1Neuroscience Program and 2Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118
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ABSTRACT |
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Luther, Jason A.,
Katalin
Cs. Halmos, and
Jeffrey G. Tasker.
A Slow Transient Potassium Current Expressed in a Subset of
Neurosecretory Neurons of the Hypothalamic Paraventricular Nucleus.
J. Neurophysiol. 84: 1814-1825, 2000.
Type I putative magnocellular neurosecretory cells of the
hypothalamic paraventricular nucleus (PVN) express a prominent
transient outward rectification generated by an A-type potassium
current. Described here is a slow transient outward current that alters cell excitability and firing frequency in a subset of type I PVN neurons (38%). Unlike most of the type I neurons (62%), the transient outward current in these cells was composed of two kinetically separable current components, a fast activating, fast inactivating component, resembling an A-type potassium current, and a slowly activating [10-90% rise time: 20.4 ± 12.8 (SE) ms],
slowly inactivating component (time constant of inactivation: = 239.0 ± 66.1 ms). The voltage dependence of activation and
inactivation and the sensitivity to block by 4-aminopyridine (5 mM) and
tetraethylammonium chloride (10 mM) of the fast and slow components
were similar. Compared to the other type I neurons, the neurons that
expressed the slow transient outward current were less excitable when
hyperpolarized, requiring larger current injections to elicit an action
potential (58.5 ± 13.2 vs. 15.4 ± 2.4 pA; 250-ms duration;
P < 0.01), displaying a longer delay to the first
spike (184.9 ± 15.7 vs. 89.7 ± 8.8 ms with 250- to
1,000-ms, 50-pA current pulses; P < 0.01), and firing
at a lower frequency (18.7 ± 4.6 vs. 37.0 ± 5.5 Hz with 100-pA current injections; P < 0.05). These data
suggest that a distinct subset of type I PVN neurons express a novel
slow transient outward current that leads to a lower excitability.
Based on double labeling following retrograde transport of systemically
administered fluoro-gold and intracellular injection of biocytin, these
cells are neurosecretory and are similar morphologically to
magnocellular neurosecretory cells, although it remains to be
determined whether they are magnocellular neurons.
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INTRODUCTION |
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The hypothalamic paraventricular
nucleus (PVN) consists predominantly of three groups of cells,
magnocellular neurosecretory cells, which regulate hormone secretion
from the posterior pituitary gland, parvocellular neurosecretory cells,
which regulate hormone secretion from the anterior pituitary gland, and
parvocellular preautonomic cells, which regulate autonomic nervous
system outflow (Liposits 1993; Swanson and
Sawchenko 1983
).
Previous studies have shown that PVN neurons can be divided into two
subtypes on the basis of distinct electrophysiological properties. Type
I neurons express a transient outward rectification (Tasker and
Dudek 1991), caused by a transient A-type potassium current
(IA) (Luther and Tasker
2000
), and have been identified as putative magnocellular
neurosecretory cells (Hoffman et al. 1991
). Type II
neurons do not express any detectable transient outward rectification
and show a small IA, and some cells
generate a variable low-threshold spike caused by a T-type calcium
current (Luther and Tasker 2000
; Tasker and Dudek
1991
). Type II neurons have been identified as putative
parvocellular neurosecretory and preautonomic cells (Hoffman et
al. 1991
; Tasker and Dudek 1991
).
The temporal firing patterns of neurosecretory cells have a profound
impact on hormone release (Bicknell and Leng 1981;
Dutton and Dyball 1979
). Under stimulated conditions,
vasopressin-secreting magnocellular neurons adopt a spiking pattern
characterized by alternating periods of activity and quiescence, termed
phasic firing (Wakerley et al. 1978
). The phasic firing
pattern causes a greater amount of vasopressin to be released per
action potential than a continuous train with the same overall
frequency (Bicknell and Leng 1981
; Dutton and
Dyball 1979
). Oxytocinergic magnocellular neurons fire in an
intermittent bursting pattern that is synchronized across neurons to
control milk ejection during suckling and uterine contractions during
child birth (Lincoln and Wakerly 1974
; Summerlee and Lincoln 1981
). The amount of oxytocin released per action potential is also increased during burst firing (Bicknell et al. 1982
).
The transient A-type potassium current
(IA) is implicated in the regulation
of temporal firing patterns in many systems through its effects on the
inter-spike interval, action potential repolarization, and postsynaptic
responsiveness (Magee et al. 1998; Rogawski
1985
; Rudy 1988
). Evidence suggests that the
IA may be important in determining the
excitability of magnocellular neurosecretory neurons and therefore may
influence the repetitive firing properties of these cells.
Fisher et al. (1998)
showed that the current density of
the IA was higher in vasopressinergic
than oxytocinergic magnocellular neurons of the supraoptic nucleus and
that the vasopressinergic neurons were less excitable, exhibiting a
longer delay to the first spike when depolarized from potentials
negative to resting membrane potential. Alonso and Widmer
(1997)
reported that Kv 4.2 potassium channel subunits, which
comprise IA ion channels, were
concentrated near synaptic contacts in magnocellular neurons of the
supraoptic nucleus, suggesting that this current might be involved in
the integration of synaptic input.
Like magnocellular neurons of the supraoptic nucleus (Bourque
1988), type I PVN neurons generate a large
IA (Li and Ferguson 1996
; Luther and Tasker 2000
). We combined whole
cell voltage- and current-clamp experiments to determine the
voltage-dependent currents responsible for the lower excitability and
distinct electrophysiological properties observed in a subset of type I
PVN neurons (Luther and Tasker 1998
).
