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Density of transient K⁺ current influences excitability in acutely isolated vasopressin and oxytocin neurones of rat hypothalamus

Thomas E. Fisher, Daniel L. Voisin and Charles W. Bourque

MS 7953 Received 26 February 1998; accepted after revision 28 May 1998.

	ABSTRACT

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1. The transient outward K⁺ current (I_TO) was studied using whole-cell recording in immunocytochemically identified oxytocin (OT; n = 23) and vasopressin (VP; n = 67) magnocellular neurosecretory cells (MNCs) acutely isolated from the supraoptic nucleus of adult rats.

2. The peak density of I_TO during steps to -10 mV was 26 % smaller in OT-MNCs (355 ± 23 pA pF^-1; mean ± s.e.m.; n = 18) than in VP-MNCs (478 ± 17 pA pF^-1; n = 52). No differences were observed in the voltage dependence of activation or inactivation.

3. Kinetic analysis revealed two components of I_TO inactivation in both OT-MNCs (₁ = 9·2 ± 0·4 ms and ₂ = 41·2 ± 1·6 ms; n = 18) and VP-MNCs (₁ = 12·4 ± 0·4 ms and ₂ = 37·1 ± 1·2 ms; n = 52). Although the density of the rapid component (₁) was not different (275 ± 13 versus 265 ± 16 pA pF^-1, respectively), the slow component (₂) was markedly smaller in OT-MNCs (183 ± 19 versus 331 ± 16 pA pF^-1 in VP-MNCs).

4. In unidentified MNCs, 0·5 mM 4-aminopyridine reduced I_TO amplitude by 29 % and decreased the latency to spike discharge by about 70 % during depolarization from -70 mV. Latency to discharge from potentials less negative than -60 mV, where I_TO is inactivated, was unaffected.

5. Comparison of latency to spike discharge in identified cells showed that OT-MNCs achieve spike threshold twice as fast as VP-MNCs when depolarized from -70 mV. The lower density of I_TO in OT-MNCs, therefore, accelerates the rate at which excitation can occur in response to depolarizing stimuli and may facilitate the occurrence of higher frequency discharges in OT-MNCs during physiological activation.

	INTRODUCTION

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Hypothalamic magnocellular neurosecretory cells (MNCs) are responsible for the release of either vasopressin (VP) or oxytocin (OT) into the bloodstream (Poulain & Wakerley, 1982). Following synthesis in MNC somata, peptides are packaged in vesicles and transported to axon terminals in the neurohypophysis (Brownstein et al. 1980) where secretion is triggered by the arrival of action potentials (Dreifuss et al. 1971). In both MNC types, action potentials are initiated at the soma as a result of interactions between afferent synaptic signals and intrinsic membrane properties (Bourque & Renaud, 1990).

In VP-MNCs in rats, hyperosmolality provokes an increase in firing rate or the emergence of phasic firing (Poulain et al. 1977; Wakerley et al. 1978), a pattern comprising alternating periods of activity (7-15 Hz) and silence lasting tens of seconds each. Although OT-MNCs also increase their firing rate during systemic hypertonicity, they rarely display phasic firing (Poulain et al. 1977; Brimble & Dyball, 1977). In females, however, OT-MNCs selectively discharge brief (2-4 s), high frequency (40-80 Hz) bursts of action potentials during lactation (Wakerley & Lincoln, 1973) and at parturition (Summerlee, 1981). Previous studies in vitro have shown that the release of both peptides increases with firing rate (Dreifuss et al. 1971) and is maximized during bursting activity (Dutton & Dyball, 1979; Bicknell & Leng, 1981; Bicknell et al. 1982). OT release, however, is facilitated over a wider range of stimulus frequencies (Bicknell, 1988) and exhibits less fatigue during maintained repetitive stimulation (Bicknell et al. 1984).

