Pka mediates the effects of monoamine transmitters on the K+ current underlying the slow spike frequency adaptation in hippocampal neurons

Neuron,

Vol. 11, 1023-1035,

December,

1993, Copyright

@ 1993 by Cell Press

PKA Mediates the Effects of Monoamine Transmitters on the K+ Current Underlying the Slow Spike Frequency Adaptation in Hippocampal Neurons Paola Pedarzani and Johan F. Storm Institute of Neurophysiology University of Oslo PB 1104 Blindern N-0317 Oslo Norway

Summary The Ca*+-activated K+ current IAHP, which underlies spike frequency adaptation in cortical pyramidal cells, can be modulated by multiple transmitters and probably contributes to state control of the forebrain by ascending monoaminergic fibers. Here, we show that the modulation of this current by norepinephrine, serotonin, and histamine is mediated by protein kinase A in hippocampal CA1 neurons. Two specific protein kinase A inhibitors, Rp-cAMPS and Walsh peptide, suppressed the effects of these transmitters on lAHP and spike frequency adaptation. The effects of the cyclic AMP analog 8CPTCAMP were also inhibited, whereas muscarinic and metabotropic glutamate receptor agonists had full effect. Intracellular application of protein kinase A catalytic subunit or a phosphatase inhibitor mimicked the effects of monoamines or 8CPT-cAMP. These results demonstrate that monoaminergic modulation of neuronal excitability in the mammalian CNS is mediated by protein phosphorylation. Introduction Protein phosphorylation is a widespread mechanism for signal transduction and regulation in the nervous system (Greengard, 1978; Nestler and Greengard, 1984; Walaas and Greengard, 1991). In invertebrate neurons, transmitter-induced phosphorylation of ion channels has been shown to mediate excitability changes, behavioral arousal, and synaptic plasticity underlying learning and memory (Castellucci et al., 1980; Kaczmarek et al., 1980; Adams and Levitan, 1982; Kandel and Schwartz, 1982; DeRiemer et al., 1985; Levitan, 1985; Klein et al., 1986; Goldsmith and Abrams, 1992; Hochner and Kandel, 1992). In the vertebrate CNS, however, it has been difficult to establish unambiguously that modulation of neuronal excitability is mediated by protein phosphorylation. Although several transmitters are known to modulate ion channels, excitability, and discharge patterns of central neurons (Madison and Nicoll, 1982; 1984; 1986a; Benardo and Prince, 1982; Haas and Konnerth, 1983; Cole and Nitoll, 1983; McCormick and Williamson, 1989; Stratton et al., 1989; Charpak et al., 1990; for reviews see Nicoll, 1988; McCormick, 1989; Nicoll et al., 1990), and phosphorylation-dependent modulation of ion channels from the brain have been demonstrated in lipid bilayers or expression systems (Costa et al., 1982; Farley

and Rudy, 1988; Rehm et al., 1989; Chung et al., 1991; Hogeret al., 1991; Reinhart et al., 1991; Catterall, 1992), it has been difficult to show that protein phosphorylation actually mediates transmitter effects on neuronal excitability in the CNS. In contrast, there is direct evidence that protein kinases mediate transmitter effects on electrogenesis in the heart (Osterreider et al., 1982; Brum et al., 1983; Trautwein and He$cheler, 1990), in smooth muscle (Kume et al., 1989), and in some peripheral neurons (Rane et al., 1989; Boland et al., 1991; Divers&Pierluissi and Dunlap, 1993). Recently, two studies in hippocampal neurons have also provided evidence for kinase-dependent modtilation of K+currents by acetylcholine (Zhang et al., 1992; Mtiller et al., 1992). One of the most prominent and best studied cases of neuromodulation in the vertebrate brain is the noradrenergic suppression of the slow Ca*+-activated K+ current, lAHp (Madison and Nicoll, 1982, 1984, 1986a, 1986b; Haas and Konnerth, 1983; Constanti and Sim, 1987; Haas and Rose, 1987; Foehring et al., 1989; McCormick and Williamson, 1989). This current underlies the spike frequency adaptation and slow afterhyperpolarization (AHP) in hippocampal and other cortical pyramidal cells (Alger and Nicoll, 1980; Hotson and Prince, 1980; Schwartzkroin and Stafstrom, 1980; Lancaster and Adams, 1986). In addition to norepinephrine, several other transmitters, including serotonin and histamine, also suppress IA~p, thus increasing the excitability of the target cells. The suppression of IAH~is probably one of the main effects of the ascending monoaminergic fiber systems: the noradrenergicprojectionsfrom locuscoeruleus(Moore and Bloom, 1979; Madison and Nicoll, 1982; 1986a; Haas and Konnerth, 1983), the serotonergic projections from the raphe nuclei (Azmitia, 1987; Andrade and Nicoll, 1987; Colino and Halliw$ll, 1987), and the histaminergic fibers from the hypothalamic mammillary region (Garbarg et al., 1974; Watanabe et al., 1983; Pollard and Schwartz, 1987; Haas and Konnerth, 1983; Haas and Greene, 1986). Thesewide ascending projections, which innervate the entire cortical mantle, are thought to control the functional state of the forebrain, leading to shifts from sleep to wakefulness, attention, arousal, and modulation of sensory perception, emotions, and cognitive functions (Chu and Bloom, 1973; Foote et al., 1980; Aston-Jones and Bloom, 1981; Livingstone and Hubel, 1981; Pollard and Schwartz, 1987; Steriade and LlinBs, 11988; McCormick, 1989; Steriade and McCarley, 1990). The suppression of lAHp is likely to be a principal mecihanism for these monoaminergictransmittereffectsatthecortical level (Madison and Nicoll, 1982; 1986a; Nicoll, 1988; McCormick, 1989; Nicoll et al., 1990). The effect of norepinephrine on the hippocampal lAHP is known to be mediated by PI fieceptors via adenylyl cyclase and cyclic AMP (CAMP; Madison and Ni-

