Reduced outward K+ conductances generate depolarizing after–potentials in rat supraoptic nucleus neurones

Authors


Abstract

  • 1Whole-cell patch clamp recordings were obtained from sixty-five rat supraoptic nucleus (SON) neurones in brain slices to investigate ionic mechanisms underlying depolarizing after-potentials (DAPs). When cells were voltage clamped around -58 mV, slow inward currents mediating DAPs (Idap), evoked by three brief depolarizing pulses, had a peak of 17 ± 1 pA (mean ± s.e.m.) and lasted for 2.8 ± 0.1 s.
  • 2No significant differences in the amplitude and duration were observed when one to three preceding depolarizing pulses were applied, although there was a tendency for twin pulses to evoke larger Idap than a single pulse. The Idap was absent when membrane potentials were more negative than -70 mV. In the range -70 to -50 mV, Idap amplitudes and durations increased as the membrane became more depolarized, with an activation threshold of -65.7 ± 0.7 mV.
  • 3I dap with normal amplitude and duration could be evoked during the decay of a preceding Idap. As frequencies of depolarizing pulses rose from 2 to 20 Hz, the times to peak Idap amplitude were reduced but the amplitudes and durations did not change.
  • 4A consistent reduction in membrane conductance during the Idap was observed in all SON neurones tested, and averaged 34.6 ± 3.3%. Small hyperpolarizing pulses used to measure membrane conductances appeared not to disturb major ionic mechanisms underlying Idap, since the slope and duration of Idap with and without test pulses were similar.
  • 5The Idap had an averaged reversal potential of -87.4 ± 1.6 mV, which was close to the K+ equilibrium potential. An elevation in [K+]O reduced or abolished the Idap, and shifted its reversal potential toward more positive levels. Perifusion of slices with 7.5–10 mm TEA, a K+ channel blocker, reversibly suppressed the Idap.
  • 6Both Na+ and Ca2+ currents failed to induce an Idap-like current during perifusion of slices with media containing high [K+]o or TEA. However, the Idap was abolished by replacing external Ca2+ with Co2+, or replacing 82% of external Na+ with choline or Li+. Perifusion of slices with media containing 1–2 μm TTX also reduced Idap by 55.5 ± 9.0 %.
  • 7These results suggest that the generation of DAPs in SON neurones mainly involves a reduction in outward K+ current(s), which probably has little or no inactivation and can be inhibited by [Ca2+]i transients, due to Ca2+ influx during action potentials and Ca2+ release from internal stores. Na+ influx might provide a permissive influence for Ca2+-induced reduction of K+ conductances and/or help to raise [Ca2+]i via reverse-mode Ca2+-Na+ exchange. Other conductances, making minor contributions to the Idap, may also be involved.

The supraoptic nucleus (SON) is composed of magnocellular neurosecretory neurones which synthesize vasopressin or oxytocin and mainly project to the neurohypophysis. In response to physiological stimuli such as dehydration, haemorrhage, vaginal dilatation and nipple suction, one or both of these magnocellular neurone types increase the release of hormones into the blood, which then act at peripheral tissue targets to retain body fluids, contract the uterus and induce milk ejection (for review see Hatton, 1990). Hormone secretion has been shown to be associated with particular neuronal activities in SON neurones. For example, phasic patterns of firing, bursts of action potentials interrupted by various periods of silence, maximize vasopressin release, while continuous patterns of firing favour oxytocin release (Bicknell & Leng, 1981). Because of its functional role in vasopressin release, phasic firing has attracted much attention from investigators over the years. Among the most important elements known to contribute to the generation of phasic firing in SON neurones is a slow depolarization following the action potential, the so-called depolarizing after-potential (DAP) (Andrew & Dudek, 1984; Andrew, 1987; Armstrong, Smith & Tian, 1994; Li, Decavel & Hatton, 1995). The DAPs last for hundreds of milliseconds to seconds and have amplitudes up to 10 mV, depending on the number of preceding spikes and membrane potential levels at which they are evoked (Andrew & Dudek, 1984; Andrew, 1987). Resembling those identified in invertebrate bursting pacemaker neurones (Thompson & Smith, 1976; Kramer & Zucker, 1985a) and other mammalian CNS neurones (Llinás, 1988; White, Lovinger & Weight, 1989), DAPs as well as phasic firing in SON neurones are abolished by depleting extracellular Ca2+, blocking membrane Ca2+ channels or chelating cytosolic free Ca2+ (Inenaga, Akamatsu, Nagatomo, Ueta & Yamashita, 1992; Li et al. 1995). Our recent study has further demonstrated that interference with Ca2+-induced Ca2+release from internal stores, with either blockade of intra-cellular ryanodine receptors or depletion of internal Ca2+ pools, reduces DAPs and eliminates phasic firing in SON cells (Li & Hatton, 1997).