OT and VP neurones thus display functionally important differences in the patterns of spike discharge generated at their somata and in excitation-secretion coupling at their axon terminals. Since OT- and VP-MNCs are similar morphologically (Silverman & Zimmerman, 1983) and in many basic electrophysiological properties, such as resting membrane potential and input resistance (Armstrong et al. 1994; Armstrong, 1995), subtle distinctions in the densities or properties of particular ion channels may underlie these differences (Hatton, 1997). Indeed, while VP-MNCs more frequently express a spike depolarizing after-potential (Armstrong et al. 1994), OT-MNCs display stronger slow outward rectification during depolarizations from negative holding potentials (Stern & Armstrong, 1995, 1997). Recently, it was observed that VP-MNCs display a greater degree of transient outward rectification than OT-MNCs during depolarization from negative membrane potentials (Stern & Armstrong, 1996). A previous study (Bourque, 1988) has shown that transient outward rectification in MNCs is largely due to the activation of a transient outward K⁺ current (I_TO). This current is similar to the inactivating K⁺ current I_A in pharmacology and biophysical properties (Connor & Stevens, 1971a; Rogawski, 1985), but differs in that it appears to be composed of both Ca²⁺-dependent and Ca²⁺-independent components (Bourque, 1988; Li & Ferguson, 1996; Hlubek & Cobbett, 1997; Fisher & Bourque, 1998). A difference in the properties or density of I_TO in OT- and VP-MNCs could be important in determining their distinct electrophysiological behaviour. Indeed, rapidly inactivating K⁺ currents have been found to be important in modulating responses to depolarizing stimuli in MNCs (Bourque, 1988) as well as in other types of neurones (e.g. Connor & Stevens, 1971b; Segal et al. 1984; Banks et al. 1996). Differences in the amplitude, kinetics, or voltage dependence of I_TO, therefore, may contribute to differences in the responsiveness of OT and VP-MNCs to depolarizing stimuli. In the present study, we characterized the properties of I_TO recorded from the somata of MNCs acutely isolated from the supraoptic nuclei of adult rats. Immunocytochemical identification of the recorded cells, as VP- or OT-containing, allowed a quantitative comparison of I_TO under voltage clamp and of electrical excitability under current clamp. Our results indicate that a reduced I_TO density in OT-MNCs accelerates the excitation of these cells upon depolarization and may therefore facilitate high frequency firing during physiological activation. These results have been reported in an abstract (Fisher et al. 1997).

	METHODS

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Preparation of MNC somata

Somata of MNCs were isolated as previously described (Oliet & Bourque, 1992; Fisher & Bourque, 1995). Briefly, unanaesthetized male Long-Evans rats (150-300 g) were killed by decapitation using a small-rodent guillotine (model 51330; Stoelting Company, Wood Dale, IL, USA). Coronal brain slices (1 mm thick) were cut, and blocks of tissue (1 mm³) containing part of the supraoptic nucleus were dissected and incubated for 90 min at 34°C in an oxygenated (100 % O₂) saline comprising (mM): NaCl, 120; KCl, 5; MgCl₂, 1; CaCl₂, 1; Pipes, 20; and D-glucose, 25, pH 7·0, containing trypsin (Type XI; 0·7 mg ml^-1). Tissue blocks were then placed in trypsin-free oxygenated Pipes saline (pH 7·35) at room temperature (for up to 8 h), until triturated with fire-polished pipettes. The dispersed cells were plated onto untreated glass-bottomed recording dishes. These dishes were prepared by drilling an 8 mm hole in the bottom of standard 35 mm plastic Petri dishes and fixing circular glass coverslips to the underside using Sylgard elastomer. All chemicals were acquired from Sigma Chemical Company.

Patch-clamp recordings

Cells were patch clamped in the whole-cell mode at room temperature. The external solution comprised (mM): NaCl, 100; TEA-Cl, 40; KCl, 3; MgCl₂, 1; Hepes, 10; CaCl₂, 2; glucose, 10; and tetrodotoxin (TTX), 0·0005; pH 7·3-7·4. The pipette solution comprised (mM): potassium gluconate, 120; MgCl₂, 3; Hepes, 10; EGTA, 1; adenosine 5'-triphosphate (disodium salt), 2; and phosphocreatine (di-Tris-HCl salt), 14; pH 7·1-7·2. Capacitative currents were minimized by electronic compensation, and leakage currents were not subtracted. Series resistance was kept as small as possible (5-8 M) and was compensated (70-80 %) electronically. Recorded voltage was corrected for a liquid junction potential of 10 mV. For the experiments on latency to excitation, TTX was omitted from the external solution and TEA was replaced by Na⁺. Membrane voltage or current (DC, 5 kHz) was recorded through an Axopatch-1D amplifier (Axon Instruments Inc.) and digitized (1·25-2 kHz) using a Labmaster interface (TL-100, Axon Instruments Inc.). Voltage commands and analyses were performed using pCLAMP software (Axon Instruments Inc.).

Isolation of I_TO in MNCs

Previous studies in hypothalamic explants (Bourque, 1988), or isolated neurones (Fisher & Bourque, 1998), have shown that non-inactivating K⁺ currents in MNCs are blocked by external TEA, whereas I_TO is unaffected by TEA, but is blocked by 4-aminopyridine (4-AP). In isolated MNCs, the I_TO may be extracted from the non-inactivating outward currents evoked from -130 mV either by subtraction (of currents evoked from a holding potential of -60 mV) or by addition of 40 mM TEA (Fisher & Bourque, 1998). Neither method results in currents that differ significantly from the 4-AP-sensitive current evoked in 40 mM TEA (Fisher & Bourque, 1998). Underlying Ca²⁺ currents do not interfere significantly with the measurement of I_TO since only 0·2 nA of inactivating Ca²⁺ current would be evoked by the voltage steps used in our experiments (Fisher & Bourque, 1995). In the present study, therefore, I_TO was simply isolated by the addition of TTX to block Na⁺ currents, and TEA to block non-inactivating K⁺ currents.