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Figure 1. Whole-Cell Recording of the Slow AHP and the Underlying Ca’+Activated K’ Current, lAHP, in CA1 Hippocampal Pyramidal Cells

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toll, 1982, 1986b). CAMP probably also mediates the effects of serotonin, via 5-HT.,-like receptors (Dumuis et al., 1988; Chaput et al., 1990; Andrade and Chaput, 1991; Bockaert et al., 1992; Zifa and Fillion, 1992), and of histamine, via HZ receptors (Haas, 1985; Haas and Greene, 1986). The effects of CAMP in many other cell types are known to be mediated by protein kinase A (PKA; Greengard, 1978; Walaas and Greengard, 1991), and it was proposed that this enzyme also mediates the modulation of limp (Nicoll, 1988; Nicoll et al., 1990). However, there has so far been no compelling evidence in direct support of this hypothesis, and recent data have called it into question. Accumulating evidence from a variety of cell types, including photoreceptors, olfactory receptors, and Drosophila muscle, indicate that CAMP and other cyclic nucleotides can modulate ion channels directly, in a kinase-independent manner (Fesenko et al., 1985; Nakamura and Gold, 1987; Delgado et al., 1991). In particular, phosphorylation-independent modulation of ion channels by CAMP has been demonstrated for the adrenergic effect on the pacemaker current in the heart (DiFrancesco and Tortora, 1991), and it was recently reported that a kinase-independent mechanism underlies the

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(A) Left: The AHP following a burst of action potentials (inset). The burst was elicited by a brief depolarizing current pulse (lower trace) injected through the recording electrode. Scale bars, from top to bottom: 2 s, 2 mV; 100 ms, 30 mV; 2 s, 150 pA. Right: The AHP current, lAHP, recorded by voltage clamp, following a depolarizing step command (upper trace). In all voltage clamp experiments, tetrodotoxin (0.5 HIM) and tetraethylammonium (5 mM) were added to the perfusing medium to promote the generation of a Ca*+ spike current during the depolarizing step (inset). Scale bars, from top to bottom: 2 s, 60 mV; 50 ms, 500 PA; 2 s, 20 pA. LB) Modulation of lAHP by several transmitter substances and by the CAMP second messenger cascade. lAHPwas suppressed by norepinephrine (5 PM; n = 13), the p receptor agonist isoproterenol (2 PM; n = II), the adenylyl cyclase stimulator forskolin (20 PM; n = 7), the membrane-permeable CAMP analog 8CPT-CAMP (100 PM; n = 20), serotonin (30 PM; n = 51, histamine (IO FM; n = 3), the muscarinic cholinergic agonist carbachol (2.5 PM; n = 6), and the metabotropic glutamate receptor agonist transACPD (20 PM; n = 3). Scale bars: 20 pA, 5 s.

suppression of the slow AHP by norepinephrine and CAMP in locus coeruleus neurons (Aston-Jones and Shiekhattar, 1992, Sot. Neurosci., abstract). In addition, the amino acid sequences of some cloned K+ channels show putative nucleotide-binding sites with a sequence homologous to that of the CAMP-binding site of PKA (Guy et al., 1991; Shabb and Corbin, 1992). Furthermore, previous attempts to prevent the noradrenergic suppression of lAHPby protein kinase inhibitors have yielded largely negative or inconclusive results. Thus, intracellular application (via microelectrodes) of specific PKA inhibitors, such as the Walsh peptide PKA inhibitor (PKI; Cheng et al., 1986), has been ineffective (e.g., J. F. S., unpublished data). Bath application of nonspecific kinase inhibitors, such as l+isoquinolinylsulfonylt2-methylpiperazine (H-7), NB-[methylamino]ethyl)-54soquinolinesulfonamide (H8), staurosporine, K-252a, sphingosine, and polymyxine, has been either negative or only partly effective (Gerber et al., 1992; Sim et al., 1992; Miiller et al., 1992; P. P., and J. F. S., unpublished data), even at such high doses and long incubation times that nonspecific effects might occur (Sako et al., 1988; Riiegg and Burgess, 1989). Given this background, the present study was un-

