Paired-pulse potentiation of 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones
1 Laboratory of Neurobiology, National Institute of Environmental Health Sciences, NIH, DHHS, PO Box 12233, Research Triangle Park, NC 27709, USA
Abstract
Diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including fast-desensitizing 7-containing receptors, are expressed in the CNS. While nAChRs appear to regulate cognitive processing and synaptic plasticity, little is known to date about how this regulation occurs, particularly in brain regions known to be important for cognition. By combining patch-clamp electrophysiology with local photolysis of caged carbachol to rapidly activate the 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices, we describe a novel transient up-regulation of channel function. The nAChRs were activated using a paired-pulse uncaging protocol, where the duration of the UV laser pulses (5–25 ms) and the interval between pulses (200 ms to 30 s) were varied. At relatively long interpulse intervals, we observed a strong (> 75%) decrease in the amplitude of the second response due to desensitization. However, when two pulses were applied at a 200 ms interval, a > 3-fold increase in the amplitude of the second response was observed, a phenomenon referred to here as paired-pulse potentiation. Interestingly, this potentiation appeared to be regulated by [Ca2+]i, and/or Ca2+-dependent processes, as it was significantly enhanced by dialysing cells with either the Ca2+ chelator BAPTA, or with peptide inhibitors of either calcineurin or PKC, and was attenuated by dialysing cells with the CaMKII inhibitor KN-93. No potentiation was observed using caged GABA or glutamate, indicating some specificity for nAChRs. Thus, rat hippocampal 7-containing nAChRs possess a newly described phenomenon of paired-pulse potentiation that may be involved in regulating synaptic plasticity in the hippocampus.
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Introduction
Nicotinic acetylcholine receptors (nAChRs) are in the superfamily of ligand-gated ion channels that also includes the serotonin 5-HT3, GABA, and glycine receptor channels. Neuronal nAChRs are widely expressed in the central and peripheral nervous system where they are involved in a variety of physiological processes, including cognition and development (Jones et al. 1999). In the brain, nAChRs have been shown both to mediate fast synaptic signalling and to regulate the release of other neurotransmitters. For example in the hippocampus, 7-containing nAChRs are located at postsynaptic sites on interneurones where they mediate fast cholinergic excitatory synaptic transmission (Alkondon et al. 1998; Frazier et al. 1998), and on presynaptic terminals where their activation increases intraterminal Ca2+ levels and facilitates glutamatergic release (Gray et al. 1996). Furthermore, they are known to participate in various forms of synaptic plasticity (Hunter et al. 1994; Fujii & Sumikawa, 2001; Ji et al. 2001; McGehee, 2002); however, the precise mechanisms involved are at present unclear.
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In the rat hippocampus, GABAergic interneurones receive cholinergic input from the medial septum-diagonal band complex (MSDB) of the basal forebrain (Frotscher & Léránth, 1985; Woolf, 1991), and express diverse subtypes of somato-dendritic nAChRs, including fast desensitizing 7-containing receptors, as well as a variety of non-7 types (Jones et al. 1999). The 7-containing nAChRs, both heterologously expressed and native to cultured neurones, have been shown to be highly Ca2+ permeable (Bertrand et al. 1993; Séguéla et al. 1993; Castro & Albuquerque, 1995; Berg & Conroy, 2002). In interneurones in rat hippocampal slices, the activation of 7-containing nAChRs significantly enhances cytoplasmic Ca2+ levels ([Ca2+]i) (Khiroug et al. 2003). Because Ca2+ regulates a variety of signal transduction cascades and plays a key role in the short- and long-term regulation of nAChRs (Berg & Conroy, 2002; Quick & Lester, 2002), this is probably one mechanism that underlies the role of nAChRs in regulating synaptic plasticity in the hippocampus (Ji et al. 2001; McGehee, 2002).
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We combined patch-clamp electrophysiology with the local photolysis of caged carbachol to rapidly and focally activate the 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices. The rapid kinetics of local photolysis of caged carbachol (Niu & Hess, 1993) dramatically reduces the effects of desensitization on peak current amplitude. By activating nAChRs using a paired-pulse uncaging protocol, we describe a novel potentiation of channel function that is dependent upon agonist exposure time as well as the interval between exposures. Furthermore we demonstrate that this potentiation phenomenon is regulated by [Ca2+]i and/or Ca2+-dependent processes, such as calcineurin, CaMKII, and PKC. This novel finding may represent a physiologically relevant form of regulation of 7-containing nAChR function in rat hippocampal slices that may play a role in synaptic plasticity in the hippocampus.
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Methods
Slice preparation
All experiments were carried out in accordance with guidelines approved by the NIEHS Animal Care and Use Committee, which includes minimizing the number of animals used and their suffering. Standard techniques were used to prepare 350 mm thick acute hippocampal slices from 14- to 21-day-old-rats (Klein & Yakel, 2004). Briefly, rats were anaesthetized with halothane and decapitated. Brains were quickly removed and placed in ice-cold oxygenated, artificial cerebral spinal fluid (ACSF) containing (mM): 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3 and 11 glucose. Upon dissection, brain chunks were glued to the stage of a vibrating blade microtome (VT1000S; Leica Microsystems, Wetzlar, Germany) and immersed in the cooled oxygenated ACSF. Slices were then used for recordings within 6 h, and after at least 1 h of recovery period.