Although previous experiments have demonstrated that an elevation in intracellular Ca2+ concentration ([Ca2+]i), due to Ca2+ influx during action potentials and Ca2+ release from internal stores, is essential for the formation of DAPs, data regarding ionic mechanisms underlying these potentials are both scarce and confusing. For example, increased membrane resistances are found to be associated with DAPs, while treatments with tetraethylammonium (TEA), a K+ channel blocker, or raised [K+]o, often fail to cancel them. Non-specific cation currents, Na+ current and/or non-inactivating K+ current have been hypothesized to play roles in generating DAPs (Bourque, 1986; Andrew, 1987; Smith & Armstrong, 1993; Armstrong & Smith, 1994). In this study, therefore, we used whole-cell voltage clamp recording methods in SON neurones in rat brain slices to reinvestigate the ionic basis of DAPs. The evidence obtained here supports the hypothesis that Ca2+-induced inhibition of outward K+ conductances is responsible for the formation of Idap and will be helpful in understanding phasic firing.

METHODS

Methods and techniques used for obtaining brain slices and whole-cell patch recordings have been described elsewhere (Li et al. 1995; Li & Hatton, 1996a, 1997). In brief, adult (50–70 days old) male Sprague-Dawley rats were decapitated using a rodent guillotine (Stoelting Co., Wood Dale, IL, USA). Their brains were removed and cooled in ice-cold (∼4 °C), oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF). The ACSF contained (mm): NaCl, 126; KCl, 5; NaHPO4, 1.3; CaCl2., 2.4; MgSO4, 1.3; NaHCO3, 26; and glucose, 10 (305 mosmol l−1, pH 7.4). Horizontal slices of the hypothalamus (250–300 mm thick) were cut using a Vibratome. After incubation at room temperature (23 °C) for at least 2 h, the shoes were transferred to a submerging recording chamber and perifused with warmed ACSF (36 °C). In some experiments, K+ concentration in ACSF was raised, or TEA (Aldrich) was added to ACSF and osmotic pressure was balanced by reducing Na+ concentration. When C concentration in ACSF was reduced from 135.8 to 10.4 mm (i.e. low Cl medium), glucuronate was added for osmotic balance.

Patch electrodes were pulled from borosilicate capillary tubing and filled with the following solution (mm): potassium gluconate, 140; MgCl2, 2; Hepes, 10; KATP, 2; Li2, GTP, 0.4 (pH 7.25). Electrodes usually had outer tip diameters of ∼2.7 μm and DC resistances around 5MΩ. Whole-cell patch clamp recordings were obtained from SON neurones using an Axoclamp-2B amplifier (Axon Instruments). Patch electrodes approached the SON under the guidance of a dissecting microscope. Once electrode tips touched neuronal membranes, an increase in voltage responses was seen, which was induced by passing hyperpolarizing current through electrodes. Gigaolim seals between electrodes and cell membranes were then obtained by applying gentle suction. When a seal was achieved, further brief suction was applied to break through the membrane. Whole-cell recordings were indicated by observation of negative membrane potentials and action potentials. KCl-agar bridges were used as reference electrodes and correction of liquid junction potential (-7mV) was applied to recorded membrane voltage.

In current clamp mode, the presence of DAPs in a neurone was first examined by applying depolarizing current pulses (5 ms, 0.1–0.2nA) to elicit action potentials at a membrane potential below spike threshold. As previously reported (Armstrong et al. 1994; Li et al. 1995), about half of SON neurones encountered displayed DAPs. In neurones with DAPs, continuous single-electrode voltage clamp was then performed to investigate membrane currents mediating DAPs (Idap). The membrane potentials were held around -58mV. Feedback gain was set at 10–50nAmV−1 and low-pass filter at 1.0kHz. The Idap was a slow, apparent inward current following brief depolarizations produced by voltage steps (5 ms, 80 mV). These short depolarizations, used to mimic action potentials, always induced fast inward (Na+ and Ca2+) and outward (K+) currents known to occur during spikes. However, it was impossible (neither was it our intention) to accurately measure those currents during short depolarizations, due to space-clamp limitations and the large amplitudes. Series resistances were evaluated by measuring instantaneous currents evoked by a hyperpolarizing pulse (10 mV, 20 ms) and ranged between 4 and 10 MΩ. Since amplitudes of IDAp obtained in the present experiments were always less than 40 pA and series resistance compensation was approximately 50%, voltage error between a true membrane potential and the measured potential would have been less than 0.2 mV. Except where otherwise indicated, each test protocol was run 8 times at an interval of at least 15 s and consecutive current traces were then averaged. Membrane input resistance was measured at the end of voltage responses evoked by hyperpolarizing current pulses (40 pA, 1 s). Idap duration is the period from the beginning of depolarizing pulses to the return of the inward current to baseline, while Idap amplitude refers to the maximal value of the inward current compared with baseline. The change in the Idap was determined by comparing ion currents obtained before and during perifusion of slices with test agents. All data acquisition and analyses were performed using an IBM compatible computer operating AXOTAPE and pCLAMP (Axon Instruments).