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Figure 2. RpcAMPS, a Specific Blocker of the CAMP-Binding Site of PKA, Inhibits the Effects of lsoproterenol and a CAMP Analog on IAHP (A) Under normal conditions, the B recep tor agonist isoproterenol (1 RM) sup pressedtheAHPcurrentwithin1 min(Control). In contrast, in a cell dialyzed with Rp-cAMPS (500 )IM in the pipette), the sup pression of lAHP was largely prevented (lower records). RpcAMPS was allowed to diffuse from the patch pipette into the cell for 20-25 min after breaking into the cell, before applying the agonists. To ensure a maximal effect, isopraterenol (1 RM) was applied for a longer time (7 min) to the cell with Rp-cAMPS than to the control cell (3 min). The first and last of the traces are shown superimposed to the right. Scale bars: 20 pA, 5 s. (B) Time course of the effects of isoproterenol (l-2 PM) on lAnr (peak amplitude, normalized; overlapping symbols represent the same percentage) In 6 cells: 3 with RpCAMPS (closed symbols; 500 NM) and 3 without RpcAMPS (open symbols) in the recording pipette. The isoproterenol application lasted between 2.5 min (solid bar) and 7 min (dashed bar) and was longer for ceils with RpcAMPS (4-7 min) than for control cells (2.5-5 min), to ensure a maximal effect. (C) Similar experiments as in (A), but using the membrane-permeable CAMP analog BCPT-CAMP (100 PM; bath applied) to activate PKA directly. Again, the suppression of lAHP was prevented in cells dialyzed with RpcAMPS (508 PM in the pipette), although 8CPT-CAMP was applied for a longer time to cells whh Rp-CAMPS (up to 8 min; dashed bar in [O]). Scale bars: 20 pA, 5 5. (D) Time course of the effect of 8CPT-CAMP on IAHP in 12 cells, 7 with (closed circles) and 5 without (open circles) Rp-CAMPS in the recording pipette. Two concentrations of Rp-CAMPS (in the pipette) were tested: 100 PM (left panel; 3cells) and 500 PM (right panel; 4 cells). Solid bars indicate the minimal application times and dashed bars show the maximal application times for the drugs.

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dertaken to reexamine the hypothesis that the suppression of lAHP by monoaminergic transmitters and CAMP in pyramidal cells is mediated by PKA, as opposed to a kinase-independent mechanism. The results provide four lines of evidence that protein phosphorylation by PKA mediates the modulation of IAH~ by norepinephrine, serotonin, and histamine in hippocampal pyramidal cells. Results Whole-Cell Recording of i,jHp and Its Modulation Wholecell recordings (Blanton et al., 1989; Edwards et al., 1989) were obtained from 128 CA1 pyramidal cells

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in hippocampal slices from 16 to 28day-old rats. More than 95% of thecells showed atypical AHPcurrent (IAHP; Lancaster and Adams, 1986) under normal conditions. In voltage clamp, lAHpwas seen as a characteristic slow tail current following a train of unclamped action potentials, or a Ca*+ current elicited by a brief depolarizing step (Figure IA, right). In current clamp, Ca2+ spikes or bursts of Na+ spikes elicited a slow AHP with a time course similar to that of IAHP(Figure IA, left). With the recording conditions used here, the Caz+ spikes and lAHp remained stable for the duration of the recording, usually about l-2 hr, sometimes up to 4 hr. In addition, IAHPmaintained its sensitivitythroughoutthe recordings to the transmitter substances norepinephrine, seroto-

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nin, and histamine, as well as other agonists, such as the B receptor agonist isoproterenol, the cholinergic agonist carbachol, and trans-I-amino-cyclopentyl-1,3dicarboxylate (trans-ACPD; 20 PM)-an agonist of metabotropic glutamate receptors (Figure 16; Stratton et al., 1989; Charpak et al., 1990). Likewise, lAHpshowed normal sensitivity to the membrane-permeable CAMP analogs 8-(4chlorophenylthio)cAMP (8CPT-CAMP; Figure IB) and &BrcAMP (n = 3; data not shown), the adenylyl cyclase activator forskolin (Madison and Nicoll, 1986b), and the ion channel blockers Cd*+ and Ba*+ (data not shown). All drug effects were reversible. These properties seemed indistinguishable from those of i,tHP as recorded with sharp microelectrodes. Thus, under the conditions used in this study, whole-cell recording in hippocampal slices allowed long-term stable recordings of lAHp and its modulation by transmitters.

Blockade of the PKA Regulatory Subunit by Rp-cAMPS Prevents the Effects of fl Receptor Agonists and CAMP Analogs We first focused on the mechanism of inhibition of lAHp by norepinephrine, via BI receptors and CAMP. In most of these experiments, we used the selective B receptor agonist isoproterenol (isoprenaline; l-5 PM), which suppresses IAHP in the same way as norepinephrine (Figure IB; Madison and Nicoll, 1986a). Besides avoiding activation of a receptors, isoproterenol has the advantage of being a poor substrate for neuronal uptake in slices (Hirayama et al., 1991). One way to test whether CAMP-dependent protein kinase (PKA) mediates the modulation of l,q+p would be to block the activation of PKA by CAMP. This can be obtained by using the nonactivating CAMP analog, Rp-CAMPS (Botelho et al., 1988). By binding to the CAMP-binding site on the regulatory subunit of PKA, it competitively inhibits its activation by CAMP; and it is resistant to breakdown by phosphodiesterases. When Rp-cAMPS (100-500 ~.LM) was included in the intracellular medium of the patch pipette, it inhibited the effect on IAHP of bath-applied isoprenaline (n = 5; Figures 2A and 28) and of the membrane-permeable CAMP analog 8CPT-CAMP (n = 7) in all the cells tested (n = 12; Figures 2C and 2D). It is not surprising that some inhibition of IAH~ remained in the presence of Rp-CAMPS (Figure 2), since Rp-CAMPS is a competitive inhibitor and can be partly displaced by high levels of CAMP. An incomplete blockof PKA-dependent processes has been observed in Aplysia neurons, even with high doses of Rp-cAMPS (Hochner and Kandel, 1992). Rp-CAMPS alone did not appear to cause any inhibition of IAHP,since the current amplitude was not significantly reduced while the cell was being dialyzed with Rp-CAMPS for up to 1 hr and was similar to that of control cells. These observations are in accordance with the hypothesis that PKA mediates the effect of norepinephrine on limp. However, they may not entirely exclude

