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Department of Anatomy and Neurobiology, University of Tennessee Medical School, Memphis, Tennessee 38163
Submitted 27 April 2004; accepted in final form 2 July 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-subunit in VP neurons, which may be related to the greater importance of this current type in VP spike repolarization. Because OT and VP neurons are not considered fast firing, but do exhibit frequency- and calcium-dependent spike broadening, Kv3-like currents may be important for maintaining spike width and calcium influx within acceptable limits during repetitive firing. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Poulain and Wakerley 1982). Many of the membrane properties, known to shape these APs, their afterpotentials, and the resulting firing patterns in these neurons, have been described. Among these are K+ currents generated by voltage- and/or Ca2+-activated K+ channels. For example, SON neurons have a prominent IA (Bourque 1988; Fisher et al. 1998; Hlubek and Cobbett 1997), an SK-mediated IAHP (Kirkpatrick and Bourque 1996), BK-mediated IC (Dopico et al. 1999; Roper et al. 2003; Stern and Armstrong 1997), and delayed rectifier-type currents (Cobbett et al. 1989; Hlubek and Cobbett 1997) that contribute to spike repolarization and spike afterhyperpolarizations. Similar currents have been found in PVN neurons (Li and Ferguson 1996; Luther et al. 2000; Nagatomo et al. 1995). However, to date, there has been little dissection of delayed rectifiers in MNCs.
In the large family of voltage-gated K+ (Kv) channels, the Kv3 subfamily includes genes of four -subunits: Kv3.1–Kv3.4, all of which evoke high-voltage–activated outward K+ currents. The specific assembly of these various subunits is pivotal for shaping AP repolarization, the AP afterhyperpolarization, and consequently the firing pattern exhibited by a given neuron type (Rudy et al. 1999). Slowly inactivating currents ascribed to Kv3.1/3.2 subunits (which typically coassemble) allow rapid repolarization and high-frequency firing, and have been identified in the globus pallidus (Baranauskas et al. 1999; Hernández-Pineda et al. 1999), fast-spiking neocortical neurons (Chow et al. 1999), hippocampal interneurons (Martina et al. 1998; Riazanski et al. 2001), and brain stem auditory neurons (Wang et al. 1998). The Kv3.4 subtype exhibits voltage dependency similar to that of the Kv3.1/3.2 subtype, but is rapidly inactivating, and participates in AP repolarization in some cell types (Baranauskas et al. 2003; Riazanski et al. 2001). Intermediate inactivation rates (<1 s) have been identified for Kv3.3 subtypes (Coetzee et al. 1999). Kv3 subunits can form hetero-oligomeric channels of intermediate, or unusual properties (Baranauskas et al. 2003; MacKinnon 1991; Weiser et al. 1994) and are regulated by phosphorylation (Beck et al. 1998) and site-specific proteolysis (Hoshi et al. 1990). All members of the Kv3 subtype exhibit a similar sensitivity to low (<1 mM) concentrations of tetraethylammonium (TEA), but some may be distinguished by differential sensitivity to other toxins, such as 4-aminopyridine (4-AP) and BDS-I (Coetzee et al. 1999; Diochot et al. 1998). An investigation of Kv3.1, 3.2, and 3.4 mRNAs indicated only scattered cells in the preoptic area (Kv3.1, 3.2) and anterior hypothalamus (Kv3.4), with no mention of the SON or PVN (Weiser et al. 1994).
In this study we characterized high voltage–gated K+ currents in identified OT and VP neurons from the SON in hypothalamic slices using voltage-clamp recordings and pharmacologic sensitivity. For clarity, we will refer to these as Kv3-like currents where appropriate. We also examined the characteristics of these currents in acutely dissociated cells, to more accurately assess their voltage dependency, and inactivation. The role of these currents in spike repolarization was then assessed in current-clamp recordings. Finally, we used immunohistochemistry to investigate whether Kv3 subtypes could be localized to the SON. Parts of this study were previously presented in abstract form (Armstrong and Teruyama 2001; Shevchenko and Armstrong 2001).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Although problematic for precise evaluation of current kinetics and voltage dependency ascribed to the presence of dendrites, the slice preparation afforded us the opportunity to reliably record K+ currents in viable adult SON neurons, and further to compare some of their properties in OT and VP neurons using established, double-labeling immunochemical procedures. Whereas single-cell polymerase chain reaction (PCR) techniques have been applied to SON neurons (Xi et al. 1999) and could be useful to identify acutely dissociated cells, there is significant co-localization of OT and VP mRNA. Although SON neurons in slices would have some dendritic tree, the influence of poor space clamp on the time course and activation voltage dependency is not severe for depolarization-activated K+ currents (Surmeier et al. 1994). Another factor to consider is the relatively large currents recorded in slices, compounding series resistance errors. As a compromise to these advantages and disadvantages, we have verified the basic characteristics of Kv3-like currents studied in slices using a sample of unidentified, acutely dissociated SON neurons.