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Electrophysiology
Whole-cell patch-clamp recordings were performed from CA1 stratum radiatum interneurones using patch pipettes (Garner 7052 or 8250 glass, with resistances of 3–4 M) filled with a solution containing (mM): 130 caesium gluconate, 2 NaCl, 4 Na2ATP, 0.4 Na2GTP, 4–5 MgCl2, 0.2 Oregon Green and 20 Hepes (pH 7.2–7.3). BAPTA (20 mM), calcineurin autoinhibitory peptide (CaN-AIP; 0.3 mM), protein kinase C inhibitor peptide (PKC-I19-31; Calbiochem; 0.2 mM), or KN-93 (Calbiochem; 5 μM, diluted from a 5 mM stock in DMSO), were added as indicated. Slices were superfused at room temperature (18–22°C) with ACSF containing TTX (1 μM) to block synaptic activity. CNB-caged carbachol, glutamate, and GABA (Molecular Probes, Eugene, OR) were added to the ACSF and delivered to the vicinity of the patch-clamped cell using an UltraMicroPump II syringe pump (WPI Inc., Sarasota, FL, USA) at the flow rate of 10 μl min–1. Quartz syringe tips (250 μm internal diameter) were placed just above the slice surface to ensure homogeneous coverage of the cell. The use of the syringe pump allowed reliable application of maximal concentrations of caged carbachol during continuous superfusion of the slice with fresh oxygenated ACSF, thus avoiding recycling of small volumes, which may cause slice damage. Cells were clamped at –70 mV (unless otherwise specified) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA). Current signals were recorded and filtered at 1 kHz and sampled at 10 kHz using pCLAMP 8.2 software (Axon Instruments). Statistical analyses were performed using Origin software (OriginLab Corp., Northampton, MA, USA). Averaged data were presented as means ± S.E.M. Statistical significance (P < 0.05) was assessed using Student's t test. Recordings were analysed only if the holding current was less than 100 pA when cells were voltage clamped at –70 mV. In some experiments (as indicated), responses were induced by pressure application of ACh (2 mM; 3–5 ms duration pulses at 10 p.s.i. pressure) via a glass pipette placed 5–10 μm from the cell body using a Picospritzer II (General Valve Co., Fairfield, NJ, USA). Atropine 1 μM was included to block muscarinic ACh receptors.
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Local photolysis
Interneurones were visualized using a fast-scanning confocal microscope (Radiance 2100; Bio-Rad, UK). Oregon Green–BAPTA (0.2 mM; Molecular Probes) was dialysed into the cell via the patch pipette, and its fluorescence was excited with 488 nm light and recorded using a 515 ± 15 nm bandpass filter. For local photolysis of caged carbachol, the 351–364 nm output of a continuous emission argon ion laser (Spectra Physics Stabilite 2017) was delivered via a multimode optical fibre (OZ Optics, Ontario, Canada) and a precision UV spot positioning device (Prairie Technologies, Middleton, WI, USA), through an Olympus 40x water-immersion objective (Khiroug et al. 2003). Focusing the UV beam (up to 1.3 mW laser power at the objective) on the interneuronal membrane yielded an uncaging spot of about 7 μm in diameter (Khiroug et al. 2003).
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Unless otherwise indicated, all chemicals were purchased from Sigma.
Results
Potentiation of 7 nAChR-mediated currents activated by local uncaging of carbachol
In acute slices of rat hippocampal CA1 stratum radiatum interneurones, nicotinic ACh receptor (nAChR)-mediated currents were elicited through rapid and local UV laser-based photolysis of caged carbachol (cCarb; 500 μM) at a holding potential of –70 mV. As previously described, rapid uncaging parameters preferentially activate 7-containing receptors, the predominant subtype of nAChRs in rat hippocampal interneurones (Khiroug et al. 2003). Using a dual-pulse uncaging protocol where the duration of both uncaging pulses was held constant at 15 ms, we examined the amplitude of the second response relative to the first by varying the interval between pulses (Fig. 1). A plot of the ratio of these amplitudes (i.e. ‘Resp2/Resp1’) versus interval produced a biphasic relationship where maximal desensitization (> 75%) was observed at a 3 s interval, and full recovery was achieved by 30 s (Fig. 1B). The relative amplitude of the second response was still partially desensitized at intervals of 500 ms and 1 s, although to a lesser extent than at 3 s. Interestingly when the interval was decreased to 200 ms, the relative amplitude of the second response was sometimes larger (i.e. potentiated) than the first response (Fig. 1).
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A, representative traces of 7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μM) by 15 ms UV laser pulses at varying intervals. The interpulse intervals are shown above the traces; for the bottom traces, the interval between the first pulse (shown on the left) and second are indicated above the hash marks. Scale bars are 50 pA and 250 ms. B, plot of the ratio of the amplitude of the response evoked by the second uncaging pulse to the first uncaging pulse (Resp2/Resp1) versus pulse interval. Data are means ± S.E.M. from 7–10 interneurones for each data point.
