 |
INTRODUCTION |
The tetradecapeptide somatostatin (SST) is distributed widely throughout the brain, including the hippocampal formation, and participates in several physiological and pathophysiological processes (Epelbaum et al. 1994
). In hippocampus, SST is thought to play a role in learning (McNamara and Skelton 1993
; Ohno et al. 1993
) and memory (Matsuoka et al. 1994
) as well as long-term potentiation (Matsuoka et al. 1991
). Decreased SST immunoreactivity has been described in patients with Alzheimer's dementia (Bissette and Myers 1992
; Morrison et al. 1985
), and SST release is increased during kindling in the hippocampus (Monno et al. 1993
; Vezzani et al. 1992
). In the brain, SST acts predominantly as an inhibitory neurotransmitter through opening of different types of K+ channels. The peptide augments an inwardly rectifying K+ conductance in cultured locus coeruleus neurons (Inoue et al. 1988
) and possibly in the dorsolateral septal nucleus (Twery and Gallagher 1989
). SST also activates a delayed rectifier in cultured neocortical neurons (Wang et al. 1989
) and increases the voltage-dependent outward M current (IM) in neurons of the nucleus tractus solitarius (Jacquin et al. 1988
) and CA1 hippocampus (Moore et al. 1988
). Among these mechanisms, only the increase in inward rectification has been shown to hyperpolarize neurons at resting potential. SST also affects Ca2+ conductances in central neurons (Rhim et al. 1996
; Viana and Hille 1996
; Wang et al. 1990
). In dissociated hippocampal pyramidal neurons (HPNs), SST reduces currents passing through N-type calcium channels (Ishibashi and Akaike 1995
), providing a possible presynaptic inhibitory mechanism by reducing Ca2+ entry and neurotransmitter release.
Several studies of hippocampal neurons have verified that SST increases IM, a time- and voltage-dependent noninactivating K+ current (Moore et al. 1988
; Schweitzer et al. 1990
; Watson and Pittman 1988
). This SST action is believed to be mediated by activation of the phospholipase A2 (PLA2) pathway and generation of arachidonic acid and its 5-lipoxygenase (5-LO) metabolite, leukotriene C4 (Schweitzer et al. 1990
, 1993
). Lipoxygenase metabolites also increase IM in other cell types (Villarroel 1994
; Yu 1995
) and mediate the SST stimulation of Ca2+-activated K+ channels in rat pituitary tumor cells (Duerson et al. 1996
). The augmentation of IM by SST in hippocampus requires activation of the 5-LO-activating protein (FLAP), and both 5-LO and FLAP are highly expressed and colocalized in HPNs (Lammers et al. 1996
). However, inhibition of the 5-LO pathway only partially blocks the inhibitory effect of SST in these cells (Schweitzer et al. 1993
); also, the hippocampal IM is thought to play a limited role at normal resting membrane potentials (Halliwell and Adams 1982
), pointing to the participation of another conductance in the hyperpolarizing effect of SST.
In the present study, we have characterized a second conductance involved in the SST effect by blocking the SST-induced augmentation of IM with MK886, a specific FLAP inhibitor that prevents the formation of leukotriene C4 (Dixon et al. 1990
). Our results show that SST opens a voltage-insensitive K+ leak current IK(L) in HPNs, an effect concomitant with, but independent of, the IM increase. The former is the predominant effect at rest, whereas the latter develops when the neuron depolarizes.
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METHODS |
Slice preparation
We used standard intracellular recording techniques in rat hippocampal slices as described previously (Schweitzer et al. 1993
). In brief, transverse hippocampal slices (taken from male Sprague-Dawley rats of 100-170 g) 350-µm thick were cut on a brain slicer and incubated in gassed (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgSO4, 2.0 CaCl2, 24 NaHCO3, and 10 glucose. Other ions and agents were added to this ACSF. In some experiments, we raised K+ to 8.5 or 12.5 mM (Na+ was decreased proportionately). Slices were submerged completely and continuously superfused with warm (30-31°C) ACSF at a constant rate within the range of 2-4 ml/min. Methods of superfusion, voltage-clamp recording, drug administration, and data analysis were as described previously (Schweitzer et al. 1993
). We dissolved MK886 (a gift of Dr. D. Piomelli) in 0.05% dimethylsulfoxide. Dimethylsulfoxide had no effect on membrane properties at this concentration (Schweitzer et al. 1993
). We purchased SST from Peninsula laboratories, tetrodotoxin from Calbiochem, and all other chemicals from Sigma.