Hypothalamic slices
The methods are essentially those of Stern et al. (1999, 2000). Female virgin rats (150–200 g; random cycling) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally [ip]) and perfused through the heart with cold artificial cerebrospinal fluid (ACSF) in which NaCl was replaced by an equiosmolar amount of sucrose (see following text). The brain was rapidly removed after decapitation, blocked, and coronal slices were cut on a vibrating microtome (Campden Instruments). Slices were placed in ACSF consisting of (in mM) 125 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 20 glucose, 2 CaCl2, 1 MgSO2, 0.4 ascorbic acid at pH 7.3–7.4, with an osmolality of 300–310 mOsm/kg H2O, and saturated with 95% O2–5% CO2. Slices were incubated in this solution 
8 h at room temperature after a 1.5-h preincubation at 32°C. A Ca2+-free modification of ACSF had Ca2+ replaced by 3 mM EGTA–NaOH and 2 mM MgCl2. Decreasing NaCl compensated for the excess of Na+ ions added with NaOH.
Dissociated cells
Dissociated cells were prepared as reported by Hlubek and Cobbett (1997). For enzymatic dissociation brain slices were preincubated in ACSF containing 0.1 mg/ml pronase for 75 min at 32°C. The SON area was triturated with a fire-polishing Pasteur pipette of about 0.2–0.5 mm inner diameter. The cell suspension was adhered to a cover glass that was then placed in the same chamber used for patch-clamp recording from slices. Cells adhered within a few minutes, after which recordings could be made under visual guidance. Recordings from dissociated MNCs in this region were based on cell diameters (>25 µm), as determined by Oliet and Bourque (1992).
Electrophysiology
Electrophysiological recordings used the whole cell configuration of the patch-clamp technique (Hamill et al. 1981). Data were obtained with an Axopatch 200B (Axon Instruments, Foster City, CA) patch-clamp amplifier. Pipettes were made with borosilicate glass capillary tubes (150T-3, Warner Instrument) and filled with an intracellular solution containing (in mM) 142.6 K+ gluconate, 7.4 KCl, 0.9 MgCl2, 10 HEPES, 0.2 EGTA, 4 ATP-Mg, and 0.3 GTP-Na, biocytin 1–2 mg/ml; pH 7.2–7.3 with KOH, osmolality 280–300 mOsm/kg. In some experiments, 2 mM BAPTA was used instead of 0.2 mM EGTA. For dissociated cells, biocytin was omitted from the patch solution because we were not able to successfully retain cells for immunohistochemical identification. The standard ACSF solution described above was used as the initial external solution for both dissociated cell and slice recordings. For voltage-clamp experiments, 0.5 µM tetrodotoxin (TTX) was applied to block sodium currents. All the pharmacological agents were applied as additives in external solution. BDS-I was dissolved in the presence of 0.03–0.1 mg/ml bovine serum albumin. All recordings, including those from dissociated neurons, were done at 33 ± 0.5°C.
The initial electrode resistance was 6–9 M
. Voltages were corrected for a liquid junction potential of –10 mV, measured empirically according to Neher (1992). The acquisition rate was 20 kHz with filtering at 2–5 kHz. The ground electrode consisted of a silver/silver chloride wire coupled to an Agar (4%) bridge saturated with 3 M KCl.
In voltage clamp, whole cell capacitance and series resistance were compensated (75–80%) and monitored after each recorded series. The recordings reported herein all had a series resistance <20 M before compensation, and were terminated if exceeding this value.
Current-clamp recordings were performed in the fast mode of the 200B amplifier. Voltage signals were digitized at 20 kHz. After each experiment, changes in the electrode tip potential were estimated by measuring the DC offset of the electrode in the bathing medium and considered acceptable if the offset was ±5 mV.
Dendrotoxin (DTX), TTX, and BDS-I were purchased from Alomone Labs. Additional BDS-I was a gift from Drs. Michel Lazdunski and Sylvie Diochot. All other chemicals for media preparation were purchased from Fisher, Sigma, or J. T. Baker Chemical companies.