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METHODS |
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Hypothalamic slice preparation
Male Sprague-Dawley rats (21-40 days old; Charles River
Laboratories, Raleigh, NC) were deeply anesthetized with 50 mg/kg ip
pentobarbital sodium (Abbott Laboratories, North Chicago, IL) and
decapitated with a rodent guillotine. The brain was removed and placed
into ice-cold (1°C) artificial cerebrospinal fluid (ACSF),
consisting (in mM) of 140 NaCl, 3 KCl, 1.3 MgSO4,
1.4 NaH2PO4, 11 D-glucose, 5 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 2.4 CaCl2, and 3.25 NaOH, pH was
7.3; osmolarity was 290-300 mOsm, and it was bubbled with 100%
O2. A 1-cm3 block of
hypothalamus was isolated from the rest of the brain by making razor
cuts rostral to the optic chiasm, caudal to the median eminence, dorsal
to the third ventricle, and lateral to the fornix. The block was glued
to the chuck of a vibrating tissue slicer (World Precision Instruments,
Sarasota, FL) and three 400-µm slices containing the PVN were
sectioned in ice-cold, oxygenated ACSF. The slices were equilibrated
for 1-2 h at room temperature in a tissue-storage chamber containing
ACSF saturated with 100% O2. Slices were
bisected along the third ventricle, and a single hemi-slice at a time
was placed on the ramp of an interface recording chamber and superfused
with oxygenated ACSF maintained at 29-30°C. The other slices were
kept in the storage chamber until needed.
Electrophysiology
Patch electrodes were pulled in multiple stages on a
Flaming-Brown P-97 horizontal puller (Sutter Instruments, Novato, CA) from borosilicate glass (1.2 mm ID, 1.65 mm OD, type KG-33, Garner Glass, Claremont, CA) to a resistance of 2-4 M. They were filled with a solution consisting (in mM) of 130 potassium gluconate, 10 HEPES, 1 NaCl, 1 CaCl2, 10 ethyleneglycol-bis-(
-amino ethyl ether)-N,N,
N',N'-tetraacetic acid (EGTA), 1 MgCl2, 2 ATP (magnesium salt), and 0.5 GTP (sodium salt); pH was adjusted to 7.2 with KOH. Biocytin (0.3%, Molecular Probes, Eugene, OR) was added as an intracellular marker. The osmolarity of the internal solution was
increased to 300-305 mOsm with D-sorbitol, which was found to decrease the series resistance of recordings without eliciting a
significant osmotic response from the neurons. Slices were
transilluminated and visualized under a dissecting microscope.
Electrodes were positioned in the PVN under visual control and advanced
through the tissue using a piezoelectric step motor (Nano-Stepper type B, Adams and List Associates, Westbury, NY). The liquid junction potential (11 mV) was corrected for according to Neher
(1992)
. Cells were discarded if they did not meet the following
criteria: action potentials
50 mV from threshold to peak, input
resistance (near resting potential)
500 M
, and resting potential
equal to or negative to
50 mV when the cell was not spontaneously
active. Series resistance compensation of
80% was routinely
employed, and changes in series resistance were monitored and
compensated for during the course of experiments. The average series
resistance at the beginning of recordings was 9.9 ± 0.5 M
, and
the average voltage error for the transient outward current was
2.2 ± 0.2 mV with pulses to
25 mV. The quality of the space
clamp was assessed by measuring the rate of activation and inactivation
of the IA activated by stepping from
progressively more hyperpolarized conditioning steps to a constant test
step. The rates of activation and inactivation of the
IA are voltage dependent and should
vary as a function of test step potential, whereas varying the
conditioning step should only affect the amplitude of the current
(i.e., more negative conditioning steps remove more inactivation and
elicit larger currents). Cells in which the activation or inactivation
rate of the current changed with the conditioning step amplitude were considered not under voltage control and were not included for analysis. All voltage-clamp traces represent an average of at least
three separate trials. Transient outward current traces were leak
subtracted by digitally subtracting the current elicited from a
conditioning step at which the transient outward current was completely
inactivated (
50 mV), which removed delayed rectifier, leak, and
capacitative currents. Recordings were made using an Axopatch 1-D
amplifier (Axon Instruments, Foster City, CA). Data were low-pass
filtered at 2 kHz with the amplifier and sampled at 5-10 kHz using
pClamp 6 data-acquisition and analysis software (Axon Instruments).
Data were recorded onto VHS video tapes using a Neuro-corder DR-484
digitizing unit (NeuroData Instruments, New York, NY). Selected traces
were saved to the hard drive of a computer using a Digidata 1200B
interface (Axon Instruments).
Isolation of potassium currents
Potassium currents were isolated with ACSF containing (in mM) 140.5 NaCl, 3 KCl, 3.7 MgCl2, 11 D-glucose, 5 HEPES, 3.25 NaOH, 0.003 tetrodotoxin (to block voltage-gated sodium currents) 0 CaCl2 and 0.2 CdCl2 (to block voltage-gated calcium currents); pH was adjusted to 7.3 with NaOH, osmolarity was 290-300 mOsm. The potassium channel blockers tetraethylammonium chloride (10 mM) or 4-aminopyridine (5 mM) were added to ACSF in some experiments, in which case, the NaCl concentration was reduced proportionately to maintain constant osmolarity. When 4-aminopyridine was used, the NaOH was replaced with NaCl and pH was adjusted with HCl. All chemicals were purchased from Sigma (St. Louis, MO) with the exception of tetrodotoxin, which was acquired from Alomone Labs (Jerusalem, Israel).
