Protein Kinase A Mediates the Modulation of the Slow Ca2+-Dependent K+ Current, IsAHP, by the Neuropeptides CRF, VIP, and CGRP in Hippocampal Pyramidal Neurons

Trude Haug and Johan F. Storm

Institute of Physiology and Neurophysiology, University of Oslo, N-0317 Oslo, Norway


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ABSTRACT
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Haug, Trude and Johan F. Storm. Protein Kinase A Mediates the Modulation of the Slow Ca2+-Dependent K+ Current, IsAHP, by the Neuropeptides CRF, VIP, and CGRP in Hippocampal Pyramidal Neurons. J. Neurophysiol. 83: 2071-2079, 2000. We have studied modulation of the slow Ca2+-activated K+ current (IsAHP) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). These peptides are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 µM) and tetraethylammonium (TEA, 5 mM), IsAHP was measured after a brief depolarizing voltage step eliciting inward Ca2+ current. Each of the peptides CRF (100-250 nM), VIP (400 nM), and CGRP (1 µM) significantly reduced the amplitude of IsAHP. Thus the IsAHP amplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 µM CGRP. We found no consistent concomitant changes in the Ca2+ current or in the time course of IsAHP for any of the three peptides, suggesting that the suppression of IsAHP was not secondary to a general suppression of Ca2+ channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and IsAHP is known to be suppressed by cAMP via the cAMP-dependent protein kinase (PKA), we tested whether the effects on IsAHP by CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of IsAHP by CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of IsAHP was reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1 µM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca2+-activated K+ current, IsAHP, in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP cascade modulating IsAHP.


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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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The slow Ca2+-activated K+ current (IsAHP) underlies the slow afterhyperpolarization and spike frequency adaptation in hippocampal and cortical pyramidal neurons (Alger and Nicoll 1980; Hotson and Prince 1980; Schwartzkroin and Stafstrom 1980). It has been suggested that IsAHP is mediated by small conductance Ca2+-activated K+ channels (SK channels) (Köhler et al. 1996; Lancaster and Adams 1986; Sah 1996; Storm 1989, 1990).

The IsAHP of hippocampal pyramidal cells can be modulated by several neurotransmitters secreted from the widespread ascending projections from the brain stem and basal forebrain. Acetylcholine, glutamate, and the monoamines norepinephrine, serotonin, dopamine, and histamine all suppress IsAHP, thus increasing the excitability of the target cells (Constanti and Sim 1987; Foehring et al. 1989; Haas and Konnerth 1983; Haas and Rose 1987; Madison and Nicoll 1982, 1984, 1986a,b; McCormick 1989; for reviews, see Nicoll 1988; Sah 1996; Storm 1990; Storm et al. 1999). This increase in excitability is thought to be part of the cellular processes leading to shifts in the functional state of the forebrain, from sleep to wakefulness, arousal and attention, and to participate in the regulation of sensory perception, emotions, cognition, learning, and memory (Foote et al. 1980; Livingston and Hubel 1981; Nicoll et al. 1990; Pollard and Schwartz 1987; Steriade and McCarley 1990).

The monoamine transmitters modulate IsAHP in hippocampal pyramidal cells via cyclic AMP (cAMP, adenosine 3',5'-cyclic monophosphate) and protein kinase A (PKA) (Blitzer et al. 1994; Haas and Konnerth 1983; Madison and Nicoll 1982, 1986a; Pedarzani and Storm 1993, 1995a,b; Pedarzani et al. 1998).

In addition to the ascending monoaminergic fibers, several peptidergic interneurons also synapse on the CA1 hippocampal pyramidal cells, and might modulate IsAHP and excitability in a similar manner. The peptides corticotropin releasing factor (CRF), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP) are found in neurons and interneurons in several parts of the brain, including the hippocampus (Chappell et al. 1986; De Souza 1987; De Souza et al. 1985a; Kresse 1995; Skofitch and Jacobowitz 1992; Usdin et al. 1994; van Rossum et al. 1997; Vertongen et al. 1997). Each of these peptides stimulates the cAMP production in several types of target cells (Etgen and Browning 1983; Gurantz et al. 1994; Lechleiter et al. 1988; Lee and Tse 1997; Magistretti and Schorderet 1984). We therefore decided to test whether these peptides also can suppress the sAHP in hippocampal pyramidal cells by activating the cAMP/PKA cascade, in a manner similar to that of the monoamine transmitters.