Data analysis

The conductance underlying I_TO (G_TO) was calculated as: G_TO = I_TO/(V - V_rev), where V_rev (reversal potential) under these conditions was previously shown to be -79 mV by tail current analysis (Fisher & Bourque, 1998). Data points for the voltage-dependent activation of G_TO were fitted to the Boltzmann equation:

G_TO(V) = GTO_,max/(1 + exp (-(V - V½)/k)),

where, G_TO(V) is the value of G_TO at a given V, and V_½ is the voltage at half-maximum activation/inactivation. Maximal G_TO (GTO,max) was defined as the value calculated from the current recorded in response to a step to +20 mV, whereas the slope factor k was determined by curve fitting. Data describing voltage-dependent inactivation were fitted to the equation:

I(V) = I_max/(1 + exp((V - V½)/k)),

where, I(V) is the current evoked by steps to -10 mV following a 500 ms prepulse to a given V, and I_max is the current recorded following a prepulse to -130 mV.

Time constants characterizing the decay of I_TO components were estimated using the least-squares fitting algorithm of pCLAMP. The program provides an estimate of current amplitude (I) as a function of time (t) according to the equation:

I(t) = A₀ + A₁exp(-t/₁) + A₂exp(-t/₂) + ... A_nexp(-t/_n).

The solution to this equation determines the sum of non-inactivating currents (A_o), and the amplitudes (A_n) and time constants (_n) that best fit the evoked current. In all of our experiments on MNCs, the decay of I_TO was best fitted as the sum of two exponentials.

All data reported in this paper are expressed as means ± standard error of the mean (S.E.M.). Comparisons between groups were made using the Mann-Whitney U test, except in Fig. 5 where measurements made before and after the addition of 4-AP were compared using Student's paired t test. Differences were considered to be significant when P < 0·05.

Immunocytochemistry

Following recording, the patch pipette was carefully displaced from the cell. Three or four spots of ink were then placed on the bottom of the dish with a marking pen such that the patch-clamped cell and surrounding cells (usually no more than 1-4 in total) were enclosed. The recorded cell, and its relation to the spots of neighbouring cells, was then sketched for later identification. Cells were fixed overnight at room temperature with 4 % paraformaldehyde in phosphate-buffered saline (PBS; 0·1 M, pH 7·3). Following three washes with PBS, the cells were placed in PBS containing 1 % normal goat serum and 0·3 % Triton X-100 for 1 h before incubation for 24 h at 4°C with dilutions of mouse monoclonal antibodies (see Acknowledgements) recognizing either OT (AI-28; 1 : 200) or VP (III-D7; 1 : 50) in PBS containing 0·3 % Triton X-100. After three rinses in PBS, cells were treated with tetramethyl rhodamine isothiocyanate-conjugated goat anti-mouse immunoglobulins diluted in PBS (1 : 200; Jackson Immunoresearch Laboratories, USA) for 2 h at room temperature, rinsed again, and subsequently viewed using a Zeiss inverted Axiovert-10 microscope (× 40 objective).

Identification of cells as VP- or OT-MNCs

After the recorded cell was identified under phase contrast (e.g. Fig. 1), the presence of immunolabelling was determined under fluorescence using a rhodamine filter set. Experiments using either no primary antibody or an inappropriate secondary antibody resulted in the absence of specific labelling. With a mixture of both antibodies, 94·6 % (401/424) of the large (> 15 µm diameter) neurones examined were immunopositive, as previously reported (Oliet & Bourque, 1992). In forty-three dishes from nine preparations, where the two antibodies were used separately, 39·8 % of the large cells were found to be positive for OT (310/779) and 57·2 % of cells were positive for VP (155/271). Recordings revealed two types of transient outward current in isolated large cells. A small subset of neurones (< 6 %) exhibited a transient outward current that was slower in onset (time to peak at -10 mV, > 10 ms versus < 5 ms in MNCs; Fisher & Bourque, 1998), more slowly inactivating ( > 60 ms) and smaller in peak amplitude (mean = 2·2 ± 0·4 nA) than I_TO in MNCs (> 4 nA). When exposed to the mixture of both antibodies, none of these cells was immunopositive (0/7), whereas each of seven neurones displaying a 'fast' current was. The small proportion of large neurones that are not MNCs can therefore be distinguished by the slow waveform of their transient outward current. The cells retained for analysis were therefore identified as MNCs jointly on the basis of a large soma and the presence of a I_TO with fast kinetics. Since the antibodies against VP and OT labelled all MNCs with apparently no overlap and patch clamping did not reduce antigenicity (see below), recorded cells immunopositive for one antibody were assumed to contain only the targeted neuropeptide, and recorded cells immunonegative for one neuropeptide were assumed to contain the other.