Cell identification
Type I putative magnocellular neurons of the PVN were
differentiated from type II, putative parvocellular neurons based
primarily on the expression of a transient outward rectification in
current clamp. Hoffman et al. (1991) demonstrated that
in the PVN, the expression of transient outward rectification in type I
neurons is positively correlated to neurophysin immunoreactivity and
large somatic diameter, suggesting that type I neurons are
magnocellular neurosecretory cells. In our experiments, the presence of
transient outward rectification was qualitatively assessed using a
current clamp protocol consisting of a series of progressively more
depolarizing current injections from a membrane potential near
100 mV
(Fig. 1A). Neurons that did
not express transient outward rectification were classified as type II
putative parvocellular neurons (Fig. 1B) and were not
included in this study. Type I and II neurons have also been shown to
differ on the basis of the functional expression of voltage-gated
currents (Luther and Tasker 2000
). All current-clamp
experiments were performed in standard ACSF containing calcium, as
described in the preceding text (Hypothalamic slice
preparation).
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Statistical methods and curve fitting
Values are expressed as means ± SE. Comparisons were made
using the Student's unpaired t-test or the Mann-Whitney
rank sum test when comparing two different normally or nonnormally
distributed data sets, respectively; the Student's paired
t-test for two data sets generated with repeated measures;
and the ANOVA or the Kruskal-Wallis ANOVA on ranks when comparing three
normally distributed or nonnormally distributed data sets, respectively
(SigmaStat 2.0, Jandel Scientific Software, San Rafael, CA).
Differences were considered significant at P < 0.05. Boltzmann and exponential fits were used to fit data plots and current
traces using the simplex fitting method (Clampfit, pClamp 6.0, Axon
Instruments). Time windows for exponential fits of current decay were
chosen to allow the IA to inactivate
completely (100 ms) and the slowly inactivating current to decay to
<15% of peak (460 ms). Statistical comparisons of goodness-of-fit were conducted using the simplex maximum likelihood estimate technique in pStat (pClamp 6.0, Axon Instruments).
Biocytin histochemistry
Following experiments, the slices were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4°C for 48 h, then washed in PBS with 20% sucrose for 1 h. The fixed slices were sectioned to 20-25 µm on a cryostat and washed in PBS for 30 min. Biocytin-filled neurons were labeled by incubating the sections for 3 h in a solution containing 5 µg/ml streptavidin conjugated with 7-amino-4-methyl-coumarin-3-acetic acid (AMCA; Vector Labs, Burlingame, CA) and 0.5% Triton-X in PBS. The sections were then washed for 10 min in PBS and examined under a fluorescence microscope using a UV/420K filter combination to find the biocytin-filled, AMCA-labeled neurons.
Fluoro-gold injection and immunohistochemistry
Rats aged 28-30 days were administered an intrajugular
injection of the retrograde tracer fluoro-gold (Fluorochrome, Denver, CO) under ketamine anesthesia as described in Leong and Ling
(1990). Briefly, rats were administered 40 mg/kg fluoro-gold
diluted in 500 µL of 0.9% NaCl with 20% lactose delivered in two
injections (250 µL each) timed 30 min apart. The rats were killed
7-10 days later for experiments.
Although fluoro-gold fluorescence was visible in retrogradely labeled neurosecretory cells in the PVN (e.g., see Fig. 10, A1 and B1), double labeling with AMCA and fluoro-gold was difficult to ascertain, so the fluoro-gold labeling was enhanced with fluoro-gold immunohistochemistry. Tissue sections containing biocytin-positive neurons were incubated in 0.1 M PBS with 2% normal sheep serum (NSS) (Vector Labs) for 10 min, and then incubated for 24 h at 4°C in a rabbit anti-fluoro-gold antiserum (lot No. 18080728, Chemicon International, Temecula, CA) diluted 1:5,000 in PBS with 1% NSS and 0.2% sodium azide. The sections were then washed three times in PBS and incubated for 1 h at room temperature in anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC; Vector Labs) diluted 1:200 in PBS with 1% NSS and 0.2% sodium azide. The sections were then washed in PBS for 10 min and mounted in elvanol and coverslipped, and the coverslips were sealed with nail polish. Mounted sections were examined under 450- to 490-nm excitation/515-nm barrier filters to detect FITC-labeled, fluoro-gold-positive neurons.
Oxytocin and vasopressin immunohistochemistry
Oxytocin and vasopressin immunohistochemistry was performed on slices in one of two ways depending on whether the animal was injected previously with fluoro-gold or not. Sections from animals not injected with fluoro-gold that contained a biocytin-filled, AMCA-labeled neuron were processed for oxytocin/vasopressin double labeling. They were incubated for 36 h in a mixture of rabbit polyclonal antibody to oxytocin-associated neurophysin (VA-10) diluted 1:2,000 and a mouse monoclonal antibody to vasopressin-associated neurophysin (PS-41) diluted 1:4,000 in PBS with 1% NSS and 0.2% sodium azide. Both primary antibodies were kindly provided by Dr. H. Gainer of the National Institutes of Health. The sections were then rinsed in PBS and incubated for an hour in anti-rabbit secondary IgG conjugated with FITC and goat anti-mouse secondary IgG conjugated with rhodamine (Vector Laboratories) diluted 1:200 in PBS with 1% NSS and 0.2% sodium azide. The sections were mounted in elvanol and coverslipped, and the coverslips were sealed with nail polish. The slides were examined under 515- to 560-nm excitation/580-nm barrier filters to detect the rhodamine-labeled, vasopressin-positive neurons or under 450- to 490-nm excitation/515-nm barrier filters to detect FITC-labeled, oxytocin-positive neurons.