the alternative possibility that the effect of CAMP could be due to a kinase-independent mechanism. Thus, the amino acid sequences of some cloned channel proteins show putative nucleotide-binding sites with a sequence homologous to that of the CAMPbinding site of PKA (Guy et al., 1991; Shabb and Corbin, 1992). If the modulation of lAHP by CAMP were kinase independent, i.e., mediated by a binding site other than that of PKA (e.g., on the channel itself), it is also possible that this binding site could be blocked by Rp-CAMPS. Hence, the experiments with RpCAMPS do not completely rule out a direct nucleotide effect. Inhibition of the PKA Catalytic Subunit with PKI Prevents the Effects of Norepinephrine and CAMP Analogs To test whether the effect of norepinephrine and CAMP is mediated by PKA, as opposed to a possible kinase-independent pathway, we used a selective blocker of the catalytic subunit of this protein kinase: PKI (Cheng et al., 1986). This peptide is a pseudosubstrate inhibitor that binds to the substrate recognition site of PKA and has little or no effect against other kinases (Cheng et al., 1986; Goldsmith and Abrams, 1992). When PKI was applied intracellularly (1 mM in the pipette), it had essentially the same effects as RpCAMPS: the modulation of lAHp by isoprenaline and by 8CPT-CAMP was inhibited, whereas the peptide itself had no detectable inhibiting effect on the current (Figure 3). As expected, PKI (molecular weight = 2221) required a longer diffusion time to be effective, compared with Rp-CAMPS, which is a smaller molecule (molecular weight = 345). Together with the effects of Rp-CAMPS, these results indicate that PKA mediates the effects of isoproterenol and CAMP analogs on IAHP. Except for this kinase, no other enzyme or signaling molecule is known or suspected to be sensitive to both Rp-CAMPS and PKI. Exogenous PKA Catalytic Subunit Mimics the Effects of Norepinephrine or CAMP Analogs If the inhibition of lAHp by transmitters is mediated by PKA, it may be possible to obtain a similar inhibition by intracellular application of the active form of this enzyme (Castellucci et al., 1980; Brum et al., 1983). This possibility was tested by including the catalytic subunit of PKA (PKA-C; 12-25 PM; Vintermyr et al., 1993) in the recording pipette. These experiments were complicated bythe tendency of the protein solution to inhibit seal formation and cause a high or variable series resistance; but two of the cells tested with PKA-C met all our criteria: a seal resistance > 5 GQ; a stable access resistance between 15 and 30 Ma; an initial IAHP amplitude of at least 20 pA; a stable recording for at least 35 min, with no significant change in seal or access resistance; and no significant rundown of the Ca*+ spike. These cells exhibited a gradual decline of IAHPto 16% and 46% of the initial amplitude (measured after 50 and 70 min, respectively; Figures

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Figure 3. PKI, a Specific Peptide Inhibitor of the Substrate-Binding Site of PKA, Reduces the Effects of lsoproterenol and a CAMP Analog on lAHP

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(A) In a control cell, isoproterenol (2 PM) rapidly suppressed IAHP (upper records), whereas the effect of isoproterenol was largely prevented in a cell dialyzed with PKI (1 mM added to the pipette solution; lower records). The first and last of the traces are shown superimposed to the right. Scale bars: 20 pA, 5 s. (B) Time course of the effects of isoproterenol(2 PM) on the lAHPamplitude in IO cells: 5 with (closed circles; 1 mM) and 5 without (open circles) PKI in the recording pipette. (C)Similar experimentg as shown in (A), but using the membrane-permeable CAMP analog 8CPT-CAMP (100 jM; bath applied) to activate PKA directly. Again, the suppression of lAHPwas prevented in cells recorded with 1 mM PKI in the pipette (lower records). Scale bars: 20 pA, 5 s. (D) Time cou rse of the effect of 8CPT-CAMP on lAHP in 9 cells, 4 with (closed circles) and 5 without (open circles) 1 mM PKI in the recording pipette. Solid bars in (B) and (D) indicate the minimal application times, and dashed bars indicate the maximal application times for the drugs. To ensure a maximal effect, drugs were often applied for a longer time to cells with PKI than to control cells.