Data analysis
To obtain activation parameters for Kv-like currents in dissociated neurons, we plotted the current and normalized conductance (G/Gmax) against voltage using TEA-subtracted currents. Current amplitude for the fast transient component was evaluated at its peak and for the persistent current was averaged over the last 50–100 ms of a 2 s depolarization pulse. The conductance (G) was calculated as G = I/(V – Vrev). The K+ current reversal potential (Vrev) was estimated at –90 mV from a series of tail currents steps to –50 to –120 mV after a 20 ms depolarization step from –50 to +20 mV. Conductance data were fitted to a Boltzmann function
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Exponential fitting of the currents (+50 mV depolarization pulse, 2 s duration) provided inactivation time constants (
) for the transient (the fastest inactivation value) and the persistent (the slowest inactivation value) components. Curve fits were made from averages of 3–6 records. Although in several neurons an intermediate was present, it was an inconsistent property across all neurons. For these neurons, the fastest and slowest values of were reported from the fits using 3 exponentials.
Current-clamp records of spikes were aligned at the rising phase and averaged (n > 3) to measure spike width. Spike widths were measured at threshold (base width) and at 1/2 spike amplitude (1/2 width). Rise times were calculated from the 10–90% region of spike amplitude. DC current injection was used to maintain a slow (<1 Hz), irregular pattern of activity near threshold.
All values are shown as means ± SD. Data analysis was performed with Clampex (8.0 or 8.2), AxoGraph 4.6 (Axon Instruments), Igor Pro 4.0 (Wavemetrics), and Excel 8.0 (Microsoft) software. Within-group statistical comparisons were analyzed with the nonparametric Wilcoxon's signed-rank test for paired samples, or for multiple comparisons, Friedman's test. Between-group comparisons of OT and VP neurons were made with the nonparametric Mann–Whitney U test. Only significant P values (<0.05) are listed. All tests were performed using Statview 5.0 software.
Immunohistochemistry
To identify the cell type after the recording session, slices were fixed 1–7 days in 0.15 M sodium phosphate–buffered 4% paraformaldehyde, and 0.2% picric acid (pH 7.2–7.4), rinsed in phosphate-buffered saline (PBS) containing 0.5% Triton-X 100, and incubated overnight at 4°C in avidin conjugated to alphamethylcoumarin (AMCA; Vector Labs). Avidin–AMCA was diluted 1:1,000 in PBS containing 0.5% Triton-X 100. After rinsing 3 times in PBS the biocytin-labeled cells were identified with UV illumination (DM-400 Nikon filter) on a Nikon Optiphot microscope.
Immunoidentification of recorded neurons was performed with previously published procedures (Stern et al. 1999; Teruyama and Armstrong 2002). Briefly VP neurons were identified with a rabbit antiserum specific for VP-neurophysin (provided by Alan Robinson, UCLA Medical School) at a 1:20,000 dilution, and then revealed by a fluorescein-conjugated goat anti-rabbit secondary antibody (1:200). OT neurons were labeled with a mouse antibody PS 36 (provided by Harold Gainer, National Institutes of Health) specific for OT-neurophysin, at a dilution of 1:1,000, and then revealed by a Texas Red–conjugated goat anti-mouse secondary antibody (1:200; Vector or Sigma Chemicals). All antibodies were diluted with PBS containing 0.5% Triton X-100. Incubation times for primary and secondary antibody cocktails were 16–24 h at 4°C. Figure 1 illustrates an immunolabeled neuron using this protocol.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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SUPRAOPTIC NEURONS POSSESS A KV3-LIKE TRANSIENT AND PERSISTENT OUTWARD K+ CURRENT.
Because MNCs are known to possess Ca2+-dependent K+ currents (Cobbett et al. 1989: Li and Ferguson 1996), including SK (Kirkpatrick and Bourque 1996) and BK varieties (Dopico et al. 1999), we tested for Kv3-type currents with reduced Ca2+ (lowering [Ca2+]o to 0, and adding 3 mM EGTA) in the ACSF. Under these conditions, MNCs exhibited a fast-activating, transient outward current, and a more slowly inactivating outward current when depolarized from –100 to +50 mV (Fig. 2). Previous studies have characterized a large IA-type (low threshold and inactivating) K+ current in MNCs (Bourque 1988; Fisher et al. 1998; Hlubek and Cobbett 1997; Li and Ferguson 1996; Nagatomo et al. 1995). To remove the influence of IA, we subtracted records taken at holding potentials of –50 mV from those taken at –100 mV. As expected, the IA-type current was effectively inactivated at –50 mV, and was insensitive to 1 mM TEA. Thus we used a holding potential of –50 mV, and by subtraction studied only the TEA-sensitive portion of the current, as shown in Fig. 3. TEA-subtracted currents were activated with a threshold of –20 to –10 mV, and all neurons contained a fast transient (finact = 10–30 ms) and a more slowly inactivating (sinact = 1–2 s) component.