Sections of slices from animals injected with fluoro-gold were prepared for double immunolabeling of fluoro-gold-filled cells and oxytocin/vasopressin neurons. Sections that contained a biocytin-filled, AMCA-labeled neuron were first processed for fluoro-gold immunohistochemistry as described in the preceding text. Oxytocin and vasopressin neurons were then labeled together as a single group by combining monoclonal antibodies for each peptide. For combined oxytocin-vasopressin immunohistochemistry, the sections were incubated for 36 h in a mixture of mouse monoclonal antibodies to oxytocin-associated neurophysin (PS-38) and vasopressin-associated neurophysin (PS-41), both diluted 1:2,000 in PBS with 1% NSS and 0.2% sodium azide. Both monoclonal antibodies were also graciously provided by Dr. H. Gainer. The sections were then rinsed three times in PBS and incubated for an hour at room temperature in anti-mouse IgG conjugated with rhodamine (Vector Labs) diluted 1:200 in PBS with 1% NSS and 0.2% sodium azide. The sections were mounted in elvanol and coverslipped, and the coverslips were sealed with nail polish. The slides were examined under a UV/420K filter combination to view the biocytin-filled, AMCA-labeled neurons, under 515- to 560-nm excitation/580-nm barrier filters to detect the rhodamine-labeled, vasopressin- and oxytocin-positive neurons, and under 450- to 490-nm excitation/515-nm barrier filters to detect FITC-labeled, fluoro-gold-positive neurons. Both oxytocin/vasopressin staining techniques routinely labeled the magnocellular neurons of the PVN; however, recorded cells identified electrophysiologically as type I putative magnocellular neurons often did not stain positive for oxytocin or vasopressin. We suspect that the oxytocin and vasopressin signals were either dialyzed or damaged during patch-clamp recordings, which routinely lasted over an hour.
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RESULTS |
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Eighty-six PVN neurons were recorded and classified as type I
neurons (Fig. 1) for this study. The recorded cells had an average resting membrane potential of 59.0 ± 1.2 mV, input resistance of 1,056.6 ± 47.9 M
, and action potential amplitude of
73.9 ± 1.0 mV.
Strong transient outward rectification in a subset of type I neurons
In response to depolarization from a hyperpolarized membrane potential, all type I PVN neurons expressed transient outward rectification that was characterized by a dampening of the charging curve and a delayed onset to the first spike (Fig. 1). A subset of type I neurons (33/86 neurons, 38%) was found that expressed a particularly strong transient outward rectification characterized by a hyperpolarizing "notch" in the membrane potential charging curve and a prolonged delay to the first spike (Fig. 2). We refer to these type I neurons as strongly rectifying type I neurons and to the remaining type I neurons as weakly rectifying type I neurons. All type I neurons fell clearly into one or the other population of a bimodal distribution based on the presence or absence of this distinct hyperpolarizing notch (Fig. 2C).
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Transient outward rectification and the associated delay to the first
spike were examined in 23 weakly rectifying neurons and 12 strongly
rectifying neurons by delivering a series of progressively more
depolarizing current steps from a membrane potential near 100 mV. The
strongly rectifying neurons required a larger amplitude current to
elicit action potentials (58.5 ± 13.2 and 15.4 ± 2.4 pA,
250-ms pulse, for strongly rectifying and weakly rectifying neurons,
respectively; P < 0.01, Mann-Whitney rank-sum test). When constant depolarizing current pulses (+50 pA, 250-1,000 ms) were
delivered from a membrane potential near
90 mV, the delay to the
first spike was longer in strongly rectifying neurons (184.9 ± 15.7 ms) than in weakly rectifying neurons (89.7 ± 8.8 ms;
P < 0.01, Student's unpaired t test; Fig.
3A). This was not due to
differences in the passive charging of the membrane because the
membrane time constants did not differ between the two cell groups. The
membrane charging curve, measured in the linear range of the
current-voltage relationship, was fitted with a single exponential
function in some cells (9/35 or 26% of weakly rectifying neurons and
4/16 or 25% of strongly rectifying neurons) and was fitted with the
sum of two exponential functions in the others. Neither the single
exponential membrane time constants (
: 51.6 ± 9.4 vs.
25.7 ± 5.6 ms, for weakly rectifying and strongly rectifying cells, respectively) nor the double exponential time constants (
1: 15.3 ± 2.1 vs. 16.7 ± 3.2 ms,
2: 74.2 ± 9.9 vs. 80.3 ± 23.6 ms,
for weakly rectifying and strongly rectifying neurons, respectively) differed significantly between groups.
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Strongly rectifying neurons were less excitable than weakly rectifying
neurons when depolarized from hyperpolarized membrane potentials. Cells
were stimulated to fire action potentials with depolarizing current
pulses ranging in amplitude from +10 to +100 pA. When depolarized from
a membrane potential of 90 to
100 mV, strongly rectifying neurons
(n = 5) fired at a lower initial frequency (calculated
from the interspike interval between the 1st 2 spikes) than weakly
rectifying neurons (n = 5; 18.7 ± 4.6 and
37.0 ± 5.5 Hz, respectively, at 100 pA; P < 0.05, Student's unpaired t-test; Fig. 3B). When
cells were depolarized from resting potential (
50 to
65 mV), the
initial firing frequency was similar in both cell types (Fig.
3C). Strongly rectifying neurons did not differ from weakly
rectifying neurons in the other electrophysiological properties
analyzed (Table 1).