CRF is a 41 amino acid peptide involved in stress responses at several levels. It functions as a hormone in the hypothalamus-pituitary axis, releasing adrenocorticotropic hormone (ACTH), and as a neurotransmitter in the CNS, mediating numerous behavioral stress responses. When applied in the brain, CRF can cause increases in arousal, locomotion, startle and fear responses, general motor activity, and body temperature and suppresses exploration, food intake, and sexual behavior (Buwalda et al. 1997; Heinrichs et al. 1995; Holahan et al. 1997; Linthorst et al. 1997). CRF has also been implicated in memory consolidation and retention (Hung et al. 1992; Lee et al. 1996; Wu et al. 1997). High levels of CRF receptors are found in several brain structures, including cerebral cortex, the limbic system, hypothalamus, amygdala, cerebellum, some brain stem nuclei, and hippocampal cortex (De Souza et al. 1985a, 1987, 1995; Lehnert et al. 1998; Potter et al. 1994). CRF-containing neurons are found in neocortex, hypothalamus, several brain stem nuclei, amygdala, dentate hilus, and in the hippocampus (Chappel et al. 1986; Kaneko et al. 1998; Smith et al. 1997). In the hippocampus, the CRF-containing neurons form axosomatic and axodendritic symmetrical synapses on pyramidal and granular cells, and some also synapse on the axon initial segment of pyramidal cells (see Baram and Hatalski 1998). CRF is mostly coexpressed with GABA in the hippocampus but is also found together with neuropeptide Y (Smith et al. 1997). In CA1 hippocampal pyramidal cells, CRF has been shown to suppress IsAHP, thereby increasing the spontaneous discharge frequency (Aldenhoff et al. 1983). This increase in excitability may help to explain the finding that CRF can induce epileptic discharges and seizures in the hippocampus and other limbic structures (Baram and Hatalski 1998).

VIP is a 28 amino acid peptide, which functions as a hormone in the gastrointestinal tract, as a vasodilator both peripherally and in the brain, and as a neurotransmitter and modulator in CNS (Fahrenkrug 1993). VIP has also been implicated in arousal and selective attention (Murphy et al. 1993) and in learning processes (Glowa et al. 1992). VIP receptors are expressed in several brain structures, like the cerebral cortex, amygdala, thalamus, hypothalamus, and hippocampus (De Souza et al. 1985b; Usdin et al. 1994), and VIP-containing neurons are found in cerebral cortex and hippocampus (Leranth and Frotscher 1983; Magistretti and Morrison 1985; Magistretti and Schorderet 1984). In the hippocampus, three types of interneurons contain VIP: interneurons at the oriens/alveus border, a subpopulation of basket cells and interneurons projecting to the stria radiatum (Acsady et al. 1996). VIP can be coexpressed with ACh, GABA (Bayraktar et al. 1997; Casini and Brecha 1992; Eckenstein and Baughman 1984), substance P in regenerating neurons (Fristad et al. 1998), and neuropeptide Y (Houdeau et al. 1997; Leblanc et al. 1987). VIP has also been shown to suppress the sAHP in CA1 and CA3 pyramidal cells (Haas and Gähwiler 1992) and to increase the cAMP level in hippocampal slices (Etgen and Browning 1983).

CGRP is a 37 amino acid peptide, made by alternative splicing of the calcitonin/CGRP gene (CGRPalpha ) (Amara et al. 1982; Rosenfeld et al. 1983) or a separate gene (CGRPbeta ) (Amara et al. 1985). Like VIP, CGRP is also involved in vasodilatation (Bulloch et al. 1998; Fergus et al. 1995), besides being a neurotransmitter in the CNS (see van Rossum et al. 1997). CGRP receptors are distributed in the cerebral cortex and hippocampus, in the amygdala, several brain stem nuclei, cerebellum, thalamus, and hypothalamus (Skofitch and Jacobowitch 1985a, 1992). CGRP-containing neurons are found in the hypothalamus, preoptic area, amygdala, thalamus, limbic forebrain, several brain stem nuclei, hippocampus (CA3 pyramidal cells), and dentate gyrus granule cells (Bulloch et al. 1998; Freund et al. 1997; Skofitch and Jacobowitch 1985b). CGRP may be coexpressed with glutamate (Freund et al. 1997), substance P, and VIP (Baffi et al. 1992; Heym et al. 1993). The presence of CGRP neurons in the pathways for olfaction, vision, audition, and nociception suggests that CGRP is involved in sensory processing (see van Rossum et al. 1997). CGRP is also reported to be involved in learning and memory processing (Kovács and Telegdy 1992, 1994a,b).