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), on currents evoked with a step from –50 to +50 mV in both VP and OT neurons. A Hill function was fit to all points in the dose–response curve (n = 6–11 for each dose). No cell was tested with more than 4 doses of TEA, but typically only 2 doses were tested on a particular cell. Figure 4 shows that the fast and slow components of the outward current were attenuated by TEA with a similar sensitivity, and this was true of both OT and VP neurons. In both cell types, the inhibition of the transient (peak) current was well fit by the Hill function up to a dose of 1 mM TEA. The 1/2 maximal value for the single fits were 0.29 ± 0.05 mM for OT neurons, and 0.21 ± 0.07 mM for VP neurons. For the persistent current, the dose–response curve was well fit only to doses 0.7 mM TEA (Fig. 4). The 1/2 maximal values for the inhibition of this component were similar between the 2 cell types: 0.2 ± 0.06 and 0.19 ± 0.11 mM, for OT and VP neurons, respectively. Whereas VP and OT neurons were similarly sensitive to doses of TEA <1 mM, the difference in the proportion of the slower current inhibited at 1 mM was much greater for VP than for OT neurons (P < 0.0025). Thus about 75% of the slow current in VP neurons was blocked at this dose.
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; Rudy et al. 1999). To compare OT and VP neurons, we examined the fastest (finact) and slowest (sinact) time constants of inactivation from TEA-subtracted currents evoked at a single step, from –50 to +50 mV. Within each cell type, we also compared currents sensitive to 0.7 or 1 mM TEA, given the dose–response differences determined above. We found no significant difference in finact or sinact between OT and VP neurons, at either dose of TEA (Table 1). We also found no dose effect for either finact or sinact within either cell type. Even though these experiments were done in nominal extracellular Ca2+, to further rule out the possibility of TEA acting on BK channels activated from intracellular Ca2+, we tested the effect of 1 mM TEA on 7 (4 VP, 3 OT) neurons recorded with 2 mM BAPTA instead of 0.2 mM EGTA in the pipette. The TEA-subtracted current with this stronger chelation showed amplitudes and time constants in both the fast (1.9–4 nA; 17–96 ms) and slow components (2.3–5.6 nA; 510–1,166 ms) within the same range of those neurons recorded in 0.2 mM EGTA.
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of the rapidly deactivating portion of the tail current was 1.2 ± 0.5 ms in VP neurons (n = 7) and 1.4 ± 0.4 ms in OT neurons (n = 7). The slower was 9.9 ± 7.7 ms in VP neurons and 9.3 ± 5 ms in OT neurons. Neither component was statistically different between cell types.
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finact of 15.4 ± 6.7 ms, the slow component with a sinact of 1.0 ± 0.25 s, values similar to those recorded in slices at this dose. Over the range of +10 to +50 mV, neither finact nor sinact showed a consistent voltage dependency (not shown). In general, the slope and half activation of the persistent current matched well to Kv3-like currents in expression systems, whereas for the transient current the 1/2 activation is 10–20 mV more hyperpolarized than Kv3.3/3.4-like currents (Coetzee et al. 1999; Fernandez et al. 2003; Rudy et al. 1999).
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Although most Kv1 family subunits underlie persistent currents, in association with accessory 
subunits some Kv1 subtypes form a transient K+ current that may be sensitive to low doses of TEA (Heinemann et al. 1996; Rhodes et al. 1997) and to DTX (e.g., Kv1.1, 1.2, and 1.6). We tested DTX at a concentration of 100 nM (n = 7) and found no significant effect on either the peak (–1.1 ± 8%) or the persistent component (–6.4 ± 7.9%) (Fig. 7). Even a dose of 1 µM failed to reduce the current (n = 2; not shown). Thus it is unlikely that the high-voltage components studied here derive from DTX-sensitive, Kv1-like currents in the SON.
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Kv3.4 currents in homomeric expression systems are strongly inhibited by the sea anemone peptides BDS-I and -II, in the nM range (Diochot et al. 1998). In 7 MNCs tested in slices (including 2 VP and 1 OT neuron), BDS-I (100 nM) reversibly blocked a small portion of the peak high-threshold current compared with controls (Fig. 8) (12.9 ± 9%; P < 0.03; n = 7). A smaller portion of the persistent current also was blocked (8.7 ± 7.8%; P < 0.02).