	RESULTS

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Immunocytochemical identification of MNCs following patch-clamp recording

The photomicrographs in Fig. 1 show representative examples of isolated MNCs that were fixed and identified as VP- or OT-containing by immunocytochemistry following whole-cell patch-clamp recording of I_TO. Fluorescent (immunopositive) neurones could be clearly distinguished from unstained cells, and the recording process did not appear to diminish the intensity of the fluorescent signal in labelled MNCs. Of the ninety cells characterized following patch-clamp recording, sixty-seven were determined to be VP-MNCs and twenty-three were OT-MNCs.

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Figure 1. Immunocytochemical identification of MNCs following whole-cell recording Following patch-clamp recording the electrode was removed, the cells were fixed, and immunocytochemical staining was performed (see Methods for details). The upper panels are fields obtained from separate Petri dishes showing small groups of cells photographed using phase-contrast microscopy. Lower panels show the same fields under conditions where fluorescence reports the presence of either anti-oxytocin (left) or anti-vasopressin (right) antibodies. Note that recording did not diminish the intensity of the fluorescent signal in the recorded cells (indicated by arrows) compared with other labelled cells in the field.

I_TO amplitude in identified MNCs

Although all MNCs recorded under voltage clamp (70/70) displayed a prominent I_TO (e.g. Fig. 2A), there was a difference in the mean peak amplitude of the current measured in OT- and VP-MNCs. As illustrated in Fig. 2B, the current evoked by steps to -10 mV was significantly less in OT-MNCs (4·587 ± 0·268 nA; n = 18) than in VP-MNCs (6·274 ± 0·252 nA; n = 52; U = 202·5, P = 0·0004). This was not due to a difference in the size of the cells recorded (Fig. 2C), since the mean input capacitance of the two groups was not different (13·3 ± 0·8 pF for OT-MNCs versus 13·6 ± 0·5 pF for VP-MNCs; U = 433, P = 0·6381). Current density was therefore significantly lower in OT-MNCs (355 ± 23 pA pF^-1) than in VP-MNCs (478 ± 17 pA pF^-1; U = 198·5, P = 0·0003). For the analysis described above, the eighteen OT-MNCs consisted of cells that were stained with anti-OT antibody (n = 12) and cells not stained with anti-VP antibody (n = 6). Likewise, the fifty-two VP-MNCs consisted of cells that were stained with anti-VP antibody (n = 29) as well as those that were not stained with anti-OT antibody (n = 23). When only cells identified by positive staining were included in the analysis, the mean values for the peak I_TO density were very similar to those given above, (OT: 348 ± 24 pA pF^-1, n = 12; VP: 471 ± 25 pA pF^-1, n = 29) and the difference between OT- and VP-MNCs remained significant (U = 77, P = 0·0054). These data support the validity of our method for the identification of MNCs using either staining or lack of staining with antibodies against VP or OT.

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Figure 2. Amplitude of ITO in identified MNCs A, currents (lower traces), recorded in the cells shown in Fig. 1, which were evoked during depolarizing steps (upper traces) applied following a 500 ms prepulse to -130 mV. B, bar histogram comparing mean peak amplitude of ITO (± S.E.M.) in identified OT- (n = 18) and VP-MNCs (n = 52; ** P < 0·001). C, comparison of the mean (± S.E.M.) input capacitance of the recorded cells. Note that a significantly larger peak ITO amplitude is recorded in the VP-MNCs (B) despite the similar size of the cells recorded.