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To further explore this potentiation phenomenon, the duration of the uncaging pulses was varied between 5 and 25 ms, while maintaining a constant 200 ms interval. As shown in Fig. 2, the amplitude of the first response and the degree of potentiation of the second response were dependent on the pulse duration (Fig. 2A). Although the peak amplitude of the first response is smaller at shorter pulse durations, the amplitude of the second pulse is dramatically potentiated as the pulse duration decreases (Fig. 2B and Table 1). The bath application of dihydro--erythroidine (DHE; 10 μM), which blocks the non-7 nAChRs in these interneurones (Fayuk & Yakel, 2004), had no significant effect on the extent of potentiation (5 cells), confirming that potentiation was due to the enhancement of 7-containing nAChRs.
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A, representative traces of 7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μM) while varying the UV laser pulse durations (5–25 ms) and maintaining a constant 200 ms interval. Scale bars are 100 pA and 100 ms. B, plot of Resp2/Resp1 ratio versus pusle duration. Data are means ± S.E.M. from 19–23 interneurones.
The kinetic values for the rise times (10–90%) and decay times (i.e. the half-time of decay) were also examined, and the data are summarized in Table 1. For the pulse durations that produced potentiation (i.e. 5 and 10 ms), the decay of the responses to the second pulse was significantly slower as compared to the first pulse. For example for 5 ms duration pulses, the half-time of decay was 8.8 ± 2 ms for the first response and 31 ± 2 ms for the second (7 cells), whereas for 10 ms pulses, these values were 13 ± 2 ms and 20 ± 3 ms, respectively. Interestingly, there were no significant differences in the rates of decay for 15 ms duration pulses. As for the rise times, they were slower as the pulse duration increased, although there were no significant differences between the first and second pulses (Table 1).
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We tested whether responses to additional uncaging pulses elicited after the second response could also be potentiated. To do this, a sequence of four equal uncaging pulses (5 ms duration pulses at an interval of 200 ms) were given, and all three subsequent responses were significantly potentiated relative to the first. In eight cells, the ratio of the amplitudes of the second, third and fourth response relative to the first were, respectively, 2.9 ± 0.8, 2.7 ± 1 and 1.6 ± 0.6.
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No potentiation of glutamate and GABA receptor-mediated currents activated by local uncaging
To assess whether the potentiation observed with 7-containing nAChRs also occurred with other ligand-gated ion channels, we elicted responses by the local uncaging of either glutamate (cGlu; 0.5 μM) or GABA (cGABA; 2 mM). Using a constant interval of 200 ms, the duration of the uncaging pulses was varied from 1 to 25 ms. As shown in Fig. 3, the amplitudes and kinetics of the second responses were not significantly different from the first for both cGlu (Fig. 3A) and cGABA (Fig. 3B) (n = 4 for each agonist), suggesting that the potentiation that we have observed appears to be selective for 7-containing nAChRs. Furthermore, the similar kinetics and amplitudes for both the first and second uncaging responses for both cGlu and cGABA demonstrate that equal amounts of agonist are released with similar time courses for both pulses. In addition, if the interval between pulses was varied (using the same range as for cCarb, 0.2–30 s; Fig. 1), the amplitudes and kinetics of the second responses were not significantly different from the first for both cGlu and cGABA (data not shown). This suggests that the potentiation that we have observed for the 7-containing nAChRs is not the result of differential amounts and/or kinetics of carbachol uncaging with the two different uncaging pulses.
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Representative traces of interneurones responding to photolysis of 500 μM cGlu (A) or 2 mM cGABA (B). Varying paired-pulse durations (1–25 ms, as indicated above traces) were delivered at a 200 ms interval (n 4 for each transmitter). Scale bars are 50 pA and 100 ms for cGlutamate, and 50 pA and 250 ms for cGABA.
Potentiation of 7 nAChR-mediated currents activated by pressure application of ACh
In order to test whether the potentiation that we have observed by the local photolysis of cCarb was observed using an alternative approach, 7-containing nAChRs were activated by the dual-pulse pressure applications of ACh (2 mM) at a 200 ms interval. When applying brief pressure pulses (3–5 ms), the amplitude of the second response was potentiated relative to the first, yielding a ratio of 3.8 ± 0.6 (n = 6; data not shown). This is in strong agreement with the degree of potentiation observed with cCarb photolysis using a 5 ms UV pulse duration. These data demonstrate that the potentiation of 7-containing nAChRs is observed with both carbachol and the natural agonist ACh, as well as local UV laser-based photolysis or pressure-applied activation techniques.
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Potentiation of 7 nAChR-mediated currents is unaffected by bath-applied agonist and holding potential
We considered the possibility that the potentiation of 7-containing nAChRs with dual cCarb uncaging pulses was due to the residual buildup of free agonist after the first uncaging pulse that remained during the second uncaging pulse. To test this, carbachol was bath-applied at 10 μM, a concentration at the base of the 7 nAChR dose–response curve (with an EC50 value, determined for 7 nAChRs expressed in Xenopus oocytes, of 285 μM; Khiroug et al. 2002) that did not induce desensitization during these experiments. Using an uncaging pulse duration of either 5 or 10 ms, with an interval of 200 ms between pulses, the bath application of carbachol had no significant effect on either the potentiation or the kinetics of either the first or second uncaging response (n = 4; data not shown). Although the residual agonist concentration may be higher than 10 μM, the bath-application of higher concentrations of carbachol led to significant desensitization, and thus could not be used.
