A Slow Transient Potassium Current Expressed in a Subset of Neurosecretory Neurons of the Hypothalamic Paraventricular Nucleus

Jason A. Luther,1 Katalin Cs. Halmos,2 and Jeffrey G. Tasker1,2

 1Neuroscience Program and  2Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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: tau  = 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . They were filled with a solution consisting (in mM) of 130 potassium gluconate, 10 HEPES, 1 NaCl, 1 CaCl2, 10 ethyleneglycol-bis-(beta -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 MOmega , 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 MOmega , 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).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Type I putative magnocellular neurons are distinguished from type II putative parvocellular neurons by the expression of a transient outward rectification. A: a representative type I neuron responds to a series of incremental depolarizing current pulses (top) delivered at a hyperpolarized membrane potential with the expression of transient outward rectification, characterized by a dampening of the membrane charging curve (right-arrow) and a delay to the 1st action potential. B: type II neurons lack a transient outward rectification. A type II neuron displays electrotonic charging of its membrane (right-arrow) in response to the same protocol. The amplitudes of the current pulses were adjusted according to the input resistance of the recorded cell, and typical values used are shown.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega , 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).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Two distinct populations of type I neurons. A: a type I neuron exhibited transient outward rectification in response to a series of depolarizing current pulses delivered at a hyperpolarized membrane potential. B: in response to the same protocol, a different type I neuron exhibited a more pronounced transient outward rectification, with a hyperpolarizing "notch" in the membrane potential (down-arrow ) and a longer latency to the first spikes. These 2 types of neurons were referred to as weakly rectifying neurons (A) and strongly rectifying neurons (B), respectively. The amplitudes of current pulses were adjusted according to the input resistance of the cell, and typical values used are shown. C: type I neurons were classified as strongly rectifying and weakly rectifying neurons based on the presence or absence, respectively, of a hyperpolarizing notch in the membrane charging curve when depolarized from a hyperpolarized membrane potential. A straight line was fitted to the voltage trace over a 10-ms window (bottom) following the threshold of the transient outward rectification (TOR). For all weakly rectifying neurons, this line had a positive slope, while for all strongly rectifying neurons, this line had a negative slope. Note that 2 strongly rectifying neurons had slopes near 0 (-0.05 and -0.03 mV/ms). Means: 0.22 ± 0.01 and -0.33 ± 0.04 mV/ms for weakly rectifying and strongly rectifying neurons, respectively. Bin width is 0.1 mV/ms.

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 (tau : 51.6 ± 9.4 vs. 25.7 ± 5.6 ms, for weakly rectifying and strongly rectifying cells, respectively) nor the double exponential time constants (tau 1: 15.3 ± 2.1 vs. 16.7 ± 3.2 ms, tau 2: 74.2 ± 9.9 vs. 80.3 ± 23.6 ms, for weakly rectifying and strongly rectifying neurons, respectively) differed significantly between groups.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. Strongly rectifying neurons expressed a longer delay to the 1st spike when depolarized from hyperpolarized membrane potentials. A: when depolarized from a membrane potential of about -90 mV with a 50-pA current injection, a weakly rectifying neuron (left) generated an action potential after an 85-ms delay, while a strongly rectifying neuron (right) responded with a 192-ms delay to the first action potential. B: when depolarized from a hyperpolarized membrane potential (about -90 mV), strongly rectifying neurons reached a lower firing frequency than weakly rectifying neurons. C: when the cells were depolarized from near resting potential, the responses were similar in both cell types.

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).


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Passive and active properties of type I PVN neurons