Voltage dependence of I_TO in OT- and VP-MNCs

Differences in I_TO amplitude measured at a single test voltage could be due to differences in the voltage dependence of activation or inactivation in the two cell types. The voltage dependence of activation of I_TO was therefore studied by stepping the membrane voltage of cells to potentials between -70 and +20 mV (10 mV increments) following a 500 ms conditioning prepulse to -130 mV. A subset of traces recorded from a MNC in response to this protocol is shown in Fig. 3A. The voltage dependence of inactivation was assessed by measuring the peak amplitude of current responses evoked by steps to -10 mV, following a 500 ms prepulse to conditioning voltages between -130 and -40 mV (10 mV increments). A subset of traces recorded from a MNC in response to this protocol is shown in Fig. 3B. In Fig. 3C, the mean activation of I_TO in VP- and OT-MNCs is plotted as normalized conductance (G_TO; see Methods) as a function of test voltage. Fitting these data to the Boltzmann equation (see Methods) indicated that neither the mid-points of activation (V½) nor the slope factors (k) were different between OT-MNCs (V½ = -29·7 ± 1·7 mV; k = 10·5 ± 0·4; n = 5) and VP-NCs (V½ = -32·9 ± 1·1 mV, U = 10, P = 0·0956; k = 10·8 ± 0·3, U = 17·5, P = 0·505; n = 9). Also plotted in Fig. 3C is the inactivation of I_TO, expressed as the relative current amplitude evoked at the test pulse as a function of conditioning voltage. The mid-point of inactivation for OT-MNCs (-74·8 ± 1·6 mV; n = 5) was not different from that for VP-MNCs (-75·0 ± 0·6 mV; U = 31, P = 0·8825; n = 13); however, the slope constant was slightly higher for OT-MNCs (k = 6·5 ± 0·3) than for VP-MNCs (k = 5·6 ± 0·2; U = 11, P = 0·0341). In all cells tested, I_TO was first detected during depolarizing steps to -60 mV, indicating an apparent threshold between -70 and -60 mV.

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Figure 3. Voltage dependence of ITO in identified MNCs A, superimposed current traces evoked by depolarizing steps to potentials between -70 and +10 mV following a 500 ms prepulse to -130 mV (see inset) in media containing TEA (40 mM) and TTX (0·5 µM). B, superimposed current traces, recorded in the same medium, evoked by test depolarizations to -10 mV following 500 ms conditioning prepulses to potentials between -120 and -50 mV (see inset). The inward currents evoked by steps from the most positive voltage shown (-50 mV) are slowly inactivating or non-inactivating calcium currents (Fisher & Bourque, 1995). C, mean (± S.E.M.) activation ( and ) and inactivation ( and ) plots for ITO in OT-MNCs ( and ) or VP-MNCs ( and ). Inactivation plots show average current at -10 mV, following steps to the indicated voltages, normalized to the amplitude recorded following a step to -130 mV. Activation plots show relative conductance as a function of voltage. Continuous and dashed lines are fits of the OT and VP data, respectively, to the Boltzmann equation (see Methods).

Kinetics of inactivation of I_TO in OT- and VP-MNCs

Differences in I_TO amplitude measured at a single test voltage could be due to differences in the inactivation kinetics of the current in the two cell types. Curve-fitting analysis (see Methods) determined that the decaying phase of the I_TO in MNCs is best described as the sum of two exponential functions with distinct amplitudes and time constants. Figure 4A shows the current evoked by stepping from -130 to -10 mV in a representative cell, as well as a superimposed curve calculated from the best fit obtained by a double exponential function. Application of this procedure provided numerical values for the time constants and amplitudes of the two components of I_TO inactivation in all identified MNCs. The mean time constants of the fast (₁) and slow (₂) components were slightly different in OT-MNCs (₁ = 9·2 ± 0·4 ms and ₂ = 41·2 ± 1·6 ms; n = 18) and VP-MNCs (₁ = 12·4 ± 0·4 ms, U = 112, P = 0·0001 and ₂ = 37·1 ± 1·2 ms, U = 276, P = 0·0099; n = 52). Moreover, a selective difference was identified in the relative densities of the fast and slow components (Fig. 4B). Thus, although the densities of the fast component were not different in OT-MNCs (275 ± 13 pA pF^-1) and VP-MNCs (265 ± 16 pA pF^-1, U = 404, P = 0·3935), the slower component was significantly smaller in OT-MNCs (183 ± 19 pA pF^-1) than in VP-MNCs (331 ± 16 pA pF^-1, U = 128·5, P = 0·0001). Note that the sum of the two amplitudes exceeds the measured peak current values since the curve-fitting program extrapolates the current amplitude to the beginning of pulse onset, whereas measurement of the observed peak current reflected partial inactivation.

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Figure 4. Kinetics of inactivation of ITO in identified MNCs A, current evoked by a step to -10 mV following a prepulse to -130 mV. Curve fitting of the decay shows that the time course of inactivation is best fitted as the sum of two exponentials (fit superimposed upon trace). The horizontal arrow at the top indicates the peak current amplitude and the arrow at the bottom points to the end of the current trace. Extensions beyond the arrows are extrapolations of the fit. B, amplitudes of fast (1) and slow (2) components of ITO in OT- () and VP-MNCs (). Bars give means (± S.E.M.); ** P < 0·001.