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We also considered the possibility that the potentiation was dependent on holding potential. In six cells, where the holding potential was varied from –40 to –70 and –90 mV (using 15 ms duration pulses with an interval of 200 ms), there was no significant change in the potentiation ratio; these values were, respectively, 1.1 ± 0.2, 1.1 ± 0.1 and 1.2 ± 0.1.
Potentiation of nAChR-mediated currents is regulated by [Ca2+]i level
Since the function of nAChRs, including 7-containing nAChRs in rat hippocampal interneurones (Khiroug et al. 2003), is regulated by intracellular Ca2+ ([Ca2+]i) levels, the effect of buffering [Ca2+]i to very low levels on the degree of potentiation was tested. Dialysing cells with the Ca2+ chelator BAPTA (20 mM) in the patch pipette solution significantly enhanced the degree of potentiation relative to control (Fig. 4). This increase in the extent of potentiation was significant for durations of 15 ms or less (for 200 ms intervals), and for intervals of 1 s or less (for 15 ms pulse durations) (Fig. 4B). The kinetics of uncaging responses in the presence of BAPTA were similar to those without BAPTA in that rise time and half-time of decay values slightly increased with the duration of the uncaging pulses, and that the rise times and decay of the second pulse were slower than the first (Table 1). Interestingly, for pulse durations of 15 ms or less, BAPTA dialysis significantly decreased the average amplitude of the first response compared to control, without significantly affecting the average amplitude of the second response (Table 1).
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A, representative traces illustrating paired-pulse potentiation evoked by a 5 ms pulse duration at a 200 ms pulse interval in a control cell (left) and in another cell dialysed with 20 mM BAPTA in the patch pipette (right). Scale bars are 50 pA and 200 ms. B, plot of Resp2/Resp1 ratio versus pulse duration using a 200 ms interval (left) or versus pulse interval using a 15 ms duration (right) for comparison of control () and BAPTA-dialysed cells (). Data are mean ± S.E.M. from 10 interneurones for BAPTA. Significant differences are indicated by an asterisk.
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Dependence of potentiation on CaN, PKC, and CaMKII
The modulatory effects of Ca2+ on the function of the 7-containing nAChRs may be direct, or indirect through Ca2+-dependent signal transduction cascades. For example, 7-containing nAChRs in chick ciliary ganglion neurones are known to be regulated by the Ca2+-dependent phosphatase calcineurin (CaN) and the Ca2+-dependent kinase CaMKII (Liu & Berg, 1999). To test whether the potentiation of the 7-containing nAChRs could be regulated by CaN, cells were dialysed with the CaN autoinhibitory peptide (CaN-AIP; 0.3 mM). Similar to the effects with BAPTA, dialysis with the CaN-AIP significantly enhanced the degree of potentiation relative to control (Fig. 5). The increase in potentiation was significant for durations of 15 ms or less for the 200 ms interval. Similar effects were observed upon dialysis with an inhibitory peptide of protein kinase C (PKC-I19–31; 0.2 mM), which also significantly enhanced the degree of potentiation relative to control for pulse durations of 15 ms or less (Fig. 5). The average current amplitude of the first response elicited for 5 ms duration pulses in the presence of either CaN-AIP or PKC-I19-31 was not significantly different from controls. Interestingly when we dialysed cells with the CaMKII inhibitor KN-93 (5 μM), there was no longer any significant potentiation for any pulse duration (Fig. 5). These data support the hypothesis that an intracellular Ca2+-dependent mechanism, perhaps through CaN, CaMKII, and/or PKC, regulates the degree of potentiation for 7-containing nAChRs in rat hippocampal interneurones in the slice.
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Plot of Resp2/Resp1 ratio versus pulse duration for control, or in the presence of either the calcineurin autoinhibitory peptide (CaN-AIP; 300 μM), or the PKC inhibitory peptide fragment 19-31 (PKC-I19-31; 200 μM), or a CaMKII inhibitor (KN-93; 5 μM). Data are means ± S.E.M. from 10 interneurones for CaN-AIP and PKC-I19-31, and 8 interneurones for KN-93.
Discussion
We have described a novel, transient potentiation of 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices. Native nAChRs were activated with the local photolysis of caged carbachol (cCarb) using a paired-pulse protocol, where the duration of the UV laser pulses (5–25 ms) and the interval between pulses (200 ms to 30 s) were varied. The rapid kinetics of photolysis dramatically reduces the effects of desensitization on peak current amplitude, which is important since 7-containing nAChRs are known to desensitize rapidly (McGehee & Role, 1995; Khiroug et al. 2002; Quick & Lester, 2002). While maintaining a constant pulse duration (15 ms), interpulse intervals 1 s resulted in a strong (> 50%) decrease in the amplitude of the second response, which can be ascribed to desensitization. However, when the interpulse interval was 200 ms (at a duration of 5 ms), a > 3-fold increase in the amplitude of the second response was observed. Similar responses were also elicited with pressure-applied ACh, demonstrating that the natural agonist can induce this potentiation phenomenon, and also that it can be observed by activating the receptors via multiple methods. In addition, if a series of uncaging pulses (i.e. 4 at 5 ms durations and 200 ms intervals) was applied, the fourth uncaging response was still potentiated relative to the first.