To reduce current noise due to synaptic inputs to the SON, blockers of glutamate (NMDA and non-NMDA) and GABAA receptors were perifused in some experiments. These drugs (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μm; RBI, Natick, MA, USA), 2-amino-5-phosphonovaleric acid (APV, 50 μm; Sigma) and bicuculline methiodide (20 mm; Sigma)), were dissolved in ACSF for perifusion. Choline chloride, CoCl2, CsCl, LiCl, tetrodotoxin (TTX) and other chemicals used were purchased from Sigma.

RESULTS

I dap , a slow inward current following brief depolarization

Whole-cell patch clamp recordings were obtained from sixty-five SON neurones. Their resting membrane potentials, amplitudes of action potentials and membrane input resistances were -59.9 ± 0.6 mV (mean ± s.e.m.), 87.2 ± 0.7 mV and 415 ± 16 MΩ, respectively. In current clamp mode, DAPs following single evoked spikes were observed in all these cells (Fig. 1A), and characterized by slow time courses for both the rise and decay, dependence on the membrane potential and spatiotemporal summation, as described previously (Andrew & Dudek, 1984; Andrew, 1987; Li et al. 1995; Li & Hatton, 1997). When neurones were voltage clamped, slow inward currents were revealed following depolarizations elicited by brief voltage pulses (Fig. 1B). The inward currents evoked by three depolarizing pulses at holding potentials (Vh) around -58 mV, reached an averaged peak of 17 ± 1 pA within hundreds of milliseconds and lasted for 2.8 ± 0.1s (n= 65), an amplitude and duration that correspond well to DAPs observed in current clamp recordings. With little variation in size, they could be stably evoked for 1–2 h before the membrane seal was broken. Similar inward current was not observed in SON neurones without DAPs. Therefore, the slow inward currents seen after brief depolarizations are designated as Idap in the present study.

Figure 1.

Depolarizing after-potentials (DAPs) and related membrane currents (Idap) observed in a supraoptic nucleus (SON) neurone

A, whole-cell current clamp record demonstrating DAPs (arrows) following action potentials evoked by brief current pulses. The DAPs were able to be superimposed, and induced spontaneous action potentials when they were large enough to reach the spike threshold (right). Note that spikes are trimmed to show the DAPs. B, current record obtained from the same neurone showing slow Idap (arrowhead) following 3 brief depolarizations (5 ms, 80 mV). The membrane potential was held at -60 mV. In B, and all following figures the insets in the right upper corner are voltage records, and both current and voltage records are trimmed.

Effects of the number of preceding pulses and dependence on membrane potentials

Although double pulses tended to evoke larger Idap than a single pulse in most neurones tested, significant differences in the amplitude and duration of Idap were not observed when one to three preceding depolarizing pulses were applied (Fig. 2A–D). A further increase in the number of depolarizing pulses (≥ 4) actually reduced the amplitude of Idap. When depolarizing pulses with a longer duration (100 ms) were applied, the Idap was also smaller but could last for a longer period (Fig. 2E). Because outward currents immediately following depolarizing pulses and concomitant reduction in Idap amplitudes were often observed with larger pulse numbers or longer durations, double or triple depolarizing pulses of 5 ms were routinely used in the present experiments in order to obtain large Idap and minimize possible contamination by other currents.

Figure 2.

Relationship between Idap and the number, or the duration, of preceding depolarizations

A, B and C show the Idap evoked by 1–3 depolarizing steps, respectively. D is a bar graph summarizing current amplitude data obtained from 9 cells. E displays the Idap evoked by a long-duration depolarization (100 ms, 80 mV). Arrowheads (B, C and E) indicate prominent outward currents induced when the number or duration of preceding depolarizations was increased. Records in A -C and E were obtained from the same SON neurone.

Voltage dependence of Idap was examined using a current subtraction method as shown in Fig. 3. First, currents were recorded by clamping the membrane potential from a Vh to various levels with (A) and without (B) three preceding depolarizing pulses. The Idap was then obtained by subtracting currents shown in B from those in A (Fig. 3C). This protocol was chosen for use since it can diminish possible influences of many voltage-dependent conductances such as hyperpolarization-activating cation current (Erickson, Bonnekleiv & Kelly, 1993), persistent Na+ current (Li & Hatton, 1996b) and non-inactivating outward rectification (Stern & Armstrong, 1995). The results showed that the Idap was absent when membrane potentials were more negative than -70 mV (Fig. 3C and D). In the range -70 to -50 mV, Idap amplitudes and durations increased as the membrane became more depolarized, with an activation threshold of -65.7 ± 0.7 mV (n= 9). The data regarding Idap at membrane potential levels above -50 mV were not available because depolarizing steps (as shown in Fig. 3B) themselves induced fast inward (Na+ and Ca2+) currents followed by the Idap. The profile of voltage dependence of Idap is consistent with our previous studies using whole–cell current clamp recording methods (Li et al. 1995; Li & Hatton, 1997).

Figure 3.

