both VNO and olfactory neurons, the TTX-sensitive
Na+ current inactivates at more depolarized
potentials (midpoint is approximately 65 mV for both VNO and
olfactory neurons) than the TTX-insensitive Na+
current (midpoint is approximately 94 mV and 110 mV in VNO and
olfactory neurons, respectively). The response properties of VNO
neurons will be dominated by the larger TTX-sensitive
Na+ current, whereas the response properties of
rodent olfactory neurons may be dominated by the TTX-insensitive
current. The greater availability of the TTX-sensitive channel at rest,
and faster recovery from inactivation, may enhance the excitability of
VNO neurons relative to olfactory neurons.
The outward K+ current in mouse VNO neurons was
carried predominantly by slowly inactivating delayed-rectifier-type
channels. This current was an order of magnitude slower in activation
kinetics than the K+ current in olfactory
neurons. Because the action potential in VNO neurons goes from 0 to +40
mV in 1-3 msec, little of the K+ current will be
activated; this may allow the cell to respond more quickly to sustained
current with additional action potentials. The negligible inactivation
of the K+ current may allow repetitively firing
without adaptation; this is in contrast to the K+
current in freshly dissociated rat olfactory neurons, which inactivates
with = 22 and 143 msec, (Lynch and Barry, 1991), and thus may
inactivate sufficiently during a maintained current injection to
prevent repolarization of the action potential and repriming of
Na+ channels.
The two components of Ca2+ current in VNO
neurons have properties similar to those of T-type and L-type
Ca2+ channels described in other cell types
(Tsien et al., 1987; Bean, 1989; Hess, 1990). In contrast, only L-type
Ca2+ current has been detected in olfactory
neurons (Trotier, 1986; Firestein and Werblin, 1987; Schild, 1989;
Trombley and Westbrook, 1991; Nevitt and Moody, 1992). Although not
well characterized, the Ca2+ current in frog VNO
neurons also appears to contain a component of T-type current (Trotier
et al., 1993). The presence of T-type current in VNO and not in
olfactory neurons indicates that it may play a role in defining the
distinct electrical properties of VNO neurons. T-type current is
activated close to the resting potential of VNO neurons (approximately
60 mV); thus, it may lower the threshold for initiation of an action
potential (Hagiwara et al., 1988).
CNG channels may not mediate transduction
The transduction of chemical into electrical signals in many
vertebrate and invertebrate sensory systems, including olfactory,
visual, and gustatory systems (Fesenko et al., 1985; Nakamura and Gold,
1987; Michel and Ache, 1992; Kolesnikov and Margolskee, 1995; Gomez and
Nasi, 1995), is accomplished by the gating of a CNG channel. Our
experiments suggest that this may not be true for transduction in the
VNO. We have been unable to detect the activation of either a
Na+ or a K+ current in
response to either cAMP or cGMP using a variety of approaches. These
included excised patch recording in which olfactory neurons produced
tens of picoamperes of current, and whole-cell recording, in which
olfactory neurons reportedly produce currents of 100-400 pA
(Kurahashi, 1990; Firestein et al., 1991a). Our results differ from a
recent report that described a CNG current in turtle VNO neurons
(Taniguchi et al., 1996). This may represent a species difference,
because in turtle the VNO is not sequestered from the main OE.
These physiological results are consistent with recent molecular
investigation of transduction in the VNO. Most of the known components
of transduction in the olfactory system are not expressed in the VNO,
including the family of odorant receptors, the G-protein
(Golf), adenylyl cyclase III, and a CNG
channel (rOCNC1; Dulac and Axel, 1995; Berghard et al., 1996). The only
transduction component that is expressed in both cell types is a second
(or 
) subunit of the CNG channel (rOCNC2) that does not form a
functional channel when expressed alone in heterologous cell types
(Bradley et al., 1994; Liman and Buck, 1994). Our current results
suggest that in VNO neurons, as well, this subunit does not form a
functional CNG channel. An unlikely possibility that we cannot rule out
is that this subunit forms a channel gated by another second messenger
or with a very unusual ion selectivity. Alternatively, if the
expression of rOCNC2 is without functional consequence, the detection
of rOCNC2 in the VNO may be the result of a transcriptional control
mechanism that has not evolved to be tightly regulated.
Although the mechanism of sensory transduction in the VNO remains
obscure, recent evidence suggests that it is likely to involve
G-proteins and G-protein-coupled-receptors. First, studies in snake
have shown that pheromone binding to VNO membranes is GTP-dependent
(Luo et al., 1994). Second, two G-proteins
(Gi2 and Go) are
found at high levels in the vomeronasal system (Shinohara et al., 1992;
Luo et al., 1994; Halpern et al., 1995; Berghard and Buck, 1996).
Third, Dulac and Axel (1995) have recently identified a family of
VNO-specific seven-transmembrane-receptors that are putative pheromone
receptors. How activation of pheromone receptors and G-proteins affects
the electrical excitability of VNO neurons remains to be elucidated.
The characterization of ion channels that we have conducted lays the
foundations for such an endeavor.
FOOTNOTES
Received March 18, 1996; revised May 1, 1996; accepted May 2, 1996.
This work was supported by a grant from the National Institutes of
Health (R03 DC 02889-01) to E.R.L. and by the Howard Hughes Medical
Institute. E.R.L. is an Associate and D.P.C. is an Investigator of the
Howard Hughes Medical Institute. We thank Don Arnold, Steve Cannon,
Jeff Holt, and Jim Morrill for helpful discussions.
Correspondence should be addressed to Emily R. Liman, Wellman 414, Massachusetts General Hospital, 50 Blossom Street, Boston, MA
02114.
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[ISI][Medline]
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