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To determine the functional contribution of Kv3-like currents in the SON, we examined the effects of TEA and BDS on action potentials (APs) in slices. To compare our results with the voltage-clamp experiments, Ca2+ was removed from the ACSF. Spike broadening in response to TEA was dose-dependent (Fig. 9). Whereas sigmoid plots were well fit to doses to 1 mM for 1/2 width, for base width these plots deviated at 1 mM in both VP and OT neurons. This was reminiscent of the dose–response curves for block of the persistent current (Fig. 4), where 1 mM TEA begins to block an additional, slow current. Although there was little difference in the 1/2 maximal dose for TEA between cell types, the amount of AP broadening, induced by TEA at base width, was greater in VP neurons. Doses of 0.4 (P < 0.002), 0.6 (P < 0.0009), and 1 mM (P < 0.003) TEA produced significantly greater broadening at the AP base in VP neurons. The difference between the 2 types at 1/2 width was significant only for 1 mM TEA (P < 0.006). TEA-sensitive currents, including Kv3 types, appear to have a greater role for AP repolarization in VP versus OT neurons. As a further control for the possibility that intracellular Ca2+ may allow some BK channel activity during spikes, we tested 1 mM TEA with 2 mM BAPTA in the pipette instead of 0.2 mM EGTA. TEA still potently broadened spike 1/2 half-width and base width in both cell types (n = 10 for each type) to degrees similar to those observed in 0.2 mM EGTA (VP: 1/2 width ratio = 194 ± 24%; base width ratio = 302 ± 107%; OT: 1/2 width 167 ± 10%; base width ratio = 177 ± 19%; P < 0.005 for both cell types).
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). If this were the reason, then it would suggest some difference in OT and VP neurons in this regard. To determine the impact of TEA-sensitive currents on firing rate, neurons were depolarized with a 60-pA, 500-ms pulse. Because of the deviation from a sigmoid at 1 mM TEA in spike broadening as well as the suppression of a slower current (see above), neurons were tested at 0.6 and 1 mM TEA, in both OT and VP neurons. As shown in Fig. 10, both 0.6 and 1 mM TEA reduced the evoked firing frequency to a similar degree, in both OT and VP neurons. No differences were found between neuron types, or between the response at 0.6 and 1 mM TEA. Interestingly, both neuron types showed a small degree of rate-dependent broadening during these evoked trains that was still present after TEA treatment. However, because TEA affected the evoked spike frequency, and spike broadening is known to be frequency dependent, we did not statistically compare rate-dependent broadening among these groups.
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Specific immunoreactivity in the SON was observed with the antibody raised against the Kv3.1b subunit (Figs. 11–13). Although some of the strongest labeling was in VP processes, the somata were also clearly labeled. Labeled processes were similar in morphology and location to both the dendrites and the axons of SON neurons (Armstrong et al. 1982). Thus many thicker processes were found in the ventral dendritic lamina, and fine-beaded processes were observed dorsally and throughout the hypothalamo-neurohypophysial pathway, including the neural lobe (Fig. 12). This reactivity was strongly suppressed with absorption of the Kv3.1b peptide (Fig. 12). Antibodies against the Kv3.4, 3.2, and 3.3 peptide did not result in detectable staining in the SON or the neurohypophysial tract. Double-labeling experiments revealed that the Kv3.1b immunoreactivity was associated more strongly with VP neurons (Fig. 13), but was weakly associated with OT neurons. Staining was also present in the MNCs within the PVN (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Weiser et al. 1994, 1995). However, the SON appears labeled in a low-power micrograph in Fig. 2b of Weiser et al. (1995), using a Kv3.1b antibody generated against the identical, c-terminal amino acid sequence (residues 567–585) as the Alomone Labs antibody used herein. Weiser et al. (1995) commented on the numerous labeled axons in the hypothalamus, citing this as a possible reason for the absence of Kv3.1b mRNA in this region. Although we also observed many labeled hypothalamic axons, the labeling of SON dendrites and somata was unambiguous.