Voltage dependence of Idap

To minimize current contamination, subtraction methods were applied to examine changes in the Idap according to the membrane potential. Current traces were obtained from a SON cell when clamping the membrane potential to various levels with (A) or without (B) preceding depolarizing steps. Subtracting B from A reveals that the Idap increases as the membrane potential becomes depolarized. Voltage dependence was not tested at membrane potential levels more positive than -50 mV, because clamping the membrane potential to those levels itself evoked action potentials followed by Idap. All records are single current traces without being averaged. D is a normalized I–V histogram summarizing data obtained from 9 SON cells, each tested at -75 to -50 mV. Abscissa, membrane potentials; ordinate, Idap amplitudes compared with that at -50 mV. The averaged activation threshold is -65.7 ± 0.7 mV.

Non–inactivation and frequency responses

In hypothalamic magnocellular neurones, most voltage–gated membrane currents, for example Ca2+ currents, transient outward K+ current, and fast Na+ current (Bourque, 1988; Fisher & Bourque, 1995; Li & Ferguson, 1996), inactivate to a certain extent during continuous depolarization. If the Idap declined due to current inactivation, subsequent depolarizing pulses given following or during Idap would evoke an inward current with reduced amplitude. In this study, the presence of Idap inactivation was examined using a protocol as shown in Fig. 4, where test pulses (b) were delivered at various periods after the maximal or near-maximal activation of Idap by conditioning pulses (a). When test pulses were applied after the preceding Idap declined, no significant differences were observed in the amplitude and duration between Idap evoked by the conditioning and test pulses (top 3 traces of Fig. 4). For example, the amplitude and duration of the test Idap were 88.7±4.1 and 95.0±3.9% (n= 7) of the conditioning Idap, respectively, with an interval of 3 s. When the interval between the conditioning and test pulses was further shortened and test pulses occurred before the decay of the preceding Idap was complete, the inward current evoked by test pulses still had an amplitude and duration similar to that of the preceding Idap (lowest 3 traces). With an interval of 0.6 s, the amplitude and duration of the test Idap were 93.1 ± 5.1 and 102.3 ± 3.5% of the conditioning ones (n= 7), respectively, (these values were obtained by measuring the conditioning Idap of the 4.6 s interval protocol shown in Fig. 4). Therefore, the test pulses can immediately activate another Idap during the decayed portion of Idap without any time delay for recovery. Our findings that the Idap could be rapidly re-activated and superimposed suggest that membrane channels responsible for Idap have little or no inactivation, and their activities probably depend on the change in intracellular ionic milieu following spikes.

Figure 4.

I AP re-activation

Current records showing that another Idap with a similar amplitude and duration could be evoked shortly after, or even during, the decay of a preceding Idap. This suggests that Idap declines are not due to current inactivation. All records are single current traces without being averaged.

Investigation of Idap frequency dependence was prompted by the finding that DAP amplitudes and durations varied little when the interval of current pulse trains used to evoke action potentials was changed (Li & Hatton, 1997). In this study, manipulation of pulse intervals of a stimulus train was applied to study the relationship between pulse frequency and Idap (Fig. 5). Both the current amplitude and duration were measured while pulse intervals of a three-pulse train were reduced. When the interval was 500 ms (2 Hz), shorter than the half-time of Idap, and subsequent pulses occurred before the inward current evidently decayed, a larger Idap with spatiotemporal current summation was observed. As pulse frequencies were increased from 2 to 20 Hz, the times to peak were reduced in all six cells tested; however, summed Idap amplitudes and durations did not significantly change. When the amplitudes and durations of Idap evoked by 2 Hz pulses were taken as the control (100%), a 5 Hz pulse-evoked Idap had an amplitude of 96.2 ± 3.6% and duration of 98.3±2.6%; while a 20 Hz pulse-evoked Idap had an amplitude of 102.2 ± 3.8% and duration of 104.2 ± 4.9% (for all, n= 6). Prominent outward currents were induced by high frequency depolarizing pulses and slightly increased latencies (relative to the last spike) of the maximal current. Responses to higher frequencies were not tested.

Figure 5.

I dap induced by pulses of varying frequency

Current traces demonstrating that the times to peak, i.e. the period from the beginning of depolarizing pulses to the maximal value of the inward current (b), were shortened as the frequency of depolarizing steps increased. Increased latencies relative to the last spike (a) of the maximal current (b) were probably due to the prominent outward currents (arrowheads) induced by high frequency depolarization. All current records are single traces without being averaged.

Reduced membrane conductance during Idap

In a total of fifty-two SON neurones, whole-cell membrane conductances before (control) and during Idap were measured using hyperpolarizing pulses (5–10 mV, 200 ms). Conductance comparison revealed a consistent reduction in the membrane conductance during Idap in each of these cells (Fig. 6A and Aa); the mean ± s.e.m. decrease was 34.6 ± 3.3% when the Idap was around its maximal value. Such decreases were found to be present both during the current peak and decay (Fig. 6D and E), and thus represent a fundamental change in the membrane conductance during Idap.