Although numerous actions have been implied for CRF, VIP, and CGRP throughout the CNS, it has not previously been examined whether CGRP modulates IsAHP or by which mechanism CRF and VIP modulates this K+ current. We have therefore tested this in CA1 hippocampal pyramidal neurons. Our results indicate that all three peptides suppress IsAHP through activation of PKA. A preliminary report has been presented in abstract form (Haug and Storm 1998).


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Transverse hippocampal slices (400 µm thick) were prepared from young Wistar rats (16-24 days old) decapitated under halothane anesthesia. The slices were kept in an interface holding chamber. During recording, the slices were superfused with extracellular medium (in mM: 125 NaCl, 25 NaHCO3, 1.25 KCl, 1.25 KH2PO4, 2.0 CaCl2, 1.5 MgCl2, and 16 glucose, saturated with 95% O2-5% CO2) at room temperature. Bicuculline free base (10 µm) dissolved in HCl, tetrodotoxin (0.5 µm), TEA (5 mM), and bovine serum albumin (BSA, 0.1 mg/ml) were routinely added to the medium. Whole cell recordings from CA1 pyramidal cells were obtained by "blind patching" (Blanton et al. 1989). The patch pipettes were filled with intracellular medium containing either 140 mM K-gluconate (19 cells) or K-methylsulphate (27 cells), and (in mM) 10 HEPES, 2 ATP, 3 MgCl2, and 0.4 guanosine triphosphate (GTP). In some experiments (see RESULTS) 1 mM Rp-cAMPS was included in the intracellular medium. The peptides were bath applied by adding them to the superfusing medium. The application was started after 15 min of whole cell recording, to allow diffusion of Rp-cAMPS into the cell. The cells were voltage clamped at a holding potential of -60 mV. The slow AHP current, IsAHP, was elicited once every 30 s by 100-ms long depolarizing step commands to 0 mV. Each pulse triggered an inward Ca2+ current, which was unclamped, presumably due to space-clamp limitations. Following the Ca2+ influx induced by the depolarizing pulses, IsAHP was seen as a slowly developing and slowly decaying tail current. In some cells, the holding current showed a small inward or outward drift during the recording (see, e.g., Fig. 3B), but in most of the cells the holding current was stable throughout the experiment.

The current traces were filtered at 10 kHz, digitized (Instrutech VR-10), and stored on videotapes. Data analysis was performed using the programs pCLAMP 6 (Axon Instruments) and Origin5.0 (Microcal). Values are reported as means ± SE. Two-tailed Student's t-test was used for statistical comparisons between groups (alpha  = 0.05).

The access resistance, monitored by the capacitative transients at the onset and end of the voltage steps, was stable throughout each of the recordings included in this study (range: 10-25 MOmega ).

The membrane potential was monitored throughout each recording (range: -73 to -50 mV). Cells that depolarized beyond -50 mV were discarded. The input resistance of the recorded cells was typically more than three times the access resistance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell recordings were obtained from 46 CA1 pyramidal cells in hippocampal slices from young rats. The cells were voltage clamped at a holding potential of -60 mV, and the slow AHP current, IsAHP, was elicited by a 100-ms-long depolarizing step to 0 mV, once every 30 s.