Role of I_TO in determining latency to excitation

The activation of transient K⁺ currents is believed to delay excitation in response to suprathreshold depolarizing pulses applied from hyperpolarized potentials in both MNCs (Bourque, 1988; Schrader & Tasker, 1997) and other neurones (e.g. Connor & Stevens, 1971a). To determine whether the 26 % smaller peak I_TO density observed in OT-MNCs could play a significant role in modulating responsiveness, we examined the effects of a low concentration of 4-AP on the latency to excitation during depolarization from various holding potentials in a group of unidentified MNCs. In preliminary voltage-clamp experiments, addition of 0·5 mM 4-AP was found to reduce I_TO by 29 ± 7 % (n = 3). This concentration of 4-AP was therefore used to test whether a reduction in the amplitude of I_TO comparable with the difference between OT- and VP-MNCs could influence the latency to excitation. The analysis, performed on five spontaneously active MNCs (no TTX added), consisted of measuring the latency to first spike upon switching from voltage clamp at a given subthreshold holding potential (V_H) to current clamp (no holding current). Figure 5 shows the effects of V_H on the latency to first spike upon release to current clamp. Consistent with the voltage dependence of I_TO inactivation (Fig. 3C), latencies were stable at potentials less negative than -60 mV, but progressively increased as V_H was made more negative. As illustrated in the same figure, causing a 29 % reduction in I_TO amplitude (0·5 mM 4-AP) reduced the latency to excitation at potentials more negative than -60 mV, but not at more positive potentials.

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Figure 5. Effect of partial blockade of ITO on latency to first spike Graph shows the mean latency to spike discharge in unidentified MNCs (± S.E.M.) plotted against the holding potential (VH). The difference between the control () and treated groups () was significant at -70 mV (P = 0·0096; Student's paired t test). Inset illustrates the protocol used for latency analysis. Following the maintenance of a constant VH under voltage clamp (> 2 s; not shown), the amplifier was switched to current clamp (Release clamp, vertical arrow). The time between the arrow and the action potential was taken as the latency to excitation.

Latency to excitation in OT- and VP-MNCs

The above data suggest that since OT-MNCs possess a smaller I_TO amplitude, and since this current is important in the control of spike latency in MNCs, there may be a shorter latency to first spike in OT- than in VP-MNCs. To test this hypothesis, latencies to first spike were determined following release from voltage clamp using a V_H of -50 mV, where differences in I_TO density have no effect, and a V_H of -70 mV, where a pronounced I_TO density-dependent delay is observed (Fig. 5). As illustrated in Fig. 6A, latency to first spike following release from a V_H of -50 mV was similar in OT- and VP-MNCs. When released from a V_H of -70 mV, however, the latency was longer in VP-MNCs. When expressed as a ratio (latency at -70 mV/latency at -50 mV; Fig. 6B) the latency to excitation was significantly longer in VP-MNCs (28 ± 3; n = 15) than in OT-MNCs (13 ± 1; n = 5; U = 6, P = 0·006).

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Figure 6. Latency to excitation in identified OT- and VP-MNCs A, voltage recordings from a spontaneously active OT-MNC (upper traces) and a spontaneously active VP-MNC (lower traces). The cells were held at either -50 mV (left) or -70 mV (right) under voltage clamp for several seconds (not shown) and were suddenly released to the current-clamp mode (beginning at each arrow, 0 pA injected). The time between the arrow and the action potential is defined as the latency to excitation. B, bar histograms showing the mean (± S.E.M.) latency to excitation following release from -70 mV expressed relative to that at -50 mV. Note that the relative latency in OT-MNCs is less than 50 % of that in VP-MNCs (* P < 0·01).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Previous studies in hypothalamic explants (Bourque, 1988) and slices (Schrader & Tasker, 1997), or in acutely isolated neurones (Hlubek & Cobbett, 1997; Fisher & Bourque, 1998), have shown that all MNCs exposed to Ca²⁺-containing saline express a prominent I_TO under voltage clamp. The presence of this current has been shown to be largely responsible for the transient outward rectification observed during depolarization from negative potentials under current clamp (Bourque, 1988) and to modify excitability when modulated by neurotransmitters (Schrader & Tasker, 1997). In a recent study, quantification of voltage responses recorded in immunocytochemically identified neurones revealed that the degree of transient outward rectification is significantly lower in OT-MNCs than in VP-MNCs (Stern & Armstrong, 1996). We therefore sought to determine (i) if differences exist in the density or properties of I_TO in OT- and VP-MNCs, and (ii) whether any observed difference might confer upon these cells a differential electrical excitability.