Voltage-clamp recordings
We performed voltage-clamp studies with an Axoclamp 2A pre amplifier, using sharp glass micropipettes filled with 3 M KCl (resistance 57-87 M
) to penetrate CA1 pyramidal neurons. We added 0.5-1 µM tetrodotoxin to the bath after impalement to block Na+-dependent action potentials and synaptic transmission. In discontinuous single-electrode voltage-clamp mode, the switching frequency between current injection and voltage sampling was 3-4 kHz. We continuously monitored electrode "settling time" and input capacitance neutralization at the headstage on an oscilloscope. Current and voltage records were filtered at 0.3 kHz and acquired by D/A sampling and acquisition software (pClamp; Axon Instruments). We fitted current relaxations and measured the current amplitudes with analysis software (Clampfit, Axon Instruments). The various problems (for example, space-clamping) associated with voltage-clamping of neurons with extended processes are discussed elsewhere (Finkel and Redman 1985
; Halliwell and Adams 1982
; Johnston and Brown 1983
) but should be minimized by the study of relative conductance changes with superfusion of drugs to equilibrium conditions.
Voltage protocols
We generated current-voltage (I-V) curves by holding the neurons at
60 mV and applying hyperpolarizing and depolarizing voltage steps (1.5-s duration, 7 s apart). We did not depolarize the neurons beyond
40 mV because of space-clamp considerations and the likelihood of activating Ca2+ currents. The I-V curves were constructed from the current values measured at the end of the voltage steps (steady state), and the values obtained in control condition were subtracted from those in the presence of the tested substance to obtain the net current induced. We also assessed several conductances found in the hippocampus: the M-current relaxation (Halliwell and Adams 1982
) seen by delivering hyperpolarizing voltage steps (1-s duration, 5 s apart) from a depolarized holding potential of about
45 mV; the Q-current relaxation (Halliwell and Adams 1982
) observed when hyperpolarizing neurons from a
60-mV holding potential; and the slow AHP-current (Lancaster and Adams 1986
) assessed as the tail-current remaining 400 ms at the offset of 1.5-s depolarizing steps from
60 to about
40 mV.
 |
RESULTS |
We recorded intracellularly from 35 HPNs with an average resting membrane potential (RMP) of
69 ± 0.5 mV (mean ± SE). Before addition of tetrodotoxin, the mean input resistance determined at the onset of a small hyperpolarizing current step was 71 ± 3 M
, and the mean action potential amplitude from threshold was 104 ± 1 mV. Three HPNs (of 38) that did not respond to SST were discarded.
SST modulates two different conductances
We generated current-voltage (I-V) curves to study the overall effects of SST on postsynaptic membrane properties. Superfusion of SST (1 µM) alone elicited an outward steady-state current associated with a conductance increase (Fig. 1). We have shown in previous studies that SST induces an outward rectification by increasing the voltage-dependent IM. When IM augmentation was prevented by superfusing MK886 (0.5-1 µM), which alone did not affect steady-state current values, SST still evoked an outward current, but of smaller amplitude (Fig. 1A). The net currents observed at the steady state for each condition are shown on Fig. 1B: the outward rectification in the depolarized range was abolished in the presence of MK886, leaving a voltage-insensitive current that remained linear within the voltage range tested (
130 to
40 mV) and reversed near the equilibrium potential for K+ ions (EK). We separated the two components that form the overall effect of SST by subtracting the SST-induced component in the presence of MK886 from that acquired in the absence of MK886, thus obtaining the net currents elicited by SST (Fig. 1C). These calculations showed that the SST-induced outward component sensitive to MK886 was voltage dependent and had a threshold of activation around
70 mV, characteristic of IM involvement. The SST component resistant to MK886 did not show voltage dependence and reversed at
102 mV. We obtained equivalent results in six neurons: in the presence of MK886, SST elicited a current of 103 ± 16 pA (holding
45 mV) that reversed at
98 ± 2 mV. Two other neurons exposed to SST that did not show an IM increase in the absence of MK886 still exhibited a voltage-insensitive current reversing near EK (
95 and
98 mV; not shown), an effect similar to that observed in the presence of MK886.

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| FIG. 1.