Effects of CRF

To test the effect of corticotropin releasing factor (CRF) on IsAHP, we first used a moderate concentration (30 nM). In two of the four cells tested with this dose, CRF partly suppressed IsAHP in a reversible manner (Fig. 1A), without any consistent change in the time course of IsAHP or in the Ca2+ current recorded during the depolarizing step (not shown). In the cell illustrated in Fig. 1A, 30 nM CRF reduced the peak amplitude of IsAHP from 58 to 23 pA, and the current recovered to an amplitude of 50 pA. In another cell, 30 nM CRF reduced the peak amplitude of IsAHP from 64 to 40 pA after 10 min of exposure, whereas the remaining two cells tested with 30 nM CRF showed no convincing reduction of IsAHP.



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Fig. 1. Protein kinase A (PKA)-dependent suppression of IsAHP by corticotropin releasing factor (CRF) in CA1 pyramidal neurons. A: CRF (30 nM, bath-applied) reversibly suppressed IsAHP. IsAHP was elicited once every 30 s by a 100-ms depolarizing step to 0 mV from a holding potential of -60 mV. The peak amplitude of IsAHP is plotted. B: CRF (250 nM) suppressed IsAHP without any measurable effect on the Ca2+ current recorded during the depolarizing step used to elicit IsAHP. The Ca2+ currents at time points 1 (---) and 2 ( · · · ) are shown superimposed in the bottom left panel. (For most of the trace, the dotted line is not visible because of complete overlap). Below the continuous chart record are shown sample records of 3 consecutive IsAHP tail currents from just before and 5 min after the CRF application. black-triangle, each depolarizing step (not visible due to filtering by the chart recorder). Bottom right panel: superimposed averages of 10 traces from 0-5 min before and 5-10 min after the CRF application. Note the slow inward tail current that developed after full suppression of IsAHP by CRF. C: in a cell recorded with the PKA-inhibitor Rp-cAMPS (1 mM) in the patch pipette, the effect of CRF (250 nM) was strongly reduced (compare with B). D: summary data from the CRF experiments. , normalized mean IsAHP amplitude of cells recorded with normal intracellular medium for 10 min in normal saline [artificial cerebrospinal fluid (ACSF); n = 6], cells with normal intracellular medium exposed to 250 nM CRF for 10 min (n = 4), and cells recorded with Rp-cAMPS-containing (1 mM) intracellular medium and exposed to 250 nM CRF for 10 min (n = 4). Each cell was recorded in whole cell mode in ACSF for 15 min before the bath application (i.e., control cells were recorded in ACSF for 20-25 min total). IsAHP amplitudes represent the average of 5 records from each cell, 5-8 min into each application, and are normalized relative to the mean amplitude just before the CRF/ACSF application (, 100%). CRF significantly reduced IsAHP, when compared with the current in ACSF, both in cells with normal intracellular medium (**) and with Rp-cAMPS (*). Nevertheless, the effect of CRF was significantly reduced in cells loaded with Rp-cAMPS (*) as compared with cells recorded with normal intracellular medium. Error bars: SEM. **P < 0.01; *P < 0.05. CRF was bath applied (horizontal bars).

Higher concentrations of CRF (100-250 nM) significantly reduced the amplitude of IsAHP inn all cells tested (n = 7; Fig. 1B), and the current did not recover after wash out of the peptide. With 250 nM CRF, IsAHP was fully abolished in two of the four cells tested (Fig. 1B), and in the two remaining cells the peak amplitude was reduced to 30 and 38% of the original amplitude. On average, the IsAHP was reduced to 17.0 ± 9.9% by 250 nM (n = 4), and to 22.3 ± 22.3% (n = 3) by 100 nM CRF. The current was measured between 5 and 8 min after each peptide application, and compared with the period 0-3 min before peptide application, and the peak amplitude values are the mean of five successive trials. During most recordings, spontaneous miniature excitatory synaptic currents (minis) were observed as fast downward deflections from the baseline current (Fig. 1B).

In some cells (n = 2/7), the high doses of CRF (100-250 nM) produced a distinct inward shift in the holding current, in addition to the effect on IsAHP. The lack of any noticeable change in the Ca2+ current (inset in Fig. 1B) indicates that the inward shift was not due to deterioration of the cell. A similar inward current in response to monoamine transmitters was previously found to be due to a PKA-independent modulation by cAMP of the hyperpolarization-activated inward current, Ih (Pedarzani and Storm 1995; Storm et al. 1999).