Figure 6.

Reduction in whole-cell membrane conductance during Idap

A, B and C, records obtained from the same SON cell showing current responses to small hyperpolarizing pulses used to measure the membrane conductance. Note the differences in the amplitude and slope of current responses acquired before (arrowheads) and during (arrows) Idap. Aa shows enlarged, superimposed segments of current trace A, demonstrating distinct slope differences and a reduced amplitude of the current response during Idap (y) in comparison with the control (x). The slope of the current response is consistent with that of Idap and, by simply moving the current response up (Ab, arrow), Idap was seen with a reasonably smooth decay. (See F for another routine method used to compare current slopes.) D and E are current records obtained from another cell showing that reduced membrane conductances could be observed both during the peak and decay of Idap, F overlapped current traces with and without small hyperpolarizing voltage pulses applied to measure membrane conductance. Voltage pulses did not change the amplitude and time course of Idap and, in particular, the slope of the current response was similar to that of Idap (asterisk). In this cell, a large (+15 pA) DC current was necessary to clamp the membrane to -59 mV, which offsets some current response to a hyperpolarizing pulse and allows direct comparison of current traces. G, traces obtained from another cell showing that large (20–40 mV) hyperpolarizing pulses diminished the reduction in membrane conductance. Conductance reduction: 10 mV pulse, 38%; 20 mV pulse, 22%; 40 mV pulse, 15%.

As described above, the Idap is voltage sensitive and membrane hyperpolarization can reduce it. It is possible, therefore, that the conductance reductions observed could have been due to hyperpolarization-induced deactivation of membrane channels responsible for generating Idap. To minimize this possibility, protocols with small hyperpolarizing steps (1–2 mV) were also used to measure membrane conductance in this study, and revealed a similar reduction in whole-cell membrane conductance (49.4 ± 3.1%, n= 8; P > 0.05 when compared with that obtained using 5–10 mV steps; Fig. 6B and C). In current responses to hyperpolarizing pulses of 1–5 mV, Idap durations with and without test pulses applied were similar (Fig. 6F, n= 36). In thirty-four of fifty-two neurones in which membrane conductances were measured during Idap decay, and in which current responses to hyperpolarizing pulses during Idap were different from the control, the slopes of current responses were found to be similar to that of Idap during which no test pulse was used (Fig. 6Ab and F). Thus, these test pulses appeared not to disturb major ionic mechanisms underlying Idap. Since large (20–40 mV) voltage responses evoked by injecting current pulses are often seen in measurement of the membrane input resistence during DAPs (Andrew, 1987; Armstrong & Smith, 1994), we examined the change in the amount by which conductance was reduced using large voltage steps, and found that conductance reduction decreased with 20–40 mV hyperpolarizing pulses (Fig. 6G, n= 4). These findings strongly suggest that the Idap is associated with reduced membrane conductance(s).

Reversal potential (Vrev) for Idap and effects of high [K+]o and TEA perifusion

Subtraction protocols, as described in the study of voltage dependence, were also applied to resolve the Vrev for Idap. The membrane potentials were clamped to various levels when the Idap reached the peak (Fig. 7Aa). Subtraction of other membrane currents, evoked by those hyperpolarizing commands (Fig.7Ab), revealed the alterations in Idap according to the membrane potential. The Idap was reduced as the membrane hyperpolarized and was converted into an outward current when membrane potentials were -98 mV (Fig. 7Ac). An averaged Vrev for Idap obtained in this study was -87.4±1.6mV (n= 11), which is close to the K+ equilibrium potential (EK, -89.4 mV).

Figure 7.

Reversal potential (Vrev) for Idap

Subtraction methods were applied to examine the Vrev for Idap in a SON neurone perifused with ACSF containing 5 mm (A, control) or 10 mm (B) K+. The membrane potential was clamped to various levels when the Idap reached the peak, yielding the resultant current traces (a), b, membrane currents evoked by hyperpolarizing voltage steps alone, c, current traces after subtraction (a–b) showing the change of Idap according to membrane potentials. All records are single current traces without being averaged. Note a reduced Idap and increased membrane fluctuations after raising [K+]o for 10 min (B). C, instantaneous I–V relationship revealing that the Idap decreased and became an outward current as the membrane was hyperpolarized. Current values of Idap were measured at the onset of different voltage steps (Ac, arrowhead), and membrane potentials at which Idap amplitudes were zero were taken as the Vrev. D, graph showing that the Vrev for Idap obtained in various [K+]o is consistent with changes in the EK (dashed line) according to [K+]o; values in parentheses indicate the number of cells. EK was calculated using the Nernst equation: -61–27 log(144/[K+]o) (mV).