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4A and 4B). Such a rundown of the AHP current was not seen under normal conditions, nor in cells recorded with the phosphate buffer used for the kinase stock solution (Figures 4A and 48; Vintermyr et al., 1983). The decline in 1~~~ in cells loaded with PKA-C could not be attributed to a reduced Ca2+ influx, since the Ca*+ current during the depolarizing step remained virtually constant. Control experiments with the Ca2+ channel blocker Cd2+ (Madison and Nicoll, 1986a) showed that a strong reduction in Ca2+current was required to give a secondary reduction in IAHp comparable to that seen with PKA-C (data not shown). The slowness of the PKA-C effect may be partly due

to the slow diffusion of this protein (imolecular weight = 38,000). In addition, since the protein solution was found to inhibit seal formation, the PKA-C experiments were performed with patch pipettes filled with normal intracellular medium in the itip and kinase solution backfilled in the shank of the pipette-a procedure that may cause a variable extra’delay in the diffusion of the kinase into the cell. The Phosphatase Inhibitor Micrwystin Mimics the Effects of Norepinephrine or CAMP If IAHP is inhibited by norepinephrine and CAMP through the activation of protein kinases, and these

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Under normal conditions, Imp was virtually constant for the duration of the recording (upper records in [A] and [Cl; open symbols in [B] and [D]; overlapping symbols represent the same percentage). In contrast, cells dialyzed with PKA-C (24 uM in [A, lower records]), or microcystin (50 PM in [C, lower records]) exhibited a gradual decay of the IA~~ amplitude over 20-90 min ([B] and [D], closed symbols). In (A) and (C), the traces are shown superimposed to the right. Scale bars: 40 pA, 3 s (A); 30 PA, 3 s (C). Microcystin concentrations in (D): 50 uM (closed diamonds) and 5 PM (closed circles). PKA-C concentrations in (B): 24 PM (closed diamonds] and 12 PM (closed circles).

kinases have a certain basal activity even in unstimulated cells, the continuous action of protein phosphatases may be needed to avoid a persistent inhibition of the current. If so, inhibition of the phosphatases maycausearundownof &in theabsenceof agonists that stimulate phosphorylation. This hypothesis was tested by adding the phosphatase inhibitor microcystin-LR to the intracellular medium (5-50 PM; molecular weight = 995; MacKintosh et al., 1990). Of the 7 cells tested, 6 showed either a clear decline in the amplitudeof lAHpduringthe recording(4cellsexposed to 5 or 50 PM microcystin; Figures 4C and 4D) or no detectable lAHp from the onset of the measurements, IO min after breaking into the cell (2 of the cells with 50 uM microcystin). In contrast, all of the 20 cells that were studied under the same conditions but without microcystin in the pipette had a clear IAHPfrom the beginning, and the current showed no significant decline (Figures 4C and 4D show data from the four control cells recorded during the series of microcystin experiments). The decline in bHp with microcystin could not be attributed to a reduced Ca2+ influx, since the Ca*+ current remained constant. These results further support the conclusion that IAH~ is inhibited by protein phosphorylation. Furthermore, the data suggest that CA1 neurons normally have an ongoing phosphorylation/dephosphorylation turnover, due to

a basal kinase activity that is balanced by phosphatases, even in the absence of applied transmitters. The experiments described so far provide four independent lines of evidence that the modulation of lAHp by norepinephrine or isoprenaline, via 8 receptors and CAMP production, is mediated by protein phosphorylation. Three of these tests (Rp-CAMPS, PKI, and PKA-C) point to PKA, whereas microcystin does not distinguish between phosphorylation by different serine-threonine kinases (MacKintosh et al., 1990). Inhibition of PKA Prevents the Effects of Norepinephrine on Spike Frequency Adaptation and the Slow AHP We next wanted to test the effects of PKA inhibitors on the modulation of the discharge pattern of the cell, since the AHP current is known to underlie spike frequency adaptation in pyramidal cells, and suppression of IAH~ by norepinephrine or other transmitters increases the number of action potentials in response to a depolarizing current pulse (Madison and Nicoll, 1982,1984; Haas and Konnerth, 1983). For these experiments, we used traditional intracellular recording with sharp microelectrodes in hippocampal slices fromadultrats(Figure5). Byusingthesametechnique and preparation as in previous studieson lA”r modulation, these experiments also served to exclude the

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(A) Rp-CAMPS (Rp) prevented the suppression of spike frequency adaptation (during a depolarizing current pulse) by norepinephrine (A,, 5 PM), serotonin (A,, 30 PM), and histamine (A,, 10 FM), but not by the cholinergic agonist carbachol (A+ 5 PM). (B) Rp-CAMPS also prevented the inhibition of the slow AHP (following a spike burst in response to a 50 ms long depolarizing current pulse) by norepinephrine (5 PM), serotonin (5 PM), and histamine (10 PM), but not carbachol (5 PM). The lower records in (A,)-(&) and in (B,), (BJ, and (BJ are all from the same cell, recorded with 67 mM RpcAMPS in the electrode tip. The upper records in (A) and (B) are from other cells, recorded without Rp-cAMPS. Scale bars: 10 mV, 550 ms (A); 10 mV, 1 or 2 s (B) (2 s in [B,] upper records and [B2] lower records; 1 s in all the other records). In (A), the depolarizing current pulses were of constant strength in each experiment (range: 0.3-0.7 nA), and in (B), the spike number during the 50 ms long depolarizing pulse (4-5) was kept constant in each experiment by adjusting the pulse strength. In both (A) and (B), the baseline membrane potential was manually clamped close to -65 mV.

possibility that the voltage-clamp results obtained with whole-cell recording were somehow atypical, e.g., due to dialyzation of the cells, or because the slices were from young animals. As expected, all the cells tested with sharp electrodes under normal conditions showed the characteristic effects of norepinephrine: reduced spike frequency adaptation (n = 4; Figure 5A1) and inhibition of the slow AHP (n = 4, Figure 5B,). In contrast, in cells recorded with RpCAMPS in the electrode, the effect of norepinephrine on the spike frequency or the slow AHP was largely suppressed (n = 4; Figures 5A, and 5B1, Rp). These

results support the hypothesis that modulation spike frequency by norepinephrine is mediated the CAMP-dependent protein kinase, PKA.