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 (tau ). 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 tau  of 20.3 ± 0.7 ms and biexponentially in 10 cells with a tau 1 of 6.8 ± 0.6 ms and a tau 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 tau  of 162.4 ± 17.5 ms and biexponentially in seven cells with a tau 1 of 28.9 ± 4.4 ms and a tau 2 of 174.1 ± 19.5 ms, with the same voltage protocol (Fig. 5B).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. Isolation of the transient outward current. A: a family of outward currents with both transient and sustained components was generated in a weakly rectifying and a strongly rectifying neuron in response to depolarizing voltage steps to between -70 and +20 mV in 5-mV increments from a 200-ms conditioning step to -100 mV. B: the same range of test steps from a 200-ms conditioning step to -50 mV elicited a family of sustained currents resembling the IK in both cells. C: by digitally subtracting the traces generated with the -50-mV conditioning step from traces generated with the -100-mV conditioning step, a transient outward current resembling the IA was isolated in both cells. The single traces (top) are expanded to show the isolated IA in response to a voltage step to -25 mV in the weakly rectifying neuron and the strongly rectifying neuron. Traces in A and B were p/8 leak subtracted. Traces in C were generated by subtracting unmodified traces (i.e., not leak subtracted) generated with the protocol in B from unmodified traces generated with the protocol in A.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. The transient outward current activated and inactivated more slowly in strongly rectifying neurons. A: plot of the mean 10-90% rise time of the transient outward current against test step potential. The rate of activation was slower in strongly rectifying neurons (filled circles) than in weakly rectifying neurons (open circles). The rate of activation of the transient outward current was voltage dependent in both cell types, becoming faster with greater depolarization. Inset: 10-90% rise time in a weakly rectifying neuron (1.5 ms) and a strongly rectifying neuron (4.2 ms), measured with steps from -100 to -25 mV. B: plot of the mean inactivation time constant (tau), on a logarithmic scale, of monoexponentially and biexponentially decaying transient outward currents against test step potential for weakly rectifying neurons (monoexponential: open circles, n = 16; biexponential: light gray circles, n = 8) and for strongly rectifying neurons (monoexponential: filled circles, n = 2; biexponential: dark gray circles, n = 7). The transient outward current of strongly rectifying neurons inactivated more slowly than that of weakly rectifying neurons at all test potentials. Inset: fitted decay phase of the transient outward current in a weakly rectifying neuron (monoexponential: tau  = 15.1 ms) and in a strongly rectifying neuron (biexponential: tau 1 = 42.6 ms, tau 2 = 140.9 ms). Currents were elicited in both cells with voltage steps from -100 to -25 mV.

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).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6. The transient outward current in strongly rectifying neurons consists of 2 kinetically separable components. A: a weakly rectifying neuron expressed a fast transient outward current in response to a voltage step from -100 to -25 mV. The introduction of a 60-ms prepulse to -40 mV reduced the current amplitude by 76% but did not change the activation or inactivation rate of the current (10-90% rise time = 2.0 vs. 3.2 ms, inactivation time constant = 19.4 vs. 20.9 ms, without and with the prepulse, respectively). B: a strongly rectifying neuron expressed a more slowly activating and inactivating transient outward current in response to the same voltage step. The prepulse reduced the current measured at the peak more strongly than the current measured after 150 ms (black-triangle). Following the prepulse, the current activated more slowly, the 10-90% rise time increasing from 4.4 to 8.4 ms. The current inactivated biexponentially without the prepulse (tau 1 = 27.8 ms and tau 2 = 148.6 ms) and monoexponentially following the prepulse (tau  = 251.5). The difference current, obtained by subtracting the current generated with the prepulse from the current generated without the prepulse, resembled the fast current seen in weakly rectifying neurons (10-90% rise time = 3.6 ms; tau 1 = 20.9 ms and tau 2 = 89.5 ms).

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 (tau 1 = 32.2 ± 10.6 ms, tau 2 = 209.5 ± 39.0 ms) to monoexponential (tau  = 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: tau 1 = 25.2 ± 2.9 ms, tau 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).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 7. Voltage dependence of activation and inactivation of the transient outward currents. A: isolated transient outward currents in a weakly rectifying neuron and in a strongly rectifying neuron showing points on the traces where amplitudes of the fast and slow components of the currents were measured at peak and 150 ms after the onset of the test step, respectively. B: voltage dependence of activation. The normalized chord conductance at peak in weakly rectifying neurons (left) and at peak and after 150 ms in strongly rectifying neurons (right) were plotted against the test step potential shown in the inset (see text for explanation). The threshold of activation (-55.7 ± 1.4 and -52.0 ± 2.0 mV) and the half-activation potential (-23.9 ± 3.3 and -25.9 ± 2.6 mV) of the fast current were similar in weakly rectifying neurons and strongly rectifying neurons, respectively. The slow current activated from a threshold of -50.2 ± 2.1 mV and reached half-activation at -32.6 ± 2.3 mV, which was not significantly different from the fast current in either cell type. C: voltage dependence of inactivation. The normalized amplitudes of the current at peak and 150 ms after the onset of the test step were plotted against the conditioning steps shown in the inset (see text for explanation). The fast current was completely inactivated (-58.1 ± 3.0 and -58.6 ± 4.9 mV) and reached half-inactivation (-80.0 ± 2.0 and -76.9 ± 4.4 mV) at similar potentials in weakly rectifying neurons and strongly rectifying neurons, respectively. The slow current reached complete inactivation at -55.0 ± 1.6 mV and half-inactivation at -80.2 ± 2.6 mV, which was not significantly different from the fast current in either cell type.