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The extent of potentiation was also dependent on the duration of the uncaging pulses, where shorter duration pulses resulted in a greater degree of potentiation. Because the amplitude of the first response was much smaller for pulse durations less than 15 ms, it appears as if potentiation is contingent upon the submaximal activation of the 7 receptors. In addition, if the receptors are maximally activated, for example with durations of 15 ms or longer, little to no potentiation is observed; this is likely to be due to the possibility that potentiation under these conditions (i.e. maximal activation of the first response) is masked by receptor desensitization. Furthermore, the amplitude of the potentiated current is not larger than the first response to a maximal stimulus, suggesting that during potentiation, there is no recruitment of new channels. Therefore, it appears as if there is a fine balance between activation, potentiation, and desensitization of 7-containing nAChRs in rat hippocampal interneurones.
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It is well established that Ca2+ can activate and regulate a variety of signal transduction cascades, and plays a key role in the short- and long-term regulation of nAChRs (Quick & Lester, 2002). In addition, Ca2+ could be exerting an effect either directly or indirectly. Previously we showed that 7-containing nAChR recovery from desensitization was strongly dependent upon [Ca2+]i (Khiroug et al. 2003). In the present study, we found that both Ca2+ and Ca2+-dependent signal transduction cascades also affected potentiation. For example, the extent of potentiation was increased several-fold by dialysing cells with high concentrations of the Ca2+ chelator BAPTA (20 mM). Interestingly it appears as if the reduction in [Ca2+]i decreases the amplitude of the first response rather than significantly altering the amplitude of the second response. However as the duration of the uncaging pulses was decreased, the amplitudes of responses in cells dialysed with BAPTA were progressively lower than control. Although this is not a true dose–response relationship, this does suggest that the efficacy of the agonist to open the channels (i.e. the gating) might be affected by [Ca2+]i. In addition, it is possible that the reduced function of the channel on the first response due to BAPTA might be counteracted by the influx of Ca2+, so that the second response is far larger (and resulting in enhanced potentiation).
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Our data suggest that Ca2+ may be having an indirect effect on nAChR channel function since inhibiting either the Ca2+-dependent phosphatase calcineurin (CaN) or protein kinase C (PKC) significantly enhanced the extent of potentiation, while inhibiting CaMKII attenuated this potentiation. In chick ciliary ganglion neurones, the function of 7 nAChRs runs down with time in a Ca2+-dependent manner, and this rundown is enhanced by inhibition of CaN and prevented by block of CaMKII (Liu & Berg, 1999). Non-7 nAChRs have also been shown to be regulated by Ca2+-dependent processes. For example for 42 receptors expressed in Xenopus oocytes, the increase of [Ca2+]i and subsequent activation of PKC promotes the recovery from desensitization (Fenster et al. 1997, 1999; Quick & Lester, 2002). In rat chromaffin cells, however, the recovery of non-7 nAChRs from desensitization was slower when [Ca2+]i increases were prolonged (Khiroug et al. 1997, 1998). At present, it is unclear how inhibiting a Ca2+-dependent phosphatase (CaN) or kinase (PKC) exerts similar effects with respect to the nAChR potentiation described here, but it is likely that it occurs by altering the Ca2+-dependent phosphorylation state of the receptor.
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We considered several possibilities to explain the molecular mechanism responsible for potentiation. The first is that residual free agonist from the first uncaging pulse remains at the time of the second pulse, thus increasing the actual agonist concentration during the second pulse, and thus activating more channels. This does not seem likely for several reasons. First, we did not see any potentiation with responses due to the uncaging of either GABA or glutamate, as might be expected if the buildup of residual agonist was responsible for potentiation. Second, the bath-application of a low dose of carbachol had no effect on potentiation; if the residual buildup of carbachol was involved in the potentiation, this bath-application of carbachol may have had an effect. Lastly, the estimated concentration required to produce a 3-fold increase in current amplitude did not appear to be attainable. The maximal effective dose of free carbachol possible using 0.5 mM cCarb (with a quantal yield of 0.8) (Milburn et al. 1989) would be 0.4 mM, a concentration which would be expected to activate responses that were 60% of maximum (based on carbachol dose–response curves for rat 7 receptors expressed in Xenopus oocytes) (Khiroug et al. 2002). Since the amplitude of the 5 ms uncaging pulses was 25% of the longest duration (i.e. 25 ms) pulse (which we will assume here achieved a carbachol concentration of 0.4 mM), we estimate that the amplitude of these brief (i.e. 5 ms) pulses activates currents to 15% of maximum (i.e. from the dose–response curve) (Khiroug et al. 2002). The approximate carbachol concentration corresponding to 15% of maximum is 120 μM. We estimate that in order to achieve a 3-fold potentiation for the second 5 ms pulse, the carbachol concentration would have to reach 300 μM. Based on these calculations, this is not possible, particularly in light of the fact that the free agonist concentration after the first pulse (which we estimated may have reached 120 μM) should have decayed to well below 20 μM (the foot of the dose–response curve) by the start of the second pulse (as the baseline current generally had returned to baseline levels). Therefore it does not seem likely that residual free agonist can explain the potentiation of the 7 nAChRs.