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Transient outward current is slower in strongly rectifying neurons
Outward potassium currents were studied in isolation in 35 weakly
rectifying neurons and 20 strongly rectifying neurons using test steps
to between 70 and +20 mV in 5-mV increments from a 200-ms
conditioning step to either
50 or
100 mV. When the conditioning step was to
100 mV, a low-threshold transient component to the current was observed that was absent when the conditioning step was to
50 mV (Fig. 4, A and
B). The current elicited from
50 mV resembled the delayed
rectifier current (IK). When the whole cell currents generated from
50 mV were digitally subtracted from the
currents generated from
100 mV, both the
IK and the leak currents were removed,
leaving the low-threshold transient component. This subtraction
protocol was used to isolate the transient outward current in all
protocols in this study. This method revealed a transient potassium
current in both weakly rectifying and strongly rectifying type I
neurons (Fig. 4C) that resembled an A-type potassium current
(IA). The mean peak amplitude of the
transient outward current in weakly rectifying neurons (1,019.0 ± 164.1 pA; n = 21) did not differ from that of the
transient outward current in strongly rectifying neurons (1,137.7 ± 196.9 pA; n = 12) measured with voltage steps from
100 to
25 mV. The transient outward current elicited in the
strongly rectifying neurons differed from that elicited in weakly
rectifying neurons, however, by its activation and inactivation
kinetics. The rate of activation of the transient outward current was
studied by measuring the 10-90% rise time of the current, which was
significantly longer in strongly rectifying neurons (2.4 ± 0.0 ms; n = 5) than in weakly rectifying neurons (1.5 ± 0.2 ms; n = 13; steps to
25 mV from
100 mV; P < 0.01, Student's unpaired
t-test; Fig. 5A).
The rate of inactivation of the transient outward current was measured
by fitting exponential functions to the decay phase of current traces
to determine the time constant of inactivation (
). The decay phase
of currents of cells in both groups was best fitted with either a
single exponential function or the sum of two exponentials. The
transient outward current in weakly rectifying neurons decayed
monoexponentially in 16 cells with a
of 20.3 ± 0.7 ms and
biexponentially in 10 cells with a
1 of
6.8 ± 0.6 ms and a
2 of 26.9 ± 4.5 ms, with voltage steps from
100 to
25 mV. The rate of inactivation
of the transient outward current was slower in strongly rectifying neurons, decaying monoexponentially in two cells with a
of
162.4 ± 17.5 ms and biexponentially in seven cells with a
1 of 28.9 ± 4.4 ms and a
2 of 174.1 ± 19.5 ms, with the same
voltage protocol (Fig. 5B).
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Transient outward current in strongly rectifying neurons consists of two separate currents
The transient outward current in strongly rectifying neurons
consists of at least two kinetically separable current components: a
fast activating and inactivating component and a more slowly activating
and inactivating component. These components could be separated
partially by taking advantage of the slow inactivation rate of the slow
component. Two voltage protocols were applied for this purpose, the
first of which consisted of the standard activation protocol (i.e., a
test step to 25 mV from a conditioning step to
100 mV). The second
protocol was identical to the first except that a short (60-ms)
depolarizing prepulse to
40 mV preceded the test step (Fig.
6).
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As expected, the inactivating prepulse eliminated the transient outward
current nearly completely (by 85.6 ± 2.1%) in weakly rectifying
neurons (n = 4; Fig. 6A). The activation and
inactivation rates of the residual transient outward current in these
cells were unchanged (10-90% rise time: 3.1 ± 0.5 and 1.7 ± 0.1 ms; inactivation rate: 16.3 ± 1.6 and 16.3 ± 1.4 ms,
with and without the prepulse, respectively), suggesting a single
current. In strongly rectifying neurons (n = 4), the
inactivating prepulse reduced the fast component of the transient
outward current substantially but had a smaller effect on the slow
component (Fig. 6B). Thus the peak current was reduced by
62.8 ± 5.1%, whereas the current measured at 150 ms was reduced
by only 32.8 ± 2.2%. Both the activation and inactivation rates
of the transient outward current in these cells were decreased by the
prepulse. Thus the 10-90% rise time increased from 3.1 ± 0.6 to
20.4 ± 12.8 ms after the prepulse, and the inactivation rate went
from biexponential (1 = 32.2 ± 10.6 ms,
2 = 209.5 ± 39.0 ms) to monoexponential
(
= 239.0 ± 66.1 ms) following the prepulse. The fast,
prepulse-sensitive component of the current was isolated by subtracting
the current generated with the prepulse from the current generated
without the prepulse (Fig. 6). This component resembled the fast
transient current observed in weakly rectifying neurons (10-90% rise
time = 2.5 ± 0.5 ms; biexponential decay:
1 = 25.2 ± 2.9 ms,
2 = 141. 4 ± 16.4 ms), except that it
was contaminated by the portion of the slow current that was
inactivated by the prepulse. Thus the elimination of the fast component
of the transient outward current in strongly rectifying neurons by the
inactivating prepulse suggests that the current is actually composed of
two separate outward currents, a rapidly activating and inactivating
current and a slowly activating and inactivating current.
The respective contributions of the fast and slow currents to the peak current were determined from the protocol shown in Fig. 6B. The percent of the peak transient current amplitude contributed by the fast current was calculated from the difference current, and the percent contribution of the slow current was calculated from the current generated with the prepulse. Both current amplitudes were measured at a time point after the onset of the test step that corresponded to the peak of the transient current generated without the prepulse. Based on this calculation, the fast current accounted for 65.5 ± 4.2% and the slow current accounted for 34.5 ± 4.2% of the peak transient outward current in strongly rectifying neurons (n = 4). This represents a conservative estimate of the respective contributions of the two currents to the peak because the fast current was probably not eliminated 100% by the prepulse, as was the case for the transient outward current in the weakly rectifying neurons (Fig. 6).