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Inhibition of PKA Prevents the Effects of Serotonin and Histamine on IAHp and Spike Frequency Adaptation Finally, we tested the modulation of IAH~ in response to activation of receptors for four other neurotransmitters: serotonin, histamine, acetylcholine, and glutamate. Both in whole-cell voltageclamp recordings (Figure 6) and in current-clamp recordings of spike

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(A) Effect of 30 uM serotonin in 2 cells, 1 with and the other without 500 uM Rp-CAMPS in the patch pipette. (6) Time course of the changes in Imp amplitude during application of serotonin (5-30 PM) in 3 cells recorded with 500 uM Rp-CAMPS in the pipette (closed symbols) and 4 cells without Rp-cAMPS (open symbols). Different doses of serotonin were tested in different cells: closed circles, 5 FM; closed diamonds, 30 uM; open circles, 5 uM (3 cells) or 30 uM (1 cell). (C) Effect of 10 uM histamine on IAHP in 2 cells, 1 with and the other without 500 PM Rp-cAMPS in the pipette. (D) Time course of the changes in IAW in response to histamine in 3 cells recorded with 500 uM Rp-CAMPS in the patch pipette (closed symbols) and 3 cells without Rp-CAMPS (open symbols). Open circles, 10 NM histamine; closed circles, 10 uM histamine (2 cells); closed diamonds, 1 uM followed by 10 uM (arrow) histamine. In (A) and (C), the traces are shown superimposed to the right. Scale bars: 20 pA, 5 s (A and C).

frequency adaptation (Figure 5A) and the slow AHP (Figure 5B), Rp-CAMPS was found to prevent the effects of serotonin (5-30 PM; n = 6; Figure 5; Figures 6A and 6B) and histamine (5-30 PM; n = 6; Figure 5; Figures 6C and 6D). In contrast, the effects of the cholinergic agonist carbachol(2.5 wM; n = 5; Figure 5) were not inhibited by Rp-cAMPS,even incellswhere it effectively suppressed the effects of norepinephrine, isoproterenol, serotonin, or histamine (the Rp records in Figures 5A,-5% are from the same cell). Similarly, the inhibition of IAHp by the metabotropic glutamate receptor (mGluR) agonist trans-ACPD (20 PM) was not prevented by Rp-CAMPS, even in cells where the isoproterenol effect was abolished (n = 3; data not shown). These results strongly suggest that both histamine and serotonin act via PKA, just like norepinephrine, whereas acetylcholine and metabotropic glutamate receptors employ other signal pathways.

Discussion The main conclusion of this study is that the suppression of lAHp by norepinephrine, serotonin, and histamine in CA1 hippocampal pyramidal cells is mediated by the CAMP-dependent protein kinase, PKA. This provides an example that modulation of neuronal excitability in the mammalian brain is mediated by protein phosphorylation, as has previously been clearly demonstrated in invertebrate neurons. The present results add to other evidence for kinasedependent modulation of ion channels in the vertebrate CNS. Several cloned channels from rat brain show putative phosphorylation sites and can alter their functional properties through phosphorylation by different kinases (Hoger et al., 1991; Catterall, 1992). Ion channels isolated from mammalian brain and reconstituted in bilayers can also be modulated by phos-