Many electrophysiological analyses of MNCs have revealed high voltage–activated, slowly inactivating K+ currents (Cobbett et al. 1989; Hlubek and Cobbett 1997; Li and Ferguson 1996; Luther et al. 2000). However, with one exception (Hlubek and Cobbett 1997), there has been no evidence for Kv3-like activity among these currents. Hlubek and Cobbett (1997) reported an additional transient outward current that activated at much higher voltages than IA, and which was strongly modulated by external Ca2+. Except for the Ca2+ dependency, this current appears similar to the transient K+ current we studied. As for the Ca2+ dependency, Hlubek and Cobbett (1997) determined that the suppression of the guinea pig transient current with Cd2+- or Ca2+-free media was not the result of a shift in voltage dependency. In the present study, the transient current was robust and present in all neurons recorded with negligible [Ca2+]o. Whether the Ca2+ dependency in guinea pig represents a real species difference from rat or is the result of other technical differences between this and the data of Hlubek and Cobbett (1997), remains to be determined. In addition, both the transient and persistence currents were still strongly affected by TEA after increasing Ca2+ buffering with 2 mM BAPTA, suggesting little contribution of BK channels to the effects described herein.
The transient, TEA-sensitive Kv3-like current
Whereas the 1/2 activation voltage of the transient K+ current in dissociated cells was 10–20 mV hyperpolarized to most Kv3-type currents in expression systems, the threshold, inactivation kinetics, and sensitivity of this current to TEA strongly imply the presence of Kv3.3/3.4 subunits (Coetzee et al. 1999; Fernandez et al. 2003; Rudy et al. 1999). Unlike the persistent current (see following text), the inhibition of the transient current with TEA was well fit by a Hill plot up to a concentration of 1 mM, where about 40% of the peak current was blocked. We determined a small but consistent effect of a low (100 nM) dose of BDS-I on the transient current, consistent with expression of the Kv3.4 subtype. The IC50 for the BDS-I block of Kv3.4 channels in expression systems is in the nM range (Abbott et al. 2001; Diochot et al. 1998), whereas the BDS-I sensitivity of natively expressed channels either can be similar (Chabbert et al. 2001) or in the low micromolar range (Abbott et al. 2001; Riazanski et al. 2001). Interestingly, coexpression of the Kv3.4 splice variant Kv3.4a with Kv3.1b type channels can produce a K+ current profile much like what we observed here, both natively in globus pallidus neurons and after transfection in HEK cells (Baranauskas et al. 2003). The coassembly of these subunits in HEK cells shifted the voltage dependency of the Kv3.4 channels negatively, resembling the pallidal neurons that were also shown to have coassembling Kv3.4a and Kv3.1 mRNA. Unfortunately, we could not verify the presence of either Kv3.3- or Kv3.4-like subunits in the SON with immunochemistry. It is difficult to conclude much with negative immunochemical results, so further work will be necessary to verify the molecular configuration underlying this current. Indeed, a single Kv3.4 subunit in heterogenic Kv3 channel clusters can dramatically alter the Kv3 current profile and impart a transient component (Weiser et al. 1994). Thus a low expression may account for the lack of reactivity. In addition, the Kv3.3 antibody we used is specific to the Kv3.3a splice variant, so it might be possible that some Kv3.3b went undetected. It is also clear that activation, and steady-state inactivation voltages, may vary widely for Kv3.3/3.4 transient currents (e.g., Chabbert et al. 2001; Martina et al. 2003). Thus we exert caution in interpreting our data as a quantitative estimate of the relative proportion of transient-type current because it is possible that a portion of the current was inactivated at –50 mV. The main point is that a high-threshold, transient current exists, and it fits a Kv3.3–3.4-type profile.
The persistent TEA-sensitive Kv3-like current
Delayed rectifier type K+ currents have often been observed in MNCs (Cobbett et al. 1989; Hlubek and Cobbett 1997; Li and Ferguson 1996; Luther et al. 2000), but there has been little characterization of their voltage dependency and precise sensitivity to TEA. This is the first report to suggest that at least a portion of the slowly decaying component of a delayed rectifier current in the SON involves Kv3.1 channels, which activate at similar voltages and show slow inactivation kinetics and high TEA sensitivity in expression systems (Coetzee et al. 1999; Rudy et al. 1999). Whereas a range of inactivation time constants may be associated with Kv1 type subunits, these channels typically activate at lower voltages than reported here, and, like Kv2 subtypes, are relatively insensitive to TEA. Furthermore, several (i.e., Kv1.1, 1.2, and 1.6) are sensitive to DTX in the low nM range. The DTX effect on many Kv1 channel subtypes is reasonably selective and preferable to another blocker, 4-AP (Stansfeld et al. 1986; Wu and Barish 1992), which at even low concentrations (
100 µM) inhibits a number of Kv1, Kv2, and Kv3 family channels. In combination with the voltage dependency, the absence of even a small effect of 100 nM -DTX would argue against the strong presence of Kv1.1, Kv1.2, and Kv1.6 channel subtypes underlying the TEA-sensitive macroscopic current we examined. As mentioned above, the small sensitivity of the persistent current to BDS-I could suggest some heterogenic expression of Kv3.1/3.2 and Kv3.4 subtypes (Baranauskas et al. 2003).