Voltage dependence of activation and inactivation of the transient outward currents
The voltage dependence of activation and inactivation of the
transient outward currents was examined in detail in nine weakly rectifying neurons and in five strongly rectifying neurons. The activation voltage dependence of the transient currents in both cell
types was examined by stepping to between 70 and +20 mV in 5-mV
increments from a 200-ms conditioning step to
100 mV and plotting the
normalized chord conductance [gchord = I/(Vstep
VK+reversal)]
against the test step potential. The current measured at peak was
compared between both groups of cells; however, as mentioned in the
preceding text the peak current represents 65 and 35% contributions
from the fast and slow current components, respectively. The voltage
dependence of activation of the slow current in strongly rectifying
neurons was determined by plotting the normalized chord conductance
measured 150 ms after the onset of the test pulse against the test step potential. The fast component decayed completely within 150 ms, such
that only the slow component remained at this time point.
The activation threshold (55.7 ± 1.4 and
52.0 ± 2.0 mV;
Mann-Whitney rank-sum test), defined empirically as the first
depolarizing step that resulted in a measurable current, and
half-activation potential (
23.9 ± 3.3 and
25.9 ± 2.6 mV; Student's unpaired t-test) of the peak current did not
differ between weakly rectifying neurons and strongly rectifying
neurons, respectively (Fig.
7B). In strongly rectifying
neurons, the slow current had a threshold (
50.2 ± 2.1 mV) and
half-activation potential (
32.6 ± 2.3 mV) that were similar to
those of the peak current in both cell types (Kruskal-Wallis ANOVA on
ranks for threshold and ANOVA for half-activation potential; Fig.
7B).
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The voltage dependence of inactivation of the transient currents was
examined by stepping to a 25-mV test step potential from 200-ms
conditioning steps to between
120 and
35 mV in 5-mV increments
(Fig. 7C). The voltage dependence of inactivation of the
transient currents was determined by plotting the normalized current
amplitude versus the conditioning step potential. The half inactivation
potential of the current (
80.0 ± 2.0 and
76.9 ± 4.4 mV)
and the potential at which the current was completely inactivated
(
58.1 ± 3.0 and
58.6 ± 4.9 mV) did not differ between weakly rectifying neurons and strongly rectifying neurons, respectively (Student's unpaired t-test, Fig. 7C). The slow
current in strongly rectifying neurons, measured 150 ms after the onset
of the test step, had a half-inactivation potential (
80.2 ± 2.6 mV) and a potential at which the current was completely inactivated
(
55.0 ± 1.6 mV) that were similar to those of the current
measured at peak in both cell types (ANOVA, Fig. 7C).
Recovery from inactivation of the transient currents
The time dependence of recovery from inactivation of the transient
outward currents was examined in eight weakly rectifying neurons and in
six strongly rectifying neurons by stepping from 50 mV to a
100-mV
conditioning step of increasing duration, followed by a test step to
25 mV to activate the current (Fig. 8A). The peak current
recovered from inactivation with similar time dependence in both cell
types, achieving 100% availability (i.e., 0% inactivation,) with
conditioning steps lasting 169.0 ± 16.0 ms in weakly rectifying
neurons and 149.3 ± 12.3 ms in strongly rectifying neurons (Fig.
8B). A plot of the normalized peak current amplitude versus
the conditioning step duration was best fitted in both cell types with
a single exponential function, the time constant of which did not
differ significantly between the two cell types (
= 40.2 ± 8.6 and 27.7 ± 2.3 ms, for weakly rectifying and strongly
rectifying neurons, respectively; Fig. 8B).
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The slow current in strongly rectifying neurons recovered from inactivation more slowly than the peak current of both cell types. The slow current took conditioning steps lasting 616.7 ± 174.0 ms to recover 100% of its availability (n = 4; P < 0.05, Kruskal-Wallis ANOVA on ranks followed by a pairwise comparison with Dunn's method). The amplitudes of the fast currents measured at peak and that of the slow current measured at 150 ms are shown plotted against the conditioning step duration in Fig. 8C.
Sensitivity of the transient currents to 4-aminopyridine and TEA
A higher sensitivity to 4-aminopyridine (4-AP) than to
tetraethylammonium chloride (TEA) is a characteristic feature of
the IA in many neurons (Dolly
and Parcej 1996; Rudy 1988
). In weakly rectifying neurons, the peak transient outward current amplitude was
decreased by 36.4 ± 8.9% in 5 mM 4-AP and by 12.9 ± 3.7%
in 10 mM TEA (n = 5; P < 0.05),
measured with steps from
100 to
25 mV (Fig.
9A). In strongly rectifying
neurons, the peak current was decreased by 48.8 ± 5.7% in 5 mM
4-AP (n = 3) and by 13.5 ± 6.6% in 10 mM TEA
(n = 6), measured with steps from
100 to
25 mV
(Fig. 9B). The current measured at 150 ms was decreased by
35.6 ± 7.7% in 5 mM 4-AP (n = 5) and by
15.0 ± 5.5% in 10 mM TEA (n = 6). Block of the
transient outward currents by both 4-AP and TEA in weakly rectifying
neurons, but by 4-AP only in strongly rectifying neurons, was generally
weaker at more depolarized levels, consistent with a voltage dependence
of blockade of A-type potassium channels seen with 4-AP (Tseng
et al. 1996
) but not TEA (Pongs 1992
). The
voltage dependence of the block by TEA in weakly rectifying neurons may
be related to the subtype composition of the channels in these cells.