AHP Current 1031

Modulation

via PKA

phorylation. In particular, different subtypes of large conductance Ca*+-activated K+ (BK) channels can be stimulated or inhibited by PKA-dependent phosphorylation (Farley and Rudy, 1988; Rehm et al., 1989; Chung et al., 1991; Reinhart et al., 1991), and a cloned species of Drosophila BK channels shows putative phosphorylation sites for PKA (Adelman et al., 1992). However, it is not yet clear what is the functional consequence of the modulation of BK channels. The BK channels are distinct from the channels underlying IAHP,which probably are of the small conductance (SK) type (Lancaster et al., 1991) and have not yet been cloned. It was recently reported that bath application of the less specific protein kinase inhibitor staurosporine partially (37%~49%) suppresses the inhibition of lAHp by a CAMP analog (8-Br-CAMP) or norepinephrine in slice cultures (Gerber et al., 1992; Sim et al., 1992). These results are consistent with the main conclusion of this study. In our hands, however, even high concentrations of staurosporine and a related kinase inhibitor (K-252a) failed to suppress the inhibition of lAHp by isoproterenol or 8CPTcAMP (P. P. and J. F. S., unpublished data). Unfortunately, the nonspecific and variable effects and partly unknown mechanism of action of this family of kinase inhibitors introduce considerable uncertainty in the interpretation of these experiments (Sako et al., 1988; Riiegg and Burgess, 1989). Tests with the nonspecific kinase inhibitors of the isoquinoline class (H-7, H-8), which are supposed to act by a similar mechanism as staurosporine (block of the ATP-binding site of various kinases), have also yielded largely negative or highly variable results in different laboratories, including ours (P. P. and J. F. S., unpublished data). For example, H-7 has been reported both to suppress and not to suppress the inhibition of IAH~by quisqualate (Baskys et al., 1990; Gerber et al., 1992), supporting the impression that results with these less specific kinase inhibitors are often difficult to interpret. In view of the present results with specific agents, however, it now seems clear that PKA mediates the CAMP-dependent inhibition of IAHP,whereas the modulation of this current via muscarinic and metabotropic glutamate receptors are largely or wholly independent of PKA. The data presented here do not, however, indicate whether the PKA-dependent modulation of lAHPis due to phosphorylation of the underlying K+channel protein or of some regulatory protein. It seems that these possibilities can only be distinguished when the AHP channels can be studied in isolation, following purification or cloning. Theconclusions of this study are in accordancewith the known ability of norepinephrine, serotonin, and histamine to stimulate CAMP production (Barbaccia et al., 1983; Green, 1983; Pollard and Schwartz, 1987; Nicoll, 1988; Zifa and Fillion, 1992) and indicate that these three transmitters all converge on PKA, thereby exerting similar effects on L++. It was recently sug-

gested that serotonin, acting via 5-HTIA receptors and thepysubunitsofaGprotein,mayfacilitatetheadrenergic stimulation of adenylyl cyclase (Andrade, 1993). However, it is likely that the main effect of serotonin on lAHp is mediated by 5-HT4-like receptors positively coupled to adenylyl cyclase (Dumuis et al., 1988; Chaput et al., 1990; Andrade and Chaput, 1991; Bockaert et al., 1992; Zifa and Fillion, 1992). Accordingly, the serotonin effect was resistant to the 0 receptor blocker propranolol (10 PM; n = 5; data not shown). Recent evidence from hippocampal neurons indicate that the muscarinic suppression of IAH? is not mediated by protein kinase C (Sim et al., 1992) as suggested previously, but rather by Ca2+/calmodulindependent protein kinase (Muller et al., 1992), whereas the suppression of IAHPby metabotropic glutamate receptors seems to involve neither protein kinase C, Ca*+/calmodulin-dependent protein kinase (Gerber et al., 1992; Muller et al.,1992), nor PKA. On the other hand, there is evidence that the muscarinic potentiation of the hippocampal delayed rectifier current, IK, is mediated by protein kinase C (Zhang et al., 1992). The physiological consequence of the potentiation of IK remains to be demonstrated, however. Action potential repolarization, which is thought to be a major function of IK (Storm, 1987a), is not accelerated by muscarinic agonists or other activators of protein kinase C (Lancaster and Nicoll, 1987; Storm, 1987b; Hu and Storm, 1991). Since none of the PKA inhibitors completely suppressed the modulation of IAHP by transmitters or CAMP analogs, it remains a possibility that the residual effect could be due to a kinase-independent action. However, in the absence of direct support for this hypothesis, it seems a more parsimonious explanation that the incomplete inhibition reflects insufficient concentrations or potencies of the inhibitors. For PKI, it is also possible that breakdown by peptidases in the cell may reduce its effectiveness. In conclusion, the present results indicate that protein phosphorylation by PKA is a final common step in the signal pathways of at least three of the major monoaminergic projections from the brain stem and basal forebrain to the hippocampus. It is likely that this mechanism also mediates the effects of norepinephrine, serotonin, and histamine in other parts of the forebrain, such as the neocortex and parts of the thalamus, where IAHP, the slow AHP, and spike frequency adaptation are also known to be suppressed by these transmitters (Constanti and Sim, 1987; Foehring et al., 1989; McCormick and Prince, 1988; McCormick and Williamson, 1989). Covalent modification of target proteins through phosphorylation is likely to be a suitable mechanism for the relatively slow and long lasting modulatory transmitter actions involved in state control of the brain (Greengard, 1978; Nicoll, 1988; McCormick, 1989; Steriade and McCarley, 1990). Finally, the present results and methods may provide ways to control for the action of protein kinase

Neuron 1032

inhibitors and activators in studies of other forms modulation, including synaptic plasticity (Greengard et al., 1991; Wang et al., 1991; Frey et al., 1993). Experimental