In two cells, the blockade of IsAHP by a high dose of CRF (100-250 nM) was also accompanied by a slow inward tail current following each depolarizing step (Fig. 1B, right, black-triangle). This inward tail current is reminiscent of the so-called slow afterdepolarization current (IsADP) observed after muscarinic or metabotropic glutamate receptor stimulation in hippocampal and neocortical pyramidal cells (Andrade 1993; Gerber and Gahwiler 1994).

CRF modulates IsAHP via PKA

Because CRF has been shown to raise the cAMP level in several cell types, we tested whether the cAMP/PKA pathway mediates the effect of CRF on IsAHP. For these tests, we used the cAMP analogue Rp-cAMPS, a competitive blocker of the cAMP binding site on PKA (Botelho et al. 1988). Rp-cAMPS (1 mM) was added to the normal intracellular medium in the patch pipette, and allowed to diffuse into the cell by maintaining the whole cell recording for 15 min before applying CRF. Previous experiments in our laboratory has shown that this method reliably blocks most of the PKA activity in CA1 pyramidal cells, as judged by the reduced effect of monoamine transmitters on sAHP (Pedarzani and Storm 1993, 1995a,b). The effectiveness of the PKA blockade was verified in the present study by test applications of the beta -adrenergic receptor agonist isoproterenol (5 µM; n = 12; not shown) which is known to suppress IsAHP by activating PKA. For comparison, the control cells recorded without Rp-cAMPS in the pipette were also maintained in whole cell mode for 15 min before applying CRF.

As illustrated in Fig. 1C, the effect of CRF was significantly reduced in cells recorded with Rp-cAMPS in the pipette, compared with the cells without Rp-cAMPS (P = 0.02). The average reduction in IsAHP induced by 100-250 nM CRF was only ~30% in cells recorded with Rp-cAMPS, in contrast to ~80% reduction in the cells recorded without Rp-cAMPS. With Rp-cAMPS in the pipette, the sAHP current amplitude, measured after 5-8 min exposure to 250 nM CRF, was reduced to 67.8 ± 13.1% (mean ± SE) of the control amplitude prior to the CRF application, (n = 4) (Fig. 1C), and 100 nM CRF reduced the current to 74.0 ± 8.7% (n = 5) of the control value (Fig. 1D).

Because some previous studies indicate that IsAHP tends to become reduced over time (run down) during whole cell recording (Velumian et al. 1997), we measured changes in the current over a control period that was equal in duration to the peptide application period. In six cells, the IsAHP amplitude was first measured at a time corresponding to the control period, 12-15 min after establishing the whole cell configuration (equal to the delay allowed for intracellular diffusion of Rp-cAMPS) and again 5-8 min later (corresponding to the time used for CRF application). We found a reduction (run down) in the IsAHP amplitude of 5 ± 4.4% over this time period (n = 6; Fig. 1D, left columns), and the change appeared to be similar for cells recorded with gluconate-based (n = 1) and with methyl sulfate-based (n = 5) intracellular media. A comparison between this reduction and the CRF-induced reduction in IsAHP with Rp-cAMPS in the pipette showed a significant difference (P = 0.046), indicating that Rp-cAMPS did not fully prevent the effect of CRF on IsAHP (Fig. 1D).

The two other peptides, VIP and CGRP, were tested with the same experiment paradigm.

VIP reduces IsAHP via PKA

VIP also significantly reduced IsAHP (Fig. 2A). When measured 5-8 min after the onset of bath application of 400 nM VIP, the peak amplitude of IsAHP was on average reduced by 78% (to 22.2 ± 11.4% of the control value before application; n = 6). There was no noticeable concomitant reduction of the Ca2+ current (bottom left panel in Fig. 2A). Unlike 100-200 mM CRF, 400 mM VIP never produced a complete suppression of IsAHP, nor was there any clear inward current in response to this dose VIP, neither in the form of a distinct shift in the holding current, nor an inward tail current following the depolarizing pulses. Higher doses of VIP were not tested.