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Anatomy
Biocytin histochemistry was combined with retrograde fluoro-gold labeling and fluoro-gold immunohistochemistry to examine the neurosecretory/nonneurosecretory nature, the somatic size, and the relative position in the PVN of the recorded neurons. The fluoro-gold immunolabel was found in 25/27 weakly rectifying neurons (93%) and in 7/8 strongly rectifying neurons (88%; Fig. 10, A and B). Both weakly rectifying and strongly rectifying neurons were large, with somata measuring 304.6 ± 30.5 and 281.8 ± 35.5 µm2, respectively (Fig. 10C). Strongly rectifying neurons were not significantly different in size from either weakly rectifying neurons or type II neurons (Kruskal-Wallis ANOVA on ranks followed by a Dunn's pairwise comparison), but their mean somatic area was closer to that of weakly rectifying than that of type II putative parvocellular neurons. Although found throughout the nucleus, weakly rectifying and strongly rectifying type I neurons, unlike type II neurons (not shown), were often located within magnocellular subdivisions of the PVN (Fig. 10D).
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Compared with sharp-electrode recordings, the use of whole cell patch-clamp recordings has resulted in a dramatic decline in the recovery of cells double labeled with biocytin and a peptide immunolabel, due presumably to either the dialysis or the degradation of the peptide signal with prolonged recordings. Therefore interpretation of negative immunolabeling of biocytin-labeled cells is problematic. Nevertheless positive immunostaining, when obtained, is still valid as an indicator of the peptidergic identity of recorded cells. We found that weakly rectifying neurons and, to a lesser extent, strongly rectifying neurons were more likely to stain positive for oxytocin, vasopressin, or the oxytocin-vasopressin antibody combination than neurons that expressed type II electrophysiology. Thus 15 of 39 weakly rectifying neurons (38%) and 3 of 16 strongly rectifying neurons (19%) stained positive for oxytocin, vasopressin, or the oxytocin-vasopressin antibody combination, in contrast to 2 of 44 type II neurons (5%) that showed oxytocin or vasopressin positivity. Although inconclusive at this point, these data suggest that the strongly rectifying neurons, like the weakly rectifying neurons, may be magnocellular neurosecretory cells.
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DISCUSSION |
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Previous studies have shown that neurons within the PVN can be
divided into two groups based on the expression of distinct electrophysiological properties, type I neurons, which correspond to
magnocellular neurosecretory cells, and type II neurons, which correspond to parvocellular neurosecretory and preautonomic cells (Hoffman et al. 1991; Tasker and Dudek
1991
). The most distinctive electrophysiological characteristic
of type I neurons is a transient outward rectification (Tasker
and Dudek 1991
), which is believed to be caused by the
activation of a large IA (Li
and Ferguson 1996
; Luther and Tasker 2000
). Here
we present evidence for a subset of type I neurons, strongly rectifying
type I neurons, which express a more slowly activating and inactivating
transient outward current that results in lower excitability following
hyperpolarization. This slow transient outward current resembled
similar currents in histamine neurons of the tuberomamillary nucleus
(Greene et al. 1990
) and in a small subset of
nonmagnocellular neurons in the region of the hypothalamic supraoptic
nucleus (Fisher et al. 1998
).
Transient outward current in strongly rectifying neurons consists of two components
Our results suggest that the transient outward current in strongly
rectifying neurons consists of a fast component and a slow component,
which correspond to two separate currents. They could be partially
separated using voltage protocols due to the difference in their
inactivation rates. Briefly, a short depolarizing prepulse to 40 mV
between the conditioning step and test step almost completely inactivated the fast current (
~ 20 ms) but had much less
effect on the slow current (
~ 240 ms). Thus the fast current
was largely removed by the prepulse, leaving only the slow current.
These protocols did not reveal a second slow component to the transient outward current in the weakly rectifying neurons. The voltage dependence of activation and inactivation, the time dependence of
recovery from inactivation, and the sensitivity to 4-AP and TEA were
determined for the single fast current in weakly rectifying neurons and
for both the peak (~65% fast component and 35% slow component) and
the slow current in strongly rectifying neurons. The peak currents in
both groups of cells and the slow current in strongly rectifying
neurons all had similar voltage-dependent properties and
pharmacological sensitivities. The slow current component activated,
inactivated, and recovered from inactivation with slower kinetics than
the peak transient current in strongly rectifying and weakly rectifying neurons.
Both cell types express IA
The fast currents in both cell types resemble the
IA by their fast rate of activation
and inactivation, their hyperpolarized voltage dependence of activation
and inactivation, and their sensitivity to block by millimolar
concentrations of 4-AP (Dolly and Parcej 1996;
Rudy 1988
). The fast transient outward current in weakly rectifying neurons resembles in its kinetics, voltage dependence, and
pharmacological sensitivity the IA
described in type I PVN neurons by Li and Ferguson
(1996)
. The strongly rectifying neurons expressed both a fast
current resembling the IA and a more
slowly activating and inactivating current.
The voltage properties and pharmacological sensitivity of the slow
transient potassium current described here in slowly rectifying type I
neurons differ from those of the transient D-type potassium current (ID). The
ID is characterized by a slower
inactivation rate (~1 s) and a slower rate of recovery from
inactivation (~1 s) (Dolly and Parcej 1996). It also
typically has a higher sensitivity to 4-AP (i.e., micromolar
sensitivity) and is insensitive to block by TEA (Christie et al.