Changing the EK affected the Idap and shifted its Vrevtoward more positive membrane potentials. An elevation in [K+]o from 5 to 10 mm reduced the Idap (n= 8 of 8; Fig. 8) and produced an averaged Vrev for Idap of -68.8 ± 1.7 mV (n= 5, Fig. 7B and C). Further raising [K+]o to 20 mm abolished the Idap in all ten cells tested (Fig. 8) and an averaged Vrev for Idap was -54.7 ± 1.6 mV (n= 6, Fig. 7D). These effects were often accompanied by unmasked fast inward currents (Fig. 8, arrowheads), which could be blocked by Na+ and Ca2+ channel blockers, TTX and Co2+.

Figure 8.

Inhibitory effects of high [K+]o on Idap

A and B, records obtained from two different SON cells before, during and after consecutive perifusion of slices with ACSF containing high [K+]o. Records shown in b and c were obtained 8–9 min after the beginning of high K+ ACSF, while records shown in Ad and Bd were obtained 10 and 13 min, respectively, after washout of the high K+ media with control ACSF (5 mm K+). When 20 mm K+ ACSF was perifused, the Idap was either abolished (Bc) or converted into an outward current (Ac). Note different Vh values in A and B. Reduced K+ currents usually unmasked fast inward (Na+ and Ca2+) currents (arrowheads), which lasted for less than 200 ms.

Perifusion of slices with 7.5–10 mm TEA, a K+ channel blocker, for 8–10 min reversibly suppressed the Idap (n=9 of 9, Fig. 9); mean ±s.e.m. suppression was 83.3 ± 7.2% when cells were voltage clamped around -58 mV. In five cells where membrane conductances were continuously monitored, TEA treatment was seen to significantly diminish the conductance reduction during Idap (from 38.7±7.4% in the control to 9.4±5.0% following treatment; P < 0.05), suggesting a specific effect on the conductance(s) mediating the Idap. Unmasked fast inward Na+ and Ca2+ currents during TEA perifusion were also observed in most of the cells tested. Perifusion of slices with Cs+ (2–3 mm), a specific blocker of inward rectifier currents, did not affect the Idap (n= 6 of 6, data not shown).

Figure 9.

TEA, a K+ channel blocker, suppresses the IAP

A and B, current traces acquired from two different SON cells before, during and after perifusion of slices with ACSF containing 10 mm TEA. Records in the middle were obtained 15 min (Ay) or 6 min (Bb) following the onset of TEA perifusion, and records on the right 23 min (Ac) or 7 min (Be) after washout of TEA with control ACSF. Blockade of K+ currents always diminished the reduction in membrane conductances, and fast inward (Na+ and Ca2+) currents (Ab, asterisk) were often revealed. Note that TEA treatment enhanced an outward (probably K+) current (Ab, arrowhead) in only 1 of 9 cells tested.

Dependence on Ca2+ and Na+ influx

Because they are eliminated by depleting extracellular Ca2+, blocking membrane Ca2+ channels, or chelating cytosolic free Ca2+ (Li et al. 1995; Andrew, 1997; Li & Hatton 1997), the DAPs in SON neurones are thought to depend on Ca2+ influx. Consistent with this hypothesis, the Idap was found in the present experiments to be abolished by replacement of external Ca2+ with the same concentration of Co2+ (n= 9, Fig. 10A). Perifusion of slices with media containing 1–2 μm TTX also reduced Idap by 55.5±9.0% (n= 10, Fig. 10B); an increase in TTX concentrations (up to 6–7 mm) did not cause further reduction. However, lowering [Na+]o by replacement of 82 % of external Na+ with choline (n= 6) or Li+ (n= 4) reversibly abolished the Idap in all ten cells tested (Fig. 10C); these effects appeared 4–6 min following choline or Li+ treatments and the Idap, often with an enhanced amplitude, could be seen 6–15 min after restoring [Na+]o to the control level.

Figure 10.

A reduction in Ca2+ (A) or Na+ (B and C) influx abolishes or reduces the IDAf

A, current traces showing replacement of external Ca2+ with the same concentration of Co2+ (2 mm), a Ca2+ channel blocker, abolished the Idap within 5 min (A, middle trace). Only partial recovery of Idap was observed 22 min following washout with control ACSF, suggesting a prolonged effect of Co2+. B, blockade of Na+ channel by adding 2 mm TTX into ACSF reduced the Idap, but prolonged treatment with a higher concentration of TTX (14 min, 6 mm) failed to cause a further reduction in the Idap (B, middle and right traces). C, lowering [Na+]o by replacing 82% of external Na+ with choline eliminated the Idap within 4 min (C, middle trace), which recovered and was enhanced 8 min after washout with control ACSF (C, right trace). D, replacement of 82% of external Na+ with Li+ also abolished the Idap within 5 min (middle trace). Partial recovery was seen following 13 min washout. Note that Li+ perifusion always (4 of 4 cells) induced current fluctuations, and a sustained inward current (not shown). E, ACSF containing low Cl (10.4 mm) failed to cancel the Idap or to diminish the reduction in membrane conductance. Current record in the middle was obtained 11 min after low Cl ACSF perifusion started, and the record on the right 6 min following washout with control ACSF. Records in A-E were obtained from five different SON neurones.