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Another consideration was whether some of the receptors had bound agonist molecules (e.g. singly liganded channels) after exposure of the first response, even if the free agonist concentration has decayed to below 20 μM. In this case, the channels might be ‘primed’ to respond more robustly to the second pulse of agonist due to increased efficacy, resulting in an apparent potentiation of the response amplitude. However this did not appear to be the case since the bath application of a low dose of carbachol (10 μM), a dose that did not induce desensitization during our experiments but which might be able to prime some channels, had no effect on either the amplitude or potentiation of 7 receptors. In addition, neither residual agonist nor priming are consistent with the fact that Ca2+ and Ca2+-dependent intracellular signal transduction cascades significantly affected the extent of potentiation. In addition, such a priming mechanism does not explain the significantly slower decay of the second (i.e. potentiated) response.
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Although clear evidence indicates that nAChRs can directly regulate synaptic plasticity in the hippocampus (Hunter et al. 1994; Fujii & Sumikawa, 2001; Ji et al. 2001; McGehee, 2002), as well as cognitive processes in humans and animal models (Levin, 2002), the cellular mechanisms involved are not fully understood. In the brain and in particular in the hippocampus, 7-containing nAChRs are located both at postsynaptic sites on interneurones where they mediate fast cholinergic excitatory synaptic transmission (Alkondon et al. 1998; Frazier et al. 1998), and on presynaptic terminals where their activation facilitates glutamatergic release (Gray et al. 1996). However, these nAChRs might also be located and function extrasynaptically in the hippocampus, and work by non-synaptic, diffuse, so called ‘volume transmission’ (Umbriaco et al. 1995).
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The medial septum/diagonal band (MS/DB) complex, via the septohippocampal pathway, is thought to be critical for the generation and/or maintenance of the hippocampal theta rhythm in vivo (Buzsaki, 2002). In the rat hippocampus, interneurones receive cholinergic input from the medial septum–diagonal band complex (MSDB) of the basal forebrain (Frotscher & Léránth, 1985; Woolf, 1991), as well as from intrinsic cholinergic interneurones (Matthews et al. 1987; Cobb et al. 1999). Septal cholinergic neurones and GABAergic interneurones fire in rhythmic bursts to entrain hippocampal interneurones (Jones et al. 1999; Buzsaki, 2002). In addition, intrinsic cholinergic interneurones can pattern network activity, via nAChRs, in the hippocampus, and nAChR activation increases theta activity in freely moving rabbits (Jones et al. 1999). The time dependence of the potentiation that we have observed in the present study is physiologically consistent with the timing of the various forms of rhythmic activity (e.g. theta rhythms; Buzsaki, 2002) and synchrony in the hippocampus directly linked with plasticity and cognitive processes. Thus this newly described phenomenon of paired-pulse potentiation could be participating in regulating rhythmic patterns of electrical activity in the hippocampus.
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Abstract
Diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including fast-desensitizing 7-containing receptors, are expressed in the CNS. While nAChRs appear to regulate cognitive processing and synaptic plasticity, little is known to date about how this regulation occurs, particularly in brain regions known to be important for cognition. By combining patch-clamp electrophysiology with local photolysis of caged carbachol to rapidly activate the 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices, we describe a novel transient up-regulation of channel function. The nAChRs were activated using a paired-pulse uncaging protocol, where the duration of the UV laser pulses (5–25 ms) and the interval between pulses (200 ms to 30 s) were varied. At relatively long interpulse intervals, we observed a strong (> 75%) decrease in the amplitude of the second response due to desensitization. However, when two pulses were applied at a 200 ms interval, a > 3-fold increase in the amplitude of the second response was observed, a phenomenon referred to here as paired-pulse potentiation. Interestingly, this potentiation appeared to be regulated by [Ca2+]i, and/or Ca2+-dependent processes, as it was significantly enhanced by dialysing cells with either the Ca2+ chelator BAPTA, or with peptide inhibitors of either calcineurin or PKC, and was attenuated by dialysing cells with the CaMKII inhibitor KN-93. No potentiation was observed using caged GABA or glutamate, indicating some specificity for nAChRs. Thus, rat hippocampal 7-containing nAChRs possess a newly described phenomenon of paired-pulse potentiation that may be involved in regulating synaptic plasticity in the hippocampus.
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Introduction
Nicotinic acetylcholine receptors (nAChRs) are in the superfamily of ligand-gated ion channels that also includes the serotonin 5-HT3, GABA, and glycine receptor channels. Neuronal nAChRs are widely expressed in the central and peripheral nervous system where they are involved in a variety of physiological processes, including cognition and development (Jones et al. 1999). In the brain, nAChRs have been shown both to mediate fast synaptic signalling and to regulate the release of other neurotransmitters. For example in the hippocampus, 7-containing nAChRs are located at postsynaptic sites on interneurones where they mediate fast cholinergic excitatory synaptic transmission (Alkondon et al. 1998; Frazier et al. 1998), and on presynaptic terminals where their activation increases intraterminal Ca2+ levels and facilitates glutamatergic release (Gray et al. 1996). Furthermore, they are known to participate in various forms of synaptic plasticity (Hunter et al. 1994; Fujii & Sumikawa, 2001; Ji et al. 2001; McGehee, 2002); however, the precise mechanisms involved are at present unclear.