1989
; Gabel and Nisenbaum 1998
; Storm 1988
; Wu and Barish 1992
). The slowly
inactivating current, therefore more closely resembles the
IA than the
ID, suggesting that strongly rectifying type I neurons may express two different types of A-like potassium channels. Slowly inactivating transient potassium currents have been reported in other cells, including hypothalamic cells, which
are similar to the slow current in strongly rectifying neurons in their
inactivation kinetics and low sensitivity to 4-AP (Fedulova et
al. 1998
; Fisher et al. 1998
; Greene et
al. 1990
; Lüthi et al. 1996
).
Are strongly rectifying neurons magnocellular neurosecretory cells?
Type I PVN neurons have been shown previously to correspond to
magnocellular oxytocinergic and vasopressinergic neurosecretory cells
(Hoffman et al. 1991). In this study, both the weakly
rectifying and strongly rectifying neurons were large compared with
type II putative parvocellular neurons, having somatic areas of ~300 µm2, which is similar to what has been reported
for magnocellular neurosecretory cells of the supraoptic nucleus
(Randle et al. 1986
; Smith and Armstrong
1990
). Both weakly rectifying neurons and strongly rectifying
neurons took up the retrograde tracer fluoro-gold when injected
intravenously, indicating that both cell types project outside the
blood-brain barrier and are therefore probably neurosecretory. Many of
both cell types were found within the magnocellular division of the
PVN, which contains primarily magnocellular neurons. The weakly
rectifying and strongly rectifying type I neurons found outside the
magnocellular subdivision of the PVN suggest that these cells may not
be magnocellular or, more likely, that their distribution is not
restricted exclusively to the magnocellular subdivision of the nucleus,
as is the case for many magnocellular neurons distributed throughout
the PVN. Type II, putative parvocellular neurons were rarely found
within the magnocellular division of the PVN (1/29, data not shown), suggesting that this region of the nucleus is highly enriched in
magnocellular neurons. Although many of both the weakly rectifying and
strongly rectifying neurons did not stain positive for oxytocin or
vasopressin, they stained positive with a higher frequency than did
type II neurons (38, 19, and 5% positive for weakly rectifying type I,
strongly rectifying type I, and type II neurons, respectively). As
described before, negative immunostaining following prolonged (30-120
min) whole cell patch-clamp recordings must be interpreted with caution
because of the apparent dialysis and/or degradation of the peptide
signal. Thus the large somatic size, the neurosecretory nature, and the
rate of oxytocin/vasopressin immunopositivity indicate that weakly
rectifying type I neurons are magnocellular neurosecretory cells and
suggest that strongly rectifying type I neurons may be a subset of
magnocellular neurosecretory cells. Consistent with this is the
observation that the supraoptic nucleus, which is comprised nearly
exclusively of magnocellular neurons, contains both weakly and strongly
rectifying neurons (unpublished observation). Nevertheless
additional double-labeling experiments are required to confirm the
magnocellular identity of the strongly rectifying neurons given the
scattered distribution of these cells within the PVN.
Physiological implications
In magnocellular neurosecretory cells of the supraoptic nucleus,
Kv4.2 immunoreactivity is concentrated near synaptic contacts, suggesting that the IA may be involved
in the integration of synaptic inputs (Alonso and Widmer
1997). The IA has been shown
to contribute to synaptic integration (Hoffman et al.
1997
) and to regulate action potential propagation through
axons (Debanne et al. 1997
). If the ion channels
underlying the transient outward currents in weakly rectifying and
strongly rectifying neurons of the PVN are also located near synaptic
contacts, the two cell types would be predicted to respond differently
to similar synaptic inputs. Our evidence suggests that the slowly
inactivating transient outward current observed in the strongly
rectifying subset of type I PVN neurons reduces cellular excitability
at hyperpolarized membrane potentials.
From our analyses, weakly rectifying neurons and strongly rectifying
neurons appear to differ primarily in their expression of
low-threshold, transient outward currents. As a result, whereas the
response properties of the two cell types are similar at resting membrane potential, they differ dramatically at hyperpolarized potentials, the strongly rectifying neurons becoming much less excitable than the weakly rectifying neurons (e.g., see Fig. 3). The
two cell types would be expected therefore to behave similarly under
resting conditions but to adopt very different behaviors under
conditions that hyperpolarize the membrane, the strongly rectifying
neurons becoming less responsive to excitatory synaptic inputs.
Potential hyperpolarizing conditions for these cells include the
closing of membrane mechanosensitive cation channels caused by a
decrease in blood osmolality (Oliet and Bourque 1993).
Indeed, both strongly rectifying and weakly rectifying type I PVN
neurons have been found to be osmosensitive (unpublished observation). Strongly rectifying neurons would be predicted to be more sensitive to
small decreases in blood osmolality because this would cause membrane
hyperpolarization, and these cells would become less responsive to
excitatory inputs under these conditions. This could cause them to
become unresponsive to an excitatory drive (e.g., from presynaptic
osmosensitive cells), thus switching off their activity and reducing
their release of oxytocin and/or vasopressin. It is likely, therefore
that these two cell types play different, albeit related, roles in the
response of the PVN to osmotic as well as other homeostatic challenges.
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FOOTNOTES |
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Address for reprint requests: J. G. Tasker, Dept. of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane University, New Orleans, LA 70118-5698.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 June 1999; accepted in final form 6 June 2000.
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REFERENCES |
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