A somatic Ca²⁺-dependent current dominates I_TO under physiological conditions

Many of the properties of I_TO in MNCs (Bourque, 1988; Hlubek & Cobbett, 1997; Fisher & Bourque, 1998) are similar to those of the transient K⁺ current I_A in other neurones (e.g. Rogawski, 1985): low voltage threshold, rapid activation and inactivation kinetics, sensitivity to 4-AP and insensitivity to TEA. Additionally, the voltage dependence of I_TO, like that of I_A (Mayer & Sugiyama, 1988), has been found to be affected by external application of inorganic divalent cations, presumably acting at an extracellular site (Li & Ferguson, 1996; Hlubek & Cobbett, 1997; Fisher & Bourque, 1998). However, experiments with sharp microelectrodes in intact MNCs (Bourque, 1988) and with whole-cell patch-clamp recording in acutely isolated MNC somata (Fisher & Bourque, 1998) have shown that the amplitude of I_TO can be reduced by application of organic Ca²⁺ channel antagonists, such as nifedipine or -conotoxin GVIA, suggesting that a component of I_TO in MNCs is dependent on Ca²⁺ influx. The composite I_TO, therefore, appears to be carried by both Ca²⁺-dependent and Ca²⁺-independent channels. A recent study in acutely isolated neurones revealed that, in Ca²⁺-free Co²⁺-containing solutions, a transient K⁺ current is expressed in OT-MNCs, but not in VP-MNCs (Widmer et al. 1997). This differential pattern of expression of the Ca²⁺-independent component of I_TO is opposite to that which would be expected to yield the enhanced transient outward rectification observed in VP-MNCs in hypothalamic explants (Stern & Armstrong, 1996). However, at 0 mV, the mean transient K⁺ current density recorded from OT-MNCs in Ca²⁺-containing solutions reported here (393 pA pF^-1) is almost 20 times greater than that reportedly present in OT-MNCs exposed to Ca²⁺-free solutions containing Co²⁺ (20 pA pF^-1; Widmer et al. 1997). This observation suggests that the Ca²⁺-dependent component of the current dominates the composite I_TO recorded in physiological solutions. In this study, therefore, we compared the density and properties of the composite I_TO recorded in OT- and VP-MNCs superfused with calcium-containing media in the absence of calcium channel blockers.

VP-MNCs express a higher density of I_TO than OT-MNCs

Voltage-clamp analysis of I_TO in immunocytochemically identified neurones isolated from adult rats showed a 26 % lower peak current density in OT-MNCs than in VP-MNCs. This suggests that an enhanced density of the Ca²⁺-dependent component of I_TO is responsible for the greater degree of transient outward rectification previously observed in VP-MNCs (Stern & Armstrong, 1996). Characterization of the decaying phase of I_TO further revealed that the difference in peak current amplitude was due to a relative difference in the amplitude of the slower of two kinetically distinct components of current inactivation. Thus, while the density of the rapidly inactivating ( 10 ms) component did not differ in the two types of cells, the density of the slow ( 40 ms) component was almost 50 % lower in OT- than in VP-MNCs (Fig. 4). It is presently unclear if distinct channel types mediate the different inactivating components of the macroscopic I_TO. From a molecular perspective, the different inactivation rates of the fast and slow components, and the different densities of the slowly decaying component in OT- and VP-MNCs could be due either to a differential expression of distinct types of K⁺ channel subunits, or to a difference in the post-translational regulation of one (or more) channel type. Interestingly, recent immunocytochemical studies have shown that Kv4.2 -subunits, which encode channels mediating transient K⁺ currents in heterologous expression systems (Pongs, 1992; Robertson, 1997), are expressed in MNC somata (Alonso & Widmer, 1997). Further studies will be required to determine the relationship between these K⁺ channel subunits and the different components of I_TO.

Reduced I_TO density shortens the delay to excitation in OT-MNCs

Modelling studies by Connor & Stevens (1971b) indicated that when suprathreshold depolarizing stimuli are applied from hyperpolarized potentials, the transient outward current I_A can dominate the early ionic current response of the cell. Since the generation of an early outward current shunts the inward current underlying the depolarizing stimulus, the rate at which the membrane capacitance becomes discharged is attenuated and the voltage approach to spike threshold is delayed. Consequently, the presence of a transient K⁺ current can introduce a lag before action potential threshold is achieved in response to depolarizing stimuli applied from negative potentials. The data presented here support the hypothesis that I_TO plays a role in regulating the excitability of MNCs by increasing the latency to excitation when the MNCs are depolarized from membrane potentials more negative than -55 mV (Fig. 5). In a group of unidentified MNCs, a 29 % decrease in peak I_TO amplitude, provoked by the application of 4-AP, caused a two-thirds decrease in the spike latency at -70 mV, but not at -50 mV (Fig. 5). Furthermore, the latency to excitation at -70 mV (normalized to that at -50 mV; Fig. 6B) was about twice as fast in OT-MNCs than in VP-MNCs, which have a 26 % larger I_TO density. The different I_TO density observed in OT- and VP-MNCs, therefore, could play an important role in regulating cellular excitability in situ. In particular, our results suggest that at membrane potentials where I_TO is not inactivated, OT-MNCs may be more readily, and rapidly, excited during synaptic activation.