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In the rat hippocampus, GABAergic interneurones receive cholinergic input from the medial septum-diagonal band complex (MSDB) of the basal forebrain (Frotscher & Léránth, 1985; Woolf, 1991), and express diverse subtypes of somato-dendritic nAChRs, including fast desensitizing 7-containing receptors, as well as a variety of non-7 types (Jones et al. 1999). The 7-containing nAChRs, both heterologously expressed and native to cultured neurones, have been shown to be highly Ca2+ permeable (Bertrand et al. 1993; Séguéla et al. 1993; Castro & Albuquerque, 1995; Berg & Conroy, 2002). In interneurones in rat hippocampal slices, the activation of 7-containing nAChRs significantly enhances cytoplasmic Ca2+ levels ([Ca2+]i) (Khiroug et al. 2003). Because Ca2+ regulates a variety of signal transduction cascades and plays a key role in the short- and long-term regulation of nAChRs (Berg & Conroy, 2002; Quick & Lester, 2002), this is probably one mechanism that underlies the role of nAChRs in regulating synaptic plasticity in the hippocampus (Ji et al. 2001; McGehee, 2002).
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We combined patch-clamp electrophysiology with the local photolysis of caged carbachol to rapidly and focally activate the 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices. The rapid kinetics of local photolysis of caged carbachol (Niu & Hess, 1993) dramatically reduces the effects of desensitization on peak current amplitude. By activating nAChRs using a paired-pulse uncaging protocol, we describe a novel potentiation of channel function that is dependent upon agonist exposure time as well as the interval between exposures. Furthermore we demonstrate that this potentiation phenomenon is regulated by [Ca2+]i and/or Ca2+-dependent processes, such as calcineurin, CaMKII, and PKC. This novel finding may represent a physiologically relevant form of regulation of 7-containing nAChR function in rat hippocampal slices that may play a role in synaptic plasticity in the hippocampus.
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Methods
Slice preparation
All experiments were carried out in accordance with guidelines approved by the NIEHS Animal Care and Use Committee, which includes minimizing the number of animals used and their suffering. Standard techniques were used to prepare 350 mm thick acute hippocampal slices from 14- to 21-day-old-rats (Klein & Yakel, 2004). Briefly, rats were anaesthetized with halothane and decapitated. Brains were quickly removed and placed in ice-cold oxygenated, artificial cerebral spinal fluid (ACSF) containing (mM): 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3 and 11 glucose. Upon dissection, brain chunks were glued to the stage of a vibrating blade microtome (VT1000S; Leica Microsystems, Wetzlar, Germany) and immersed in the cooled oxygenated ACSF. Slices were then used for recordings within 6 h, and after at least 1 h of recovery period.
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Electrophysiology
Whole-cell patch-clamp recordings were performed from CA1 stratum radiatum interneurones using patch pipettes (Garner 7052 or 8250 glass, with resistances of 3–4 M) filled with a solution containing (mM): 130 caesium gluconate, 2 NaCl, 4 Na2ATP, 0.4 Na2GTP, 4–5 MgCl2, 0.2 Oregon Green and 20 Hepes (pH 7.2–7.3). BAPTA (20 mM), calcineurin autoinhibitory peptide (CaN-AIP; 0.3 mM), protein kinase C inhibitor peptide (PKC-I19-31; Calbiochem; 0.2 mM), or KN-93 (Calbiochem; 5 μM, diluted from a 5 mM stock in DMSO), were added as indicated. Slices were superfused at room temperature (18–22°C) with ACSF containing TTX (1 μM) to block synaptic activity. CNB-caged carbachol, glutamate, and GABA (Molecular Probes, Eugene, OR) were added to the ACSF and delivered to the vicinity of the patch-clamped cell using an UltraMicroPump II syringe pump (WPI Inc., Sarasota, FL, USA) at the flow rate of 10 μl min–1. Quartz syringe tips (250 μm internal diameter) were placed just above the slice surface to ensure homogeneous coverage of the cell. The use of the syringe pump allowed reliable application of maximal concentrations of caged carbachol during continuous superfusion of the slice with fresh oxygenated ACSF, thus avoiding recycling of small volumes, which may cause slice damage. Cells were clamped at –70 mV (unless otherwise specified) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA). Current signals were recorded and filtered at 1 kHz and sampled at 10 kHz using pCLAMP 8.2 software (Axon Instruments). Statistical analyses were performed using Origin software (OriginLab Corp., Northampton, MA, USA). Averaged data were presented as means ± S.E.M. Statistical significance (P < 0.05) was assessed using Student's t test. Recordings were analysed only if the holding current was less than 100 pA when cells were voltage clamped at –70 mV. In some experiments (as indicated), responses were induced by pressure application of ACh (2 mM; 3–5 ms duration pulses at 10 p.s.i. pressure) via a glass pipette placed 5–10 μm from the cell body using a Picospritzer II (General Valve Co., Fairfield, NJ, USA). Atropine 1 μM was included to block muscarinic ACh receptors.