A specific inhibitor of the 5-lipoxygenase (5-LO) pathway, MK886, reveals a second somatostatin (SST) effect. A: selected current traces of a representative hippocampal pyramidal neuron (HPN) held at 58 mV and subjected to 3 different voltage steps sequentially applied and superimposed at each condition (voltage protocol on right). Application of 1 µM SST alone (SST 1st) for 3 min evoked a marked outward steady-state current at depolarized potentials. After washout of SST (bringing currents levels back to control) and addition of the 5-LO activating protein (FLAP) inhibitor MK886 (1 µM) to the superfusate, a second application of SST (SST 2nd; 1 µM, 4 min) in the continued presence of MK886 induced an outward current of smaller amplitude in the depolarized range. ···, control holding current at 58 mV; resting membrane potential (RMP) was 68 mV. B: net steady-state currents (subtracted from control) calculated from the cell shown in A. SST alone (SST 1st, ) elicited a current showing outward rectification. Currents values returned near control on washout of SST and addition of MK886 ( ), which itself did not affect steady-state currents. Second application of SST in the presence of MK886 ( ) induced a lesser outward current that reversed at 102 mV and showed no voltage dependence. C: separation of the 2 effects elicited by SST (same cell). Subtraction of "washout in MK886" from "SST 2nd" gave the MK886-resistant component of the SST effect ( ). Further subtraction of the MK886-resistant component from the first SST application isolated the MK886-sensitive component ( , ···) from the overall effect.
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To ensure that repeated applications did not alter the SST response (e.g., desensitization), we tested an additional six neurons by performing the first application of the peptide with MK886 already in the bath. With this protocol, we obtained results equivalent to those with sequential applications of SST: all six cells displayed a voltage-insensitive outward current (97 ± 14 pA at
45 mV) that reversed at
97 ± 2 mV (not shown). These results indicate that SST activates a voltage-insensitive outward conductance. The opening by SST of this conductance carried most of the outward current in the hyperpolarized range below
55 mV (Fig. 1C), therefore largely contributing to the SST-induced hyperpolarization at resting potential in HPNs, around
69 mV. Thus the zero current potential (i.e., the RMP) was shifted
3.9 ± 0.3 mV by SST in the absence of MK886 and
3.0 ± 0.2 mV in the presence of the inhibitor, suggesting that this conductance is the major mechanism for hyperpolarization by SST at rest.
AHP, Q, or M currents are not involved in the second SST effect
We assessed the IM amplitude on HPNs exposed to SST to verify that MK886 blocked the peptide-induced augmentation of IM and to rule out any participation of this current. We depolarized the neurons and deactivated IM by applying hyperpolarizing voltage steps (Fig. 2A). Eight neurons that presented an increase of the IM relaxation in the presence of SST (164 ± 11%) were subsequently bathed in MK886. A second application of SST had little effect on IM in all eight neurons (110 ± 9%) when the inhibitor was present. Exposing the slices to MK886 before SST also prevented the peptide-induced augmentation of IM (105 ± 7%) in 7 of 8 neurons.

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| FIG. 2.
Second SST effect is not due to an alteration of IM, sIAHP, or IQ. A: neuron held at 44 mV and subjected to 2 hyperpolarizing voltage steps to deactivate IM. First application of SST (SST 1st; 1 µM) evoked a large outward holding current (230 pA; ···, control holding current) associated with an IM relaxation increase. Second application of SST in the presence of 1 µM MK886 (SST 2nd) elicited a smaller outward holding current (120 pA) with no IM increase. Bottom: magnified IM relaxations ( 10-mV voltage step, aligned for comparison). B: neuron held at 58 mV; sIAHP observed 400 ms after the end of a depolarizing voltage step to 42 mV (dotted circle on voltage protocol indicates portion of current records shown). SST (1 µM), whether in the absence or presence of MK886, did not alter sIAHP. Two current traces displayed for each drug condition: top, slowly decaying sIAHP after repolarization of the membrane to 58 mV; bottom, holding current at 58 mV without voltage step (traces aligned for comparison). C: neuron held at 59 mV (RMP was 69 mV); a voltage step to 113 mV was applied to activate IQ (dotted circle on voltage protocol indicates portion of current records shown). Application of 1 µM SST did not alter IQ amplitude (relaxations aligned for comparison), whether in the presence or absence of MK886.
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Because the MK886-insensitive SST effect appeared to be voltage insensitive, we examined the slow Ca2+-dependent K+ AHP current (sIAHP). This current is of small amplitude and shows no voltage dependency. We assessed sIAHP as the slowly decaying tail current that appears when the neuron is repolarized from a sufficiently depolarized potential. Because we were looking for a change after drug application, we did not depolarize neurons beyond
40 mV to maintain good clamp control. As seen in Fig. 2B, SST did not affect sIAHP. Ten neurons bathed in the presence of MK886 showed a sIAHP amplitude of 103 ± 4% of control when exposed to SST.