Whereas low doses of TEA (0.05–0.7 mM) suppressed the persistent current consistent with a single site of action, at 1 mM TEA a distinctly larger portion of this current was blocked. This was especially marked in VP neurons, where the amount of persistent current blocked jumped from about 20% at 0.7 mM to about 75% at 1 mM. This would be consistent with additional high-threshold K+ channel subtypes in MNCs. Based on this deviation, it might be assumed that non–Kv3-like currents account for much of the persistent current in MNCs. There are many possibilities for these additional currents, but Kv2 family tail currents natively expressed in GP neurons show an IC50 of about 0.8 mM TEA (Baranauskas et al. 1999). Alternatively, it is striking that the expression of Kv3.1b immunoreactivity is much greater in VP than in OT neurons, despite a roughly similar inhibition of persistent current at the lower doses of TEA. Future studies need to examine more precisely the molecular configuration of Kv subunits (e.g., presence of splice variants), and relate these more precisely to activation, deactivation, and inactivation parameters in identified OT and VP neurons, under conditions with optimal space clamp and series resistance. As stated previously, the difficulty posed by coexpression of OT and VP mRNA offsets the advantages offered by dissociated cells to address these issues.
Functional significance of Kv3 channels for AP repolarization and firing
The presence of Kv3-like activity in neurons has previously been associated with large K+ currents in fast spiking cells, such as cortical interneurons, or some brain stem auditory neurons (Rudy and McBain 2001). Whereas MNCs demonstrate both repetitive and burst firing under different conditions, the maximal firing rates achieved under most physiological challenges in vivo are typically 10–20 Hz (Poulain and Wakerley 1982; Poulain et al. 1988). The exception would be the brief (3–4 s) and periodic (10–20 min) OT cell bursts that occur during lactation, and can reach a frequency of 30–80 Hz (Richard et al. 1988). In either case, the maximal firing rates achieved are strongly related to the maximal facilitation of hormone release from terminals in the neural lobe (Bicknell 1988). In vitro, both cell types are capable of firing at rates from 50 to 100 Hz for only brief periods (<1 s), when under strong intracellular depolarization (Stern and Armstrong 1996; Teruyama and Armstrong 2002). Sustained firing rates in both OT and VP neurons types are largely limited by prominent calcium-dependent afterhyperpolarizing potentials that produce spike frequency adaptation (Armstrong et al. 1994; Bourque and Brown 1987; Greffrath et al. 1998), and these channels are diminished in fast-spiking neurons (Erisir et al. 1999). Thus it is interesting that, similar to their role in fast-spiking neurons (Rudy and McBain 2001), inhibition of the Kv3-like channels with low doses of TEA suppressed depolarization-induced firing rates in MNCs. Because calcium-dependent afterhyperpolarizations in MNCs typically require spike trains for strong activation and the effects of TEA are present after the first spike (Fig. 10), Kv3-like channels would serve to maximize the initial firing rate response in MNCs.
We found a strong, submillimolar TEA sensitivity to spike width in both OT and VP neurons with a 1/2 maximal dose (for single fits) similar to that observed for suppressing the Kv3-like currents in voltage clamp, suggesting Kv3-like currents participate in spike repolarization in MNCs. Furthermore, at a low dose (100 nM) the more specific Kv3.4 channel blocker, BDS-I, also broadened spikes at 1/2 width. The latter result may suggest a more specific role for Kv3.4 activity in spike decay rather than the afterhyperpolarization. Together, these data support that Kv3.1- and Kv3.4-like currents participate in AP repolarization in MNCs. At this point, it is unknown whether these subunits exist as homomeric or heteromeric channels in SON neurons. In general, Kv3 subtypes are rapidly activating, rapidly deactivating, and have a high threshold, giving rise to questions as to how they can make a significant contribution to APs that remain depolarized for only brief periods. However, given sufficient channel density, these features ensure that Kv3 subtypes primarily affect the repolarization phase of APs (Rudy and McBain 2001). In addition, recent studies show that heteromeric expression of Kv3.4 and Kv3.1 subunits allows a stronger contribution of Kv3 types to AP repolarization by shifting the 1/2 activation voltage negatively (Baranauskas et al. 2003), much like what we observed for the peak TEA-sensitive current in dissociated cells. Interestingly, much like the difference in block at 1 mM TEA of the fast versus the persistent K+ current, the dose response for TEA broadening of the base, but not the half-width, of the AP deviated at 1 mM. Again, this may indicate the participation of other Kv families in AP repolarization. Their stronger contribution to width near the end of spike repolarization is reminiscent of the inferred contribution of BK-type Ca2+ channels to APs in these neurons after iberiotoxin application, which affects spike width only at the base (Dopico et al. 1999; Roper et al. 2003). The similar actions of TEA on both 1/2 and base spike width with 2 mM BAPTA versus 0.2 mM EGTA further suggests little contribution of BK channel activation from spikes when omitting Ca2+ from the ACSF. It is thus likely that a Kv channel of different activation and/or inactivation kinetics from Kv3 types or BK channel mediated currents also makes a contribution to spike repolarization in MNCs.