Transverse

of

Procedures

hippocampal slices (400 pm thick) were prepared from young Wistar rats (16-28 days old), decapitated under halothane anaesthesia. During recording, the slices were superfused with extracellular medium that contained 125 mM NaCI, 25 mM NaHCOJ, 1.25 mM KCI, 1.25 mM KH*PO,, 2.0 mM CaC12, 1.5 mM MgCI,, and 16 mM glucose and was saturated with 95% 02, 5% CO*, at 23OC-26OC. Bicuculline (IO PM) was routinely added to the medium to suppress spontaneous inhibitory synaptic currents. In voltage-clamp experiments, tetrodotoxin (0.5 PM) and tetraethylammonium (5 mM) were added to the bath to block Na+channelsand some K+channels.TheAHP current is resistant to this dose of tetraethylammonium. Whole-cell giga-seal recordings wereobtained from CA1 pyramidal cells in the slice, using the”blind” method (Blanton et al., 1989; Edwards et al., 1989). The patch pipettes were filled with a solution containing 140 mM potassium gluconate, IO mM HEPES, 2 mM ATP, 1 mM MgCI,, and 0.1 mM GTP (pipette resistance, 3-6 Ma). Using an Axopatch ID amplifier (2 kHz low-pass filter), the cells were voltage clamped at a slightly depolarized holding potential (-50 to -60 mV), to increase the driving force for IAHP. A depolarizing step (100 ms), of sufficient amplitude to elicit a robust (unclamped) Ca*+ action current, was applied once every 30 s. Series resistance compensation was not used. The access resistance (range, 15-30 MQ and the amplitude and time course (shape and duration) of the Cal+ current during the step were monitored and showed only minimal variations during each of the recordings included in this study; i.e., for each cell, these parameters keptwell within the limits needed to maintain a steady lAHP amplitude under control conditions. Current-clamp recordings (Figure 5) were obtained from CA1 pyramidal cells in slices from adult Wistar rats(150-3OOg), using sharp microelectrodes filled with 2 M potassium acetate (resistance, 60-80 M62) and connected to an Axoclamp 2A amplifier in bridge currentclamp mode. Substances were applied extracellularly, by adding them to the superfusing medium, or intracellularly, by adding them to the pipette solution. Stock solutions of norepinephrine or isoprenaline were made fresh each day and kept on ice to avoid oxidation. In experiments with intracellular application of RpCAMPS or PKI, we used relatively high concentrations compared with the reported K. values (Bothelo et al., 1988; Cheng et al., 1986), both in our patch pipettes (100-500 PM Rp-CAMPS, 1 mM PKI) and in the microelectrodes (67 mM Rp-cAMPS), since lower concentrations gave only a partial inhibition. This is probably due to limited diffusion, since the blind patch method in slices gives relatively high series resistances. (In contrast, Diver&Pierluissi and Dunlap [1993], using larger patch pipettes in cultured neurons, obtained effects with lower concentrations of PKI). In addition, the depolarizing holding current that was injected to enhance the amplitude of the AHP or lAHPand the depolarizing steps used to elicit the spikes or I,,HP will tend to retain the negatively charged Rp-cAMPS or PKI in the pipette. PKI may also be partly degraded by peptidases in the cell. A similar requirement for high concentrations has been found in Aplysia neurons: Hochner and Kandel (1992) report that 80 mM Rp CAMPS in a microelectrode(8-12 M62) and injection with negative current produceda60% inhibitionoftheserotonineffects;Goldsmith and Abrams (1992) found that pressure injection of >500 PM of PKI was required to get reliable effects. Finally, it has been reported that excessive concentrations of Rp-cAMPS preparations may cause a slight activation of PKA, probably due to contamination bycAMP(Van Haastertetal., 1987), butthis is unlikely to have been a problem in our experiments since we did not see any significant rundown of the AHP, IAHP,or the spike frequency adaptation, or any other apparent side effect of Rp-CAMPS or PKI.

All drugs were obtained from Sigma, except Rp-cAMPS (BIOLOG Life Science Institute), microcystin (Calbiochem), transACPD (Tocris Neuramin), and tetrodotoxin (Research Biochemicals inc.). PKA-C was prepared from bovine heart, as described (Vintermyr et al., 1983) by Dr. S. 0. Doskeland’s laboratory, University of Bergen. PKI (sequence: lTYADFIASCRTG@RNAIHDamide) was synthesized in Dr. Paul Greengard’s laboratory, Rockefeller University. The less specific protein kinase inhibitors, which are mentioned briefly in the Introduction and the Discussion, but not used in the main part of this study, were obtained from the following sources: staurosporine from Calbiothem; H-7 from Sigma (three different batches, one of hhich was not the correct compound) and from Seikagaku Corp., Tokyo, Japan; K-252a was a gift from Dr. Paul Gordon; sphingosine and polymyxine from Sigma.

Acknowledgments We thank Dr. S. 0. Ddskeland for providing PKA-C, Drs. P. Greengard, A. Czernik, and A. Nairn for providing PKI, Dr. D. Qgreid for a sample of Rp-cAMPS, and Dr. P. Gordon for a sample of K-252a. We are especially grateful to Dr. I. Walaas for helpful advice. We also thank Drs. H.-C. Genieser, T. Jansen, 0. Paulsen, K. Tasken, and D. ogreid for helpful discussions and Dr. P. Andersen for lab space and equipment. This work was supported by the Norwegian Research Council (NAVF/RMF). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

July 29, 1993; revised

October

12, 1993.

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Added

5-Hydroxytryptamine

receptors.

in Proof

While this study was in progress, it was reported that the protein kinase inhibitor staurosporin partially blocks the CAMPdependent modulation of IA”P in hippocampal neurons (Gerber et al., 1992; Miiller et al., 1992; Sim et al., 1992) and that the phosphatase inhibitor okadaic acid causes a slow rundown of IAHP (Muller et al., 1992). These results, as well as those of the present study, are in accordance with the protein kinase A-dependent modulation of lAHP previously reported by Nicoll (1988).