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 (tau  = 40.2 ± 8.6 and 27.7 ± 2.3 ms, for weakly rectifying and strongly rectifying neurons, respectively; Fig. 8B).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 8. Recovery from inactivation of the transient outward currents. A: recovery from inactivation was examined by stepping from -50 mV, which completely inactivated the transient outward current in both cell types, to a -100-mV conditioning step of variable duration followed by a test step to -25 mV (top). The fast transient outward currents in both the weakly rectifying and strongly rectifying neurons recovered completely from inactivation in <200 ms. B: plot of the mean normalized peak current amplitude vs. the conditioning step duration. The fast transient outward current, measured at peak, recovered from inactivation with a similar time dependence in both cell types, recovering 100% of its amplitude with conditioning steps lasting 169.0 ± 16.0 ms in weakly rectifying neurons and 149.3 ± 12.3 ms in strongly rectifying neurons. The plots of normalized amplitude vs. conditioning step duration were best fitted with single exponential functions in weakly rectifying (tau  = 40.2 ± 8.6 ms) and in strongly rectifying neurons (tau  = 27.7 ± 2.3 ms), which were not significantly different from one another. C: in strongly rectifying neurons, the slow current (measured at 150 ms) recovered from inactivation more slowly, requiring longer conditioning steps to recover 100% of its amplitude (616.7 ± 174.0 ms).

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.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 9. Sensitivity of the transient outward currents to potassium channel blockers. A: the transient outward current in weakly rectifying neurons is more strongly reduced by 4-aminopyridine (4-AP) than by TEA. A1: the transient outward current in a weakly rectifying neuron was reduced by 11% in 10 mM TEA and by 40% in 10 mM TEA and 5 mM 4-AP, measured with voltage steps from -100 to -25 mV. A2: plot of the mean percent block by 5 mM 4-AP (n = 5) and 10 mM TEA (n = 5) of the transient outward current at different test step potentials in weakly rectifying neurons. B: the fast and slow transient outward currents in strongly rectifying neurons were more strongly reduced by 4-AP than by TEA. B1: the peak transient outward current in a strongly rectifying neuron was unaffected by application of 10 mM TEA and was reduced by 46% with the addition of 5 mM 4-AP, measured with voltage steps from -100 to -25 mV. B2: plot of the mean percent block of the peak outward current and the current at 150 ms caused by application of 5 mM 4-AP (n = 3) and 10 mM TEA (n = 6) at different test step potentials in strongly rectifying neurons (B2). Block of the transient currents by TEA and 4-AP was voltage-dependent in both cell types, with weaker block at more depolarized potentials.

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).



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 10. Both weakly rectifying neurons and strongly rectifying neurons are large neurosecretory cells. A weakly rectifying neuron (A1) and a strongly rectifying neuron (B1) were filled with biocytin and labeled histochemically with 7-amino-4-methyl-coumarin-3-acetic acid (AMCA, right-arrow). Other fluoro-gold labeled cells, as well as blood vessels, are weakly fluorescent before fluoro-gold immunohistochemistry. The same biocytin-labeled, weakly rectifying and strongly rectifying neurons were both immunoreactive for fluoro-gold following fluoro-gold immunohistochemistry (A2 and B2, right-arrow). C: the mean somatic area of strongly rectifying neurons, estimated as the product of the long and short somatic diameters, was not statistically different from that of weakly rectifying neurons or type II putative parvocellular neurons, although there was a trend toward larger values than those for type II neurons. The mean somatic area of weakly rectifying neurons was larger than that of type II neurons. (*P < 0.05, Kruskal-Wallis ANOVA on ranks followed by Dunn's pairwise comparison). D: maps of recovered biocytin-labeled, weakly rectifying neurons (top) and strongly rectifying neurons (bottom) at 3 rostrocaudal levels through the paraventricular nucleus (PVN). PVN maps were defined based on the distribution of oxytocin and vasopressin immunoreactive cells. Many weakly rectifying and strongly rectifying neurons were located within the magnocellular divisions of the PVN, while other cells were found more medially in the nucleus. am, anterior magnocellular division; pm, posterior magnocellular division.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (tau  ~ 20 ms) but had much less effect on the slow current (tau  ~ 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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society