We also tested a possible effect of SST on the noninactivating, inwardly rectifying Q current (IQ; also called Ih), a mixed K+-Na+ conductance that predominates in the hyperpolarized range. We observed IQ at the onset of hyperpolarizing steps, as the slow inward relaxation that increased with the intensity of the voltage command. The application of SST did not affect IQ, whether in the absence or presence of MK886 (Fig. 2C), even at voltage steps to
130 mV where the current is nearly fully activated. On average, 10 neurons exposed to SST in the presence of MK886 displayed an IQ relaxation of 103 ± 2% of control; only 2 of these 10 cells presented a small increase (10-20%) of the relaxation. Therefore, IQ is unlikely to play a significant role in SST actions on HPNs.
SST augments a voltage-insensitive K+ leak conductance
To identify the second conductance evoked by SST, we generated I-V curves in neurons superfused with ACSF containing different concentrations of extracellular K+ ([K+]o) to shift the reversal potential in a more depolarized direction. This approach had two goals: first, to establish that this conductance was carried solely by K+ and second, to assess a possible inward-rectifying behavior that would not be revealed using the standard 3.5 mM [K+]o. All experiments were conducted in the presence of MK886.

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| FIG. 3.
Influence of [K+]o on the SST response. A: selected current recordings from a neuron exposed to 1 µM SST in 2 different [K+]o in the continued presence of MK886. Neuron held at 61 mV (voltage protocol, bottom left); RMP was 70 mV. Application of SST in artificial cerebrospinal fluid (ACSF) containing 3.5 mM K+ (SST-K3) elicited an outward current at potentials positive to 90 mV (see I-V plot in B). SST then was washed out and [K+]o switched to 8.5 mM, inducing an inward current and input conductance increase at all potentials (Cont-K8). We then reapplied SST, which elicited a pronounced effect. B: graph of SST-induced steady-state currents in a neuron successively bathed in 3 different [K+]o: 3.5 ( ), 8.5 ( ), and 12.5 mM ( ). SST was applied for 3-5 min and washed out for 19-24 min for each [K+]o. Reversal potentials were 99, 75, and 61 mV, respectively, consistent with a pure K+ conductance. Note the very large inward current induced by SST at the most hyperpolarized potentials due to the increased driving force for K+, as well as the nonrectifying pattern of the response with the slope proportionately increasing with [K+]o. Curves fitted by linear regression.
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Figure 3 shows a neuron repeatedly exposed to SST in ACSF containing sequentially raised [K+]o. Application of SST in the standard 3.5 mM [K+]o induced a current equivalent to that described earlier. Raising [K+]o to 8.5 mM increased the cell conductance and elicited an inward current in control conditions (Fig. 3A) due to the more depolarized equilibrium potential of K+ conductances that participate in setting the RMP. The application of SST in 8.5 mM [K+]o evoked a pronounced inward steady-state current in the hyperpolarized range, concomitant with a large conductance increase. Plotting the net steady-state current induced by SST against the membrane potential (Fig. 3B) displayed such large inward currents with 8.5 or 12.5 mM [K+]o as well as the more depolarized reversal potentials of the SST-induced current. The plot of the I-V relationships was best fit by a linear regression, indicating that the current response remained nonrectifying throughout the voltage range tested at each [K+]o. Also the slope of the linear regression increased proportionately with [K+]o, a feature not expected of K+ inward rectifiers.

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| FIG. 4.
SST effect is carried solely by K+. A: mean ± SE reversal potentials of the SST current plotted against the logarithm of [K+]o (3.5, 8.5, and 12.5 mM). ···, theoretical value of the K+ equilibrium potential calculated from the Nernst equation, assuming an intracellular K+ concentration of 150 mM. Numbers in parentheses indicate numbers of neurons. B: plot of the SST-induced conductance in 3.5 ( ; n = 14) or 8.5 ( ;n = 6) mM [K+]o. GSST calculated as Isst/(V Vrev) where Isst = SST-induced current, V = command potential, and Vrev = reversal potential. Note the absence of voltage dependence as reflected by the linearity of the curves.