The differential effects of TEA on VP and OT neuron APs could relate to the stronger expression of Kv3.1b immunoreactivity in VP neurons. Whereas the similar 1/2 maximal dose for TEA in the 2 cell types indicates participation of Kv3 channels in both cell types, the stronger effect of TEA on the AP base width in VP neurons, even at submillimolar doses, suggests Kv3 channels, probably of the Kv3.1b subtype, have a greater importance to VP neurons. In this regard it is worth noting that not only are many of the parameters of activation and inactivation of Kv3-like channels unknown between the 2 cell types, we did not estimate current density attributed to the probable errors contributed by dendritic components to whole cell capacitance measurements. Thus although we observed qualitatively and even quantitatively similar current profiles in slices for both cell types, for the reasons discussed above regarding space clamp and series resistance, the currents could be quantitatively different. However, examining the effects on spike width does not suffer from these same pitfalls, and presumably the differential ability of TEA to affect spike width reflects a difference among the currents underlying spikes in the 2 cell types.
Both VP and OT neurons undergo a similar degree of spike broadening during repetitive firing (Stern and Armstrong 1996; Teruyama and Armstrong 2002). This frequency-dependent broadening is strongly calcium-dependent (Bourque and Renaud 1985; Hlubek and Cobbett 2000) and may be important for facilitating the calcium influx into somata and dendrites that is necessary for the local, calcium-dependent release of VP, OT, and co-localized peptides like dynorphin (Ludwig 1998). Previous studies have shown that frequency-dependent broadening is controlled by a rate-dependent inactivation of K+ currents, including IA-type currents, as well as an increase in Ca2+ influx in MNCs (Hlubek and Cobbett 2000; Kirkpatrick and Bourque 1991; O'Regan and Cobbett 1993). In cortical pyramidal neurons, increases in spike width similar to those seen during rate-dependent broadening in MNCs (approximately doubling the width) provide a proportional increase in calcium entry (Stewart and Foehring 2001). Frequency-dependent spike broadening is blocked by 1 mM 4-AP, but is present, albeit with much altered time course, during a large dose (5 mM) of TEA (Hlubek and Cobbett 2000). Given the importance of IA to activity-dependent broadening and its stronger association with VP neurons (Fisher et al. 1998; Stern and Armstrong 1997), it is somewhat surprising that no differences have been reported in broadening between the 2 neuron types in normal animals. In pregnancy and lactation, however, frequency-dependent broadening is more pronounced in OT than in VP neurons (Stern and Armstrong 1996; Teruyama and Armstrong 2002). The currents responsible for this change during reproductive state are unknown, although if changes in IA and/or calcium currents underlie this increase, the diminished role of Kv3-like currents in OT neurons may be favorable to this enhanced broadening. Similarly, if high-voltage calcium channel density were higher in VP neurons, the increased presence of Kv3-like channels would allow considerable restraint of rate-dependent broadening, and thus spike frequency fidelity, without sacrificing the calcium influx necessary to activate the several calcium-dependent potentials that underscore burst activity in these neurons (Roper et al. 2003).
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: W. E. Armstrong, Department of Anatomy and Neurobiology, University of Tennessee Medical School, 855 Monroe Avenue, Memphis, TN 38163 (E-mail: warmstrong{at}utmem.edu).
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) and TEA-treated (
) current in Bc. Insets in Ba and Bb show voltage protocols used to evoke the currents.
, persistent component) for VP neurons (a; n = 7) and OT neurons (b; n = 9).