The voltage dependence of I_TO predicts a role in regulating excitability

Previous work has shown that the resting potential of both types of MNC lies near -65 mV, both in hypothalamic explants at 34-37°C (Bourque & Renaud, 1990; Armstrong et al. 1994) and in isolated cells at room temperature (Oliet & Bourque, 1992). Since a significant proportion of the current is not inactivated at this potential (Fig. 3C), it is likely that I_TO could play an important role in regulating the excitability of MNCs under resting conditions. Interestingly, previous studies have shown that the membrane potential of MNCs becomes hyperpolarized under hypotonic conditions and depolarized during increases in osmolality (Oliet & Bourque, 1993). The contribution of I_TO to the electrical responsiveness of MNCs, therefore, could presumably be modulated by the osmotic state of the animal. Under hypotonic conditions, for example, hyperpolarization of the cells would enhance the magnitude of outward currents activated during the rising phase of excitatory postsynaptic potentials (EPSPs), thereby reducing the probability of excitation. Conversely, inactivation of I_TO resulting from membrane depolarization would facilitate EPSP-dependent spike discharge and contribute to the excitation of MNCs under hypertonic conditions (Bourque et al. 1994). The differential excitability of OT- and VP-MNCs that results from a lower expression of I_TO in OT-MNCs, therefore, might be enhanced under hypotonic conditions and reduced during systemic hypertonicity.

Possible role of I_TO during phasic firing in VP-MNCs

Electrophysiological studies in vivo have shown that physiological stimuli that provoke VP release elicit the expression of phasic activity in VP-MNCs (e.g. Brimble & Dyball, 1977; Poulain et al. 1977; Wakerley et al. 1978; Poulain & Wakerley, 1982), a pattern of activity that optimizes hormone secretion from neurosecretory terminals in the neurohypophysis (Dutton & Dyball, 1979; Bicknell & Leng, 1981; Bicknell, 1988). Intracellular recordings in vitro have shown that transitions between silent and active periods during phasic firing are associated with the emergence of a depolarizing plateau potential that moves the membrane potential from a voltage more negative than -60 mV to a value near, or positive to, -55 mV (Bourque et al. 1998). By affecting the responsiveness to excitatory synaptic drive, changes in steady-state inactivation of I_TO resulting from such changes in membrane potential may contribute to the overall differences in electrical excitability that are associated with active and quiescent episodes during phasic firing in VP-MNCs.

OT-MNCs are specialized for high frequency firing

Recent studies have indicated that OT-MNCs may express electrophysiological properties which favour the production of high frequency bursts of action potentials (Stern & Armstrong, 1996, 1997; Hatton, 1997). Indeed, while synchronous high frequency bursts (> 40 Hz) are not observed in VP-MNCs under physiological conditions (Poulain & Wakerley, 1982), such discharges are produced by OT-MNCs both during lactation (Wakerley & Lincoln, 1973) and at parturition (Summerlee, 1981). The generation of brief synchronous high frequency bursts in OT-MNCs is necessary to provoke the release of OT as a bolus into the systemic circulation. While the basis for synchronicity remains unknown, the high frequency discharges of OT-MNCs are believed to result from synaptic activation (Wakerley & Ingram, 1993). Since a reduced I_TO may permit faster excitatory responses to synaptic activation, the lower density of this current in OT-MNCs may represent a functional specialization designed to facilitate the production of high frequency discharges.

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Acknowledgements

We thank Dr A. J. Silverman for her generous supply of antibodies (produced by Anna Hou-Yu) and S. H. R. Oliet for his comments on the manuscript. Our work was supported by an operating grant and Scientist Award from the MRC of Canada to C. W. B., by fellowships from the Canadian Heart and Stroke Foundation and the Fonds de la Recherche en Santé du Québec to T. E. F. and by a fellowship award from Le Programme Lavoisier (Ministère des Affaires Étrangeres, France) to D. L. V.

Corresponding author

C. Bourque: Division of Neurology, Montreal General Hospital, 1650 Cedar Avenue, Montreal, QC, Canada H3G 1A4.

Email: mdbq{at}musica.mcgill.ca

Authors' present addresses

T. E. Fisher: 1-117A Medical Sciences Building, Mayo Clinic, Rochester, MN 55905, USA.

D. L. Voisin: INSERM U-378, Institut Francois Magendie, 1 rue Camille, Saint-Saens, 33077, Bordeaux Cedex, France.

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