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The average reversal potential (Vrev) of the SST-induced current was
97 ± 1 mV in 3.5 mM [K+]o (n = 18). Vrev shifted positively by 23 mV to
74 ± 2 mV when [K+]o was raised to 8.5 mM (n = 6) and by 37 mV to
60 ± 3 mV in 12.5 mM [K+]o (n = 3). The theoretic reversal potentials as calculated by the Nernst equation for [K+]o of 3.5, 8.5, and 12.5 mM are
98,
75, and
65 mV, respectively (Fig. 4A). Our experimental values are therefore very close to those expected for a conductance carried solely by K+. We also calculated the conductance increase induced by SST, GSST, by dividing the SST-induced current by the driving force for K+ ions (V
Vrev). Figure 4B shows this parameter throughout the voltage range tested using a [K+]o of 3.5 and 8.5 mM. The conductance did not present inward or outward deviations and remained constant at all potentials, demonstrating the voltage-independent nature of the SST action. The amplitude of GSST was 1.88 nS in 3.5 mM [K+]o and 5.54 nS in 8.5 mM [K+]o, a 2.95-fold conductance augmentation for a 2.43-fold increase of [K+]o, indicating that GSST increased proportionately with [K+]o.

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| FIG. 5.
Effect of external Ba2+ on the SST response. A: continuous current recordings of a HPN held at 65 mV in 3.5 mM [K+]o and 1 µM MK886. Application of 1 µM SST (bar) elicited an outward current that maximized within 2 min of application and returned to control level after 10 min of washout of the peptide. Addition of 150 µM Ba2+ ( ) induced an inward current but only slightly affected the response to a 2nd application of SST (bar). After washout of SST, subsequent addition of 2 mM Ba2+ ( ) induced an additional inward current, and another application of SST ( ) was without effect. RMP was 70 mV; ···, pre-SST current level; bar indicating drug application adjusted to effect onset. B: plot of SST-induced currents in the absence and presence of 150 µM Ba2+ (3.5 mM [K+]o, MK886). First application of SST elicited a voltage-insensitive current ( ). After washout and addition of 150 µM Ba2+ in the ACSF, a 2nd SST application evoked a similar voltage-insensitive response ( ), although of slightly smaller amplitude. Curves fit by linear regression.
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Effect of barium and cesium
We further characterized this SST-evoked voltage-insensitive conductance by assessing the effect of extracellular Cs+ and Ba2+, two ions that affect K+ conductances, in the presence of MK886. Figure 5A shows the continuous current record of a neuron held near resting potential. A first application of SST elicited a slowly developing outward current that recovered to control level upon washout of the peptide. Addition of 150 µM Ba2+ in the medium elicited an inward current, but a second application of SST in the continued presence of Ba2+ still induced an outward current comparable to that evoked by the first application. Analysis of the average I-V relationships (n = 4) revealed only a moderate, voltage-independent diminution of the SST effect in the presence of 150 µM Ba2+ (85 ± 17 pA compared with 100 ± 11 pA in control ACSF,
45-mV holding potential). The SST response remained linear over the voltage range tested (Fig. 5B). We observed a similar voltage-independent diminution (19%) of the SST effect in the presence of low Ba2+ when the superfusate contained 8.5 mM [K+]o (n = 2; not shown).
However, a high concentration of Ba2+ (2 mM) completely abolished the SST response on five cells. Addition of 2 mM Ba2+ alone elicited a marked inward current, and subsequent application of SST did not elicit any effect (Fig. 5A). Construction of I-V relationships verified that SST had no effect, regardless of potential (not shown). This result was obtained whether we performed the first application of SST with 2 mM Ba2+ already in the bath or when we added 2 mM Ba2+ between successive SST applications (to ensure that neurons were SST-responsive).
Superfusing ACSF containing 2 mM Cs+ completely abolished the IQ relaxation and greatly decreased the input conductance at hyperpolarized potentials (Fig. 6A). A more limited decrease of input conductance was observed in the depolarized range. Under these conditions, SST still elicited an outward current over the depolarized range (n = 3), similar to that observed in absence of Cs+ (Fig. 6, B and C). The SST-induced inward component, however, was abolished by Cs+ in 3.5 mM [K+]o (Fig. 6, A and B). When we applied SST in ACSF containing 8.5 mM K+ (n = 2), the outward current again was not affected by Cs+ and reversed at the expected EK. However, analysis of the I-V relationships showed that in 8.5 mM [K+]o, Cs+ affected the SST inward current only partially at potentials below
100 mV, revealing a voltage-dependent block of the SST current that rectified outwardly (Fig. 6C). These experiments further rule out participation of IQ: this current is blocked completely by 2 mM Cs+ and insensitive to 2 mM Ba2+.