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Fig. 2. PKA-dependent suppression of IsAHP by vasoactive intestinal peptide (VIP). A-C: experimental protocol and data presentation are the same as in Fig. 1, B-D. A, top trace: continuous chart recording from a cell recorded with normal intracellular medium and exposed to 400 nM VIP (bath applied; horizontal bar). Middle traces: three successive IsAHP records from just before and 5 min after VIP application. Bottom left: the Ca2+ current at time points 1 and 2. Bottom right: IsAHP 0-5 min before and 5-10 min after VIP application (each trace is an average of 10 records). B: corresponding chart recording of a cell loaded with 1 mM Rp-cAMPS. Note the reduced effect of VIP on IsAHP (compare with A). C: summary data for cells exposed to VIP (n = 6 + 9), compared with data from cells only exposed to saline (ACSF, n = 6). VIP significantly reduced IsAHP, and Rp-cAMPS significantly reduced the effect of VIP. In each case, VIP (400 nM) was applied to the bath for 10 min.

After intracellular application of Rp-cAMPS, by 15 min of recording with 1 mM Rp-cAMPS in the recording pipette, the effect of VIP on IsAHP was significantly reduced (Fig. 2B). With Rp-cAMPS in the pipette, the amplitude of IsAHP was on average reduced by only 30% (to 70.2 ± 6.4%; n = 9), a reduction significantly smaller than the 78% reduction in the group of cells recorded without Rp-cAMPS.

As was observed with CRF, the VIP-induced reduction of the amplitude was significantly larger than the run down observed in the cells recorded for an equal period of time without any peptide application (Fig. 2C), indicating that Rp-cAMPS failed to fully block the effect of VIP.

CGRP reduces IsAHP via PKA

CGRP also significantly reduced IsAHP (Fig. 3A). When measured 5-8 min after the onset of bath application of 1.0 µM CGRP, the amplitude of IsAHP was reduced by 60% (to 40.4 ± 8.6% of the value before the application; n = 8). There was no measurable change in the calcium current (Fig. 3A, bottom left panel).



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Fig. 3. PKA-dependent suppression of IsAHP by calcitonin gene-related peptide (CGRP). A-C: experimental protocol and data presentations are the same as in Fig. 1, B-D. A, top trace: continuous chart recording from a cell recorded with normal intracellular medium and exposed to 1 µM CGRP (bath applied, horizontal bar). Middle traces: 3 successive IsAHP records from just before and 5 min after CGRP application. Bottom left: Ca2+ current at time points 1 and 2. Bottom right: IsAHP 0-5 min before and 5-10 min after CGRP application (each trace is an average of 10 records). B: corresponding chart recording of a cell loaded with 1 mM Rp-cAMPS. Note the reduced effect of CGRP. C: summary data for cells exposed to CGRP (n = 8 + 6), compared with data from cells only exposed to saline (ACSF, n = 6). CGRP significantly reduced IsAHP, and Rp-cAMPS significantly reduced the effect of CGRP. In each case, 1 µM CGRP was applied to the bath for 10 min.

Addition of Rp-cAMPS to the medium in the recording pipette blocked the effect of CGRP (Fig. 3B) virtually completely. With Rp-cAMPS in the pipette, the amplitude of IsAHP amplitude was on average reduced by <7%, to 93.3 ± 14.3% (n = 6), which is not significantly different from the observed run down in the cells only exposed to saline (Fig. 3C).

Effects of Rp-cAMPS on IsAHP under basal conditions

To assess the effect of Rp-cAMPS on the amplitude of IsAHP under basal conditions, we also compared IsAHP in cells with and without Rp-cAMPS in the pipette, measured after 15 min of whole cell recording, but before the peptide applications. In cells with Rp-cAMPS in the pipette, the amplitude of IsAHP was found to be significantly larger (53.3 ± 6.7 pA; n = 18) than in cells recorded without Rp-cAMPS (21.7 ± 4.5 pA; n = 12), indicating that IsAHP is tonically modulated via PKA even under basal conditions, as reported previously (Pedarzani et al. 1998).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main results of the present study are that each of the three neuropeptides (CRF, VIP, and CGRP) suppresses the slow Ca2+-dependent K+ current, IsAHP, via a PKA-dependent pathway in rat CA1 hippocampal pyramidal cells. CRF and VIP have already been shown to reduce the sAHP in CA1 hippocampal pyramidal cells (Aldenhoff et al. 1983; Haas and Gähwiler 1992), and CRF suppresses AHPs in cerebellar Purkinje neurons (Fox and Gruol 1993) and dentate granule neurons (Smith et al. 1997). However, to our knowledge it has not been tested previously whether CGRP has modulatory effects in hippocampal neurons, nor was it known whether CGRP has any effect on sAHP currents in other cells. It also appears to be the first time that involvement of PKA in the modulation of ion channels by CRF, VIP, and CGRP has been tested in CNS neurons.