To examine whether Cl conductance is involved in Idap generation, we perifused SON neurones with a low (10.4 mm) Cl medium, which shifts the chloride equilibrium potential from -93.8 to -25.4 mV. If the Idap resulted from a reduction in Cl conductance, treatment with this medium should reverse or at least eliminate the Idap. This appears not to be the case, however, because low Cl medium was found not to reduce, but actually enhance the Idap by 40–50% (n = 3 of 3, Fig. 10D). Such augmentation was accompanied by a prominent increase in the conductance reduction (from ∼38 to ∼80%). Therefore, it is possible that in the control ACSF Cl influx might participate in Idap generation, with an inhibiting effect. The reason that basic membrane conductances were inhibited by the low Cl medium containing glucuronate remains to be clarified.

DISCUSSION

Several lines of evidence obtained from the present experiments suggest that DAP generation in SON neurones mainly involves a reduction in outward K+ conductance(s). First, the Idap was associated with a decrease in membrane conductance. To minimize the possibility that reduced membrane conductance had resulted from deactivation of voltage-dependent current(s) during hyperpolarizing pulses, we used voltage pulses that were as small as possible to measure the membrane conductance and found a similar conductance reduction. Furthermore, the findings that current responses to small hyperpolarizing pulses before and during Idap were different and that hyperpolarizing pulses did not greatly change time courses and current slopes strongly argue against this possibility. If voltage pulses had instantaneously closed major membrane channels responsible for generating the Idap, current responses during Idap would have resembled that obtained in the control (Kramer & Zucker, 1985b). If voltage pulses had slowly closed these channels, the slope of current responses during Idap would have been different from that of Idap during which no test pulse was used (see Fig. 6Ab and F). These results are also consistent with increased membrane input resistance during DAPs observed in previous experiments using sharp-electrode recording methods (Andrew, 1987; Armstrong & Smith, 1994). Second, the Idap was dependent on K+ concentration gradients across the membrane. The Vrev for Idap was close to Ek. Raising [K+]o reduced or abolished the Idap and shifted the Vrev toward membrane potentials predicted by the Nernst equation. A positive shift of the Ecl by lowering [Cl]o, however, failed to eliminate or reduce the Idap. Third, perifusion of slices with ACSF containing low concentrations of K+ channel blockers reversibly reduced the Idap. Finally, Ca2+ and Na+ influx could not induce a slow inward current like the Idap after K+ efflux was suppressed, further suggesting an indispensable role for K+ conductance(s) in Idap generation.

The results regarding involvement of a K+ conductance(s) obtained from the present experiments are inconsistent with those from previous studies, in which TEA treatments or raising [K+]o (Andrew, 1987) were found not to affect the DAPs. Although determining exact reasons for these disparate findings is not within the scope of this study, some important factors may be considered to have at least contributed to them. For example, DAPs following a train of action potentials, instead of one to three single spikes, were analysed in those early experiments. More membrane conductances can participate in, or influence, DAP formation when depolarization is large and persists for a long period. In those current clamp studies, a reduction in K+ efflux by TEA treatment or raising [K+]o induced prolonged Ca2+ and Na+ influx, whose amounts alone would probably have been large enough to produce DAPs. In addition, the recording techniques used (sharp-electrode or whole-cell patch recording methods) could have contributed to these disparities.

Consistent with voltage dependence of DAPs observed in our recent studies (Li et al. 1995; Li & Hatton, 1997), the Idap began to be seen when membrane potentials were more positive than -70 mV with an averaged activation threshold of -65.7 mV. Idap amplitude and duration increased when membrane potentials became more depolarized. The finding that Idap without reduced amplitudes could be evoked before the preceding Idap ended suggests that Idap decline does not result from current inactivation or reduced driving force due to extracellular K+ accumulation. Therefore, voltage dependence of the Idap probably reflects the availability of related K+ conductance(s) to be suppressed at various voltage levels. Many types of outward K+ currents, such as M-current and delayed outward current (Adams, Brown & Constanti, 1982; Li & Ferguson, 1996), can be activated and then persist with little inactivation while the membrane is depolarized. The more positive the membrane potential is held, the more K+ efflux through membrane channels and, thus, the larger the apparent inward currents that can be generated by suppressing K+ efflux. Conductance availability might at least partially explain why prominent spatiotemporal summation of DAPs was only observed when current clamp recording methods were used. Membrane depolarization due to a preceding DAP(s) would cause larger K+ efflux, suppression of which generates larger DAPs. In a voltage clamp study, however, membrane potentials are held and K+ conductance(s) available probably vary minimally. It was observed, therefore, that the 7dap could not increase its amplitudes just after several brief depolarizing pulses were applied. Another obvious reason that more depolarizing pulses or longer depolarizing duration did not result in further summation of Idap is that larger outward (Ca2+-dependent K+) currents (Li & Ferguson, 1996) were activated and masked the Idap.