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| FIG. 6.
Effect of external Cs+ on the SST response. A: current recordings of a HPN held at 61 mV (voltage protocol, bottom right) in 3.5 mM [K+]o and MK886. Cs+ (2 mM) strongly decreased the cell conductance in the hyperpolarized range and blocked the IQ relaxation. Subsequent application of SST (1 µM) elicited an outward current in the depolarized range but had no effect in the hyperpolarized range. RMP was -72 mV. B: plot of the SST current derived from A. SST elicited a voltage-independent outward current, but the reversal to the inward direction was completely prevented by Cs+. ···, average SST response in Cs+-free ACSF. C: plot of the SST current from a different neuron bathed in 8.5 mM [K+]o, MK886, and Cs+. SST elicited a current that showed no voltage dependence in the potential range positive to 100 mV but that strongly rectified in the outward direction between 100 and 140 mV (··· is average SST response in Cs+-free ACSF). This negative slope conductance is probably due to a voltage-dependent block of IK(L) by Cs+.
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DISCUSSION |
In this study, we have characterized a new conductance, different from IM and inwardly rectifying currents, that is affected by SST in a central neuron. This conductance is carried by K+ and does not show voltage sensitivity; we have classified it as a voltage-independent leak current. Voltage-clamp studies have described the modulation of single K+ conductances by SST in brain neurons. We now report that in CA1 hippocampus, SST simultaneously augments two different K+ conductances in a same neuron: the voltage-insensitive IK(L) and the voltage-dependent IM.
Second SST effect is not due to an alteration of the M, Q, or AHP currents
Neurotransmitters affect several conductances that influence the activity of HPNs (Storm 1990
). Noninactivating K+ currents, such as the M and leak currents and the cationic Q current, are active at or near resting potential and thus readily regulate neuronal excitability. SST inhibits HPNs by augmenting IM (Moore et al. 1988
), an effect involving leukotriene C4 (Schweitzer et al. 1990
). We could neutralize this action of SST by treatment with the FLAP inhibitor MK886 (Lammers et al. 1996
). In basal condition, MK886 had no effect on steady-state currents or the conductances examined but completely blocked the IM augmentation and the associated voltage-dependent conductance increase elicited by SST. As we used MK886 throughout this study, the SST effect we describe is different from, and occurs independently of, the IM augmentation.
We also investigated a possible action of SST on IQ, the inwardly rectifying cationic conductance that predominates in the hyperpolarized range (Halliwell and Adams 1982
) and that may influence the resting potential of HPNs (Maccaferri et al. 1993
). Studies conducted in other neuronal types have demonstrated that SST augments inwardly rectifying conductances (Inoue et al. 1988
; Mihara et al. 1987
), although these studies implicated pure K+ conductances. We did not see a consistent effect of SST on IQ amplitude, whether in the presence or absence of MK886. Furthermore, when SST was applied in the presence of the FLAP inhibitor, we rather observed a trend (in only 2 cells) for an IQ increase. Because of the depolarized reversal potential of this mixed conductance (around
30 mV), such augmentation would elicit an inward steady-state current around resting potential, an effect opposite to the observed outward current. The unlikely participation of IQ is reinforced by the lack of effect of Cs+ on the SST current at potentials positive to
95 mV, as this ion completely blocks IQ, and by the pure K+ Nernstian relationship of the SST current.
Another candidate is the slow AHP-current seen in HPNs (Lancaster and Adams 1986
); this is a small Ca2+-dependent K+ conductance of the "SK" type (Sah 1996
). The voltage-insensitive nature of sIAHP and the reported opening of a Ca2+-dependent "BK" channel by SST in a pituitary cell line (White et al. 1991
) prompted us to investigate this candidate. Although sIAHP inactivates over a few seconds, it may have influenced the steady-state current values we obtained by applying voltage steps of 1.5-s duration. We did not observe an effect of SST on sIAHP when MK886 was present in the superfusate, and continuous records near rest (see Fig. 5) showed a slowly developing effect of SST that cannot be accounted for by sIAHP. SST also reportedly hyperpolarized CA1 neurons loaded with the Ca2+ chelator bis-(o-aminophenoxy)-N,N,N
,N
-tetraacetic acid or in the absence of extracellular Ca2+ (Xie and Sastry 1992
), two procedures that eliminate Ca2+-dependent K+ conductances. An augmentation of the K+-delayed rectifier current by SST as described in cultured neocortical neurons (Wang et al. 1989
) also can be ruled out, as this current slowly inactivates and is observed only at potentials positive to
45 mV in HPNs (Storm 1990
).