We chose to study the effects of these peptides because they are widely distributed in the brain and implicated in processes known to involve changes in hippocampal excitability, such as learning, memory, and stress responses (for references, see INTRODUCTION). Furthermore, all three peptides have been shown to elevate the cAMP level in neurons (Etgen and Browning 1983; Gurantz 1994; Lee and Tse 1997; Magistretti and Schorderet 1984; Wang et al. 1998).

It was shown previously that norepinephrine and other monoamines modulate the IsAHP in CA1 pyramidal cells, by activating PKA (Pedarzani and Storm 1993, 1995a,b, 1996). The suppression of IsAHP by the monoamine transmitters, as well as by acetylcholine, is probably an important effector mechanism for the global state control of the cerebral cortex by the long and widely branched fiber projections that constitute the monoaminergic and cholinergic ascending activation systems from the brain stem and basal forebrain.

In contrast, the distribution of the neuropeptides CRF, VIP, and CGRP suggest that they act more locally, perhaps at selected pyramidal cells or subcellular compartments. For example, CRF is expressed in hippocampal neurons synapsing onto the soma and axon initial segment of pyramidal and granular cells and is often coexpressed with GABA or neuropeptide Y (Baram and Hatalski 1998; Smith et al. 1997). VIP is found in various hippocampal interneurons, including some basket cells (Acsady et al. 1996), often coexpressed with ACh or GABA (Bayraktar et al. 1997; Casini and Brecha 1992; Echstein and Baughman 1984) and neuropeptide Y (Houdeau et al. 1997; Leblanc et al. 1987), whereas CGRP is found in CA3 and dentate gyrus (Bulloch et al. 1998; Freund et al. 1997; Skofitch and Jacobowitch 1985b), coexpressed with glutamate, substance P, and VIP (Baffi et al. 1992; Freund et al. 1997; Heym et al. 1993). Thus these peptides may modulate the excitability and activity of pyramidal cells in a local manner, in part by suppressing IsAHP.

Sah and Bekkers (1996) found that isoproterenol, which suppresses IsAHP, lowered the threshold for induction of long-term potentiation in the CA1 area, and suggested that this effect was mediated by suppression of sAHP channels in the proximal dendrites. Thus ascending monoaminergic fibers may facilitate hippocampal LTP induction, and possibly LTP-dependent learning processes, in a global manner during arousal and attention. If so, the neuropeptides CRF, VIP, and CGRP would be expected to modulate LTP via sAHP modulation in a more specific, detailed and local manner, largely under the control of local interneurons and local network activity, using the same PKA-dependent biochemical machinery as the global monoaminergic systems.

This local peptidergic control would thus add to the complex convergent modulation of IsAHP, which also includes a steady-state balance between phosphatase and A-kinase activity (Pedarzani et al. 1998), and cross-talk between different G protein-coupled receptor types (Andrade 1993; Pedarzani and Storm 1995). It remains to be tested whether the CRF, VIP, and CGRP receptors participate in this type of synergistic or antagonistic interaction with other receptor types or between themselves.

CRF, VIP, and CGRP all reduced the IsAHP amplitude by >50%, but CGRP seemed to be less efficient than the two other peptides in suppressing the IsAHP. Estimated KD values from rat CNS ranges from 1 to 20 nM for CRF (De Souza 1987; Kapcala and De Souza 1988; Miyata et al. 1999; Perrin et al. 1993), 0.28 to 2.9 nM for VIP (Li et al. 1990; Zang and Qiu 1995), and 15 to 224 pM for CGRP (Chatterjee and Fisher 1991; Owji et al. 1996; Yoshizaki et al. 1987). Thus the bath concentrations that were effective in our and previous slice experiments (0.03-0.5 µM CRF, 0.1-1 µM VIP, and 0.5-1 µM CGRP) (Aldenhoff et al. 1983; Haas and Gähwiler 1992; Magistretti and Schorderet 1984) are high compared with the estimated KD values, and these values alone cannot readily account for the apparent lower potency of CGRP compared with CRF and VIP.