Although membrane events like DAPs are also seen in neurones of other CNS regions, for example, the spinal cord, brainstem, thalamus, hippocampus and neocortex (Dudek, Deadwyler, Cotman & Lynch, 1976; Wong & Prince, 1981; Harada & Takahashi, 1983; Stevens, Gallagher & Shinnich-Gallagher, 1984; Jahnsen & Llinás, 1984), differences between these events and DAPs observed in SON neurones are striking. In general, those DAP-like events have larger amplitudes, last only for a relatively short time (< 200 ms), and are associated with an increase in membrane conductance. T-type Ca2+ current (Llinás, 1988; White et al. 1989; Zhang, Valiante & Carlen, 1993), persistent Na+ current (Azouz, Jensen & Yaari, 1996), Ca2+-activated nonspecific cation current (Kramer & Zucker, 1985a), or Na+-Ca2+ exchange (Friedman, Arens, Heinemann & Gutnick, 1992) have been suggested to be responsible for their generation. In contrast, DAPs in SON cells are abolished by perifusion of slices with either Ca2+-free medium, or L-type Ca2+ channel blockers, but not with T–type Ca2+ channel blockers (Bourque, 1986; Andrew, 1987; Li & Hatton, 1997). Intracellular diffusion of Ca2+ chelators, BAPTA or Ca2+-binding proteins, eliminates the DAPs. We propose, therefore, that DAP generation in SON neuroendocrine cells involves a rapid increase in [Ca2+]i, due to Ca2+ influx via high-threshold channels following an action potential(s) and Ca2+ release from internal stores, which then suppresses a K+ conductance(s). Cytosolic free Ca2+ is known to modulate Na+ and K+ channel gating and induce inward current (Partridge & Swandulla, 1988). Our hypothesis is supported by recent evidence that an elevation in [Ca2+]i or intracellular application of pre-activated Ca2+-dependent phosphatase suppresses a non-inactivating, voltage-gated K+ conductance (M-current) in sympathetic neurones (Marrion, 1996; Selyanko & Brown, 1996).

In this study, we have also observed a reduction in /dap following perifusion of slices with TTX. Taken together with previous findings showing that TTX partially inhibits the DAPs (Andrew, 1987; Smith & Armstrong, 1993), a role for Na+ influx in the generation of DAPs is suggested. Indeed, the Idap was completely abolished by replacing external Na+ with either choline, a Na+ channel-impermeable cation, or Li+, which can pass through Na+ channels but does not activate many intracellular Na+-dependent processes. Na+ entering the cell during action potentials is extruded rapidly, but the Idap is a slow inward current. Na+, as well as Ca2+, influx after reducing K+ efflux was found to induce inward currents with a very short duration. Therefore, involvement of Na+ as a major carrier in Idap formation seems unlikely. It is possible that a rapid elevation in [Na+]i following action potentials participates in the formation of Idap via providing a permissive influence in Ca2+-induced inhibition. In addition, intracellular Na+ has been shown to raise [Ca2+]i by enhancing ‘reverse-mode’ Ca2+-Na+ exchange across the membrane and inducing Ca2+ release from internal stores (Barcenas-Ruiz, Beuckelmann & Wier, 1987; Nuss & Houser, 1992), an effect which favours the generation of DAPs.

The DAPs in SON neurones have been demonstrated to be essential for the formation of phasic patterns of firing, which promote vasopressin release from the neurohypo-physis (Bicknell & Leng, 1981; see Hatton, 1990). They are also subject to modulation by putative neurotransmitters such as histaniine and noradrenaline (Randle, Bourque & Renaud, 1986; Smith & Armstrong, 1993). Investigation of the ionic basis of DAPs is important for understanding neuronal firing activity and its modulation in these neurones. Although a reduction in K+ conductance(s) is suggested to play a major role, many other conductances/mechanisms certainly also participate in generating Idap. At any point in time, the amplitude of Idap represents an algebraic sum of evoked currents, some of which are inward currents and other outward ones. Further experiments are needed to describe the characteristics of K+ conductance(s) related to DAP generation and determine how a rise in [Ca2+]i can suppress them. An interesting finding in this study is the Idap frequency response, i.e. that the times to peak of Idapwere shortened but the durations remained relatively constant as the frequency of depolarizing pulses increased from 2 to 20 Hz. It is likely that Idap amplitudes are proportional to Ca2+ transients, while their durations at least partially reflect Ca2+ diffusion from the submembrane space into the deep cytoplasm, the effects of endogenous Ca2+-binding proteins, sequestration of Ca2+ internal stores and Ca2+ extrusion from the cell (Tsien & Tsien, 1990; Henzi & MacDermott, 1992; Li et al. 1995; Li & Hatton, 1997). Simultaneous measurement of [Ca2+]i during Idap will be helpful to clarify this issue.

Acknowledgements

We thank J. T. Kitasako for technical assistance. This work was supported by NIH research grants NINDS NS16942 and NS09140.

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