SST does not affect an inwardly rectifying K+ conductance in HPNs
Several inhibitory neurotransmitters such as
-aminobutyric acid and serotonin augment inwardly rectifying K+ conductances in hippocampus (Colino and Halliwell 1987
; Sodickson and Bean 1996
). Cloning and expression techniques also have established the existence of several inwardly rectifying K+ channel families that are expressed in hippocampus, including "strong" and "weak" rectifiers (Doupnik et al. 1995
). Strong inward rectifiers, such as GIRK1 (G protein-coupled inward rectifier) or HIR (hippocampal inward rectifier) (Karschin et al. 1994
; Périer et al. 1994
), exhibit obvious inward rectification and pass little or no measurable outward current. Another property of this family is that the voltage dependence of channel gating shifts in parallel with EK when [K+]o is modified (Hille 1992
). In our studies, the amplitude of the SST-induced conductance, GSST, showed no deviation in the inward or outward direction, indicating that the current passed similarly in both the hyperpolarized and depolarized range. Further, GSST and the slope of the I-V plot increased proportionately with [K+]o, as opposed to the situation with inward rectifiers. On the other hand, weak inward rectifiers allow some K+ to flow outward and may present only vague or no inward rectification. Although our data rule out the involvement of strong rectifiers, the criteria defining weak inward rectifiers appear uncertain. For example, BIRK1 (brain inward rectifier) channels present in hippocampus exhibit a linear I-V relationship in the hyperpolarized range and pass current in the outward direction (Bredt et al. 1995
), resembling the conductance we described in this study. The inward component of BIRK1 channels, however, is blocked completely by 50 µM Ba2+, a feature shared by all inward rectifiers but unlike the SST current we found in HPNs.
Ba2+ routinely is used to characterize K+ conductances. Whereas millimolar concentrations block all K+ conductances (Storm 1990
), lower concentrations (10-100 µM) more specifically inhibit inward rectifiers with little or no effect on voltage-insensitive leak currents (see Williams et al. 1988
). In our hands, low concentrations (100-150 µM) of Ba2+ had only a small and voltage-independent effect on the SST-induced current. Similar Ba2+ concentrations blocked the augmentation of a K+ inward rectification by baclofen in CA3 neurons (Sodickson and Bean 1996
), opioids in nonpyramidal CA1 neurons (Wimpey and Chavkin 1991
) and SST in locus coeruleus neurons (Inoue et al. 1988
). Because the slight Ba2+ reduction of the SST effect in HPNs did not show voltage dependence, it is likely due to a just-measurable nonspecific sensitivity of IK(L) to this ion. Indeed, the peptide effect was prevented completely by 2 mM Ba2+.
Cs+ voltage-dependently inhibits K+ conductances in central neurons (Constanti and Galvan 1983
; Williams et al. 1988
). Similarly, 2 mM Cs+ blocked the SST effect in a voltage-dependent manner. In 3.5 mM [K+]o, Cs+ abolished the SST-induced inward component with little effect in the outward range. In 8.5 mM [K+]o, however, Cs+ only partially inhibited the SST-induced inward component, and the I-V plot displayed a negative slope conductance at potentials more negative than
105 mV. In cortical neurons, the presence of Cs+ also caused a negative slope conductance affecting both leak and inwardly rectifying K+ conductances (Constanti and Galvan 1983
). In our study, the development of a negative slope conductance only in 8.5 mM [K+]o may be due to a decrease of the blocking effect of Cs+ at higher [K+]o (Hagiwara et al. 1976
).
SST hyperpolarizes HPNs by augmenting a voltage-insensitive K+ leak current
Manipulating [K+]o revealed that SST acted on a pure K+ conductance: the reversal potential of the SST-induced effect obtained in different [K+]o matched very closely the theoretic Nernstian reversal potential for a conductance carried solely by K+ ions. Because of the absence of rectification and the differential Ba2+ sensitivity of the SST effect we described, we classify this conductance as a leak (or resting) current, a term used for voltage-insensitive conductances controlled by neurotransmitters (Storm 1990
). The SST effect we report here is different from that observed in locus coeruleus (Inoue et al. 1988
) and peripheral neurons (Mihara et al. 1987
), where the peptide opens inwardly rectifying K+ channels as a mechanism of hyperpolarization.