The peptide-induced reduction was largely blocked by the PKA blocker Rp-cAMPS for all three peptides. Nevertheless, CRF and VIP still produced a significant reduction of the IsAHP when the PKA was blocked by Rp-cAMPS, although the reduction was significantly smaller than without Rp-cAMPS. A similar incomplete blockade by Rp-cAMPS was also observed for monoamine transmitters and is most likely due to an incomplete inhibition of the intracellular PKA activity by Rp-cAMPS (Pedarzani and Storm 1993). However, we cannot exclude the possibility that CRF and VIP also partly works through other pathway(s). In other cell types, some effects of VIP, CRF, and CGRP are mediated through non-PKA-dependent pathways, and these peptides can also activate multiple pathways in the same cell. For example, VIP modulates N-type Ca2+ channels through PKC in sympathetic neurons (Zhu and Yakel 1997), activates PKC in astrocytes (Gressens et al. 1998), and couples to a Gi protein in lung epithelial cells (Diehl et al. 1996). CRF is thought to inhibit adenylate cyclase through PKC in rat Leydig cells (Ulisse et al. 1990) and increase cytosolic free Ca2+ concentration in human epidermoid cells (Kiang et al. 1994). CGRP receptors stimulate phospholipase C (PLC) in human bone cells (Drissi et al. 1998) and is shown to be coupled to both PKA and PKC (protein kinase C) in nonneuronal cells (Gressens et al. 1998). However, in CA1 pyramidal cells, the suppression of IsAHP seems to be mainly mediated by PKA.

The partially clamped inward Ca2+ spike current did not change noticeably during the reduction of the IsAHP. In accordance with the reasoning for monoaminergic effects of the sAHP (Madison and Nicoll 1982, 1986; Pedarzani and Storm 1993), this observation suggests that the three peptides inhibit IsAHP at a step subsequent to Ca2+ influx. This conclusion also agrees with that of Aldenhoff et al. (1983), who observed that CRF did not affect the Ca2+ spikes eliciting the sAHP in current-clamp experiments. However, the lack of spatial and point voltage control for the Ca2+ currents precludes confidence as to whether the underlying K+ channels or upstream Ca2+ channels are the primary targets of the modulation. Thus it remains possible that the peptides suppress only a part of the Ca2+ current, without noticeably changing the Ca2+ spike, which may be dominated by peptide-insensitive Ca2+ channels. Being a regenerative event, the largely unclamped Ca2+ spike is rather insensitive to suppression of a minority of the underlying channels. Furthermore, Ca2+ channels are known to be modulated by these peptides in other cell types. For example, VIP is known to inhibit N-type calcium current in rat sympathetic neurons (Ehrlich and Elmslie 1995; Zhu and Ikeda 1994; Zhu and Yakel 1997) and induces an increase of intracellular [Ca2+]i in a locus coeruleus-derived cell line via PKA activation (Bundey and Kendall 1999), whereas CGRP modulates dihydropyridine-sensitive Ca2+ channels in rat hypothalamus neurons (Tsuda et al. 1992).

We conclude that CRF, VIP, and CGRP suppress the slow Ca2+-activated K+ current IsAHP in CA1 hippocampal pyramidal neurons, largely by activating the cAMP-dependent protein kinase PKA, but it is not clear whether the underlying K+ channels or upstream Ca2+ channels are the primary targets of the modulation. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP/PKA cascade modulating IsAHP. By this mechanism, they can modulate the excitability and functional state of the hippocampus and probably play a similar role also in other parts of the cortex and forebrain.


    ACKNOWLEDGMENTS

This work was supported by the Norwegian Research Council and the Jahre and Nansen foundations.


    FOOTNOTES

Address for reprint requests: J. F. Storm, Institute of Physiology and Neurophysiology, University of Oslo, PB 1104 Blindern, N-0317 Oslo, Norway.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 October 1999; accepted in final form 14 December 1999.


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