The decrease of IK(L) participates in the depolarizing action of muscarinic agonists in HPNs (Benson et al. 1988
; Madison et al. 1987
). SST may increase the same conductance to hyperpolarize these neurons. Leak or resting currents sensitive to high Ba2+ concentrations are believed to control the resting potential in HPNs (Storm 1990
), but this mechanism is not well understood. The recent cloning of a K+-selective leak channel (Goldstein et al. 1996
) may provide new clues. The SST-induced augmentation of IM may have only a modest influence on the RMP. By comparing the SST effect in the absence and presence of MK886, we found that the outward component due to IM augmentation played a minor role (23%) in the SST-induced hyperpolarization at potentials around
70 mV. Thus IK(L) accounts for most of the peptide effect on HPN resting potentials in the slice preparation. Whether SST augments a constitutively active conductance or opens channels closed under basal conditions remains to be determined.
Thus the two main mechanisms for the hyperpolarizing effect of SST in central neurons at rest involve the leak current that we describe here and the inward rectifier observed in the locus coeruleus. Inward rectifiers, however, conduct little current at potentials positive to EK and therefore may have a limited role at potentials positive to
100 mV. The combined modulation of IK(L) and IM gives SST an extended inhibitory action throughout a wide range of physiologically active potentials in HPNs. The peptide concomitantly hyperpolarizes these neurons by augmenting IK(L) and potentiates a mechanism to oppose depolarizations by increasing IM.
Transduction mechanisms
We previously found that activation of PLA2 and generation of arachidonic acid and its metabolites mediate the SST effect in HPNs (Schweitzer et al. 1990
). Arachidonic acid itself modulates numerous K+ conductances (Meves 1994
; Piomelli 1994
). The complete lack of effect of SST when applied with PLA2 inhibitors (Schweitzer et al. 1993
) but the persistence of IK(L) in the presence of lipoxygenase and cyclooxygenase inhibitors, suggests that arachidonic acid augments IK(L). Five SST receptors have been cloned and referred to as sst1-5 (Reisine and Bell 1995
; Schindler et al. 1996
). In cell lines expressing sst4, SST releases arachidonic acid (Bito et al. 1994
), possibly via phosphorylation of cytosolic PLA2 (Sakanaka et al. 1994
). In contrast, the SST-induced opening of BK channels in pituitary cells via 5-LO metabolites of arachidonic acid (Duerson et al. 1996
) may occur through protein dephosphorylation (White et al. 1991
). The characterization of the type of PLA2 involved, as well as the role of phosphorylation/dephosphorylation processes, will provide further insight into the SST transduction mechanism in HPNs.
Because sst4 is linked to arachidonic acid release and is found mostly in hippocampus, especially in CA1 (Bito et al. 1994
; Pérez and Hoyer 1995
), this receptor subtype is presently the best candidate for mediating the effects of SST in HPNs. More than one receptor subtype could be involved in producing the two separate K+ conductance effects of SST. The sst4 receptor could trigger the activation of PLA2 to release arachidonic acid and elicit the IK(L) increase, and a different receptor subtype may be involved to activate FLAP and subsequently 5-LO, leading to leukotrienes and IM augmentation. Interestingly, sst3 and sst4 mRNAs can be coexpressed in the same neurons, especially in CA1 hippocampus (Pérez and Hoyer 1995
). Further studies await development of more specific receptor agonists and antagonists to clarify these mechanisms.
Conclusion
We have shown that SST inhibits HPNs by augmenting IM and IK(L). The activation of IK(L) is independent of the IM increase and is the major mechanism to hyperpolarize HPNs at rest, whereas IM augments when neurons depolarize, thus clamping the membrane to counteract excitatory events. Such inhibitory mechanisms may play a crucial role in response to hippocampal epileptiform activity, as postulated from results of in vivo studies where the enhanced release of SST during kindling may reduce excitability and protect surrounding tissue (Manfridi et al. 1991
; Vezzani et al. 1992
). Our previous studies showed that two different ligands (SST and acetylcholine) could act reciprocally on the M channel in a mammalian central neuron (Moore et al. 1988
; Schweitzer et al. 1993
). The present data further suggest that SST and acetylcholine could affect both the leak and M conductances in opposing directions. The interactions of these two transmitters on HPN excitability is currently under investigation.