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Local photolysis
Interneurones were visualized using a fast-scanning confocal microscope (Radiance 2100; Bio-Rad, UK). Oregon Green–BAPTA (0.2 mM; Molecular Probes) was dialysed into the cell via the patch pipette, and its fluorescence was excited with 488 nm light and recorded using a 515 ± 15 nm bandpass filter. For local photolysis of caged carbachol, the 351–364 nm output of a continuous emission argon ion laser (Spectra Physics Stabilite 2017) was delivered via a multimode optical fibre (OZ Optics, Ontario, Canada) and a precision UV spot positioning device (Prairie Technologies, Middleton, WI, USA), through an Olympus 40x water-immersion objective (Khiroug et al. 2003). Focusing the UV beam (up to 1.3 mW laser power at the objective) on the interneuronal membrane yielded an uncaging spot of about 7 μm in diameter (Khiroug et al. 2003).
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Unless otherwise indicated, all chemicals were purchased from Sigma.
Results
Potentiation of 7 nAChR-mediated currents activated by local uncaging of carbachol
In acute slices of rat hippocampal CA1 stratum radiatum interneurones, nicotinic ACh receptor (nAChR)-mediated currents were elicited through rapid and local UV laser-based photolysis of caged carbachol (cCarb; 500 μM) at a holding potential of –70 mV. As previously described, rapid uncaging parameters preferentially activate 7-containing receptors, the predominant subtype of nAChRs in rat hippocampal interneurones (Khiroug et al. 2003). Using a dual-pulse uncaging protocol where the duration of both uncaging pulses was held constant at 15 ms, we examined the amplitude of the second response relative to the first by varying the interval between pulses (Fig. 1). A plot of the ratio of these amplitudes (i.e. ‘Resp2/Resp1’) versus interval produced a biphasic relationship where maximal desensitization (> 75%) was observed at a 3 s interval, and full recovery was achieved by 30 s (Fig. 1B). The relative amplitude of the second response was still partially desensitized at intervals of 500 ms and 1 s, although to a lesser extent than at 3 s. Interestingly when the interval was decreased to 200 ms, the relative amplitude of the second response was sometimes larger (i.e. potentiated) than the first response (Fig. 1).
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A, representative traces of 7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μM) by 15 ms UV laser pulses at varying intervals. The interpulse intervals are shown above the traces; for the bottom traces, the interval between the first pulse (shown on the left) and second are indicated above the hash marks. Scale bars are 50 pA and 250 ms. B, plot of the ratio of the amplitude of the response evoked by the second uncaging pulse to the first uncaging pulse (Resp2/Resp1) versus pulse interval. Data are means ± S.E.M. from 7–10 interneurones for each data point.
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To further explore this potentiation phenomenon, the duration of the uncaging pulses was varied between 5 and 25 ms, while maintaining a constant 200 ms interval. As shown in Fig. 2, the amplitude of the first response and the degree of potentiation of the second response were dependent on the pulse duration (Fig. 2A). Although the peak amplitude of the first response is smaller at shorter pulse durations, the amplitude of the second pulse is dramatically potentiated as the pulse duration decreases (Fig. 2B and Table 1). The bath application of dihydro--erythroidine (DHE; 10 μM), which blocks the non-7 nAChRs in these interneurones (Fayuk & Yakel, 2004), had no significant effect on the extent of potentiation (5 cells), confirming that potentiation was due to the enhancement of 7-containing nAChRs.
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A, representative traces of 7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μM) while varying the UV laser pulse durations (5–25 ms) and maintaining a constant 200 ms interval. Scale bars are 100 pA and 100 ms. B, plot of Resp2/Resp1 ratio versus pusle duration. Data are means ± S.E.M. from 19–23 interneurones.
The kinetic values for the rise times (10–90%) and decay times (i.e. the half-time of decay) were also examined, and the data are summarized in Table 1. For the pulse durations that produced potentiation (i.e. 5 and 10 ms), the decay of the responses to the second pulse was significantly slower as compared to the first pulse. For example for 5 ms duration pulses, the half-time of decay was 8.8 ± 2 ms for the first response and 31 ± 2 ms for the second (7 cells), whereas for 10 ms pulses, these values were 13 ± 2 ms and 20 ± 3 ms, respectively. Interestingly, there were no significant differences in the rates of decay for 15 ms duration pulses. As for the rise times, they were slower as the pulse duration increased, although there were no significant differences between the first and second pulses (Table 1).
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We tested whether responses to additional uncaging pulses elicited after the second response could also be potentiated. To do this, a sequence of four equal uncaging pulses (5 ms duration pulses at an interval of 200 ms) were given, and all three subsequent responses were significantly potentiated relative to the first. In eight cells, the ratio of the amplitudes of the second, third and fourth response relative to the first were, respectively, 2.9 ± 0.8, 2.7 ± 1 and 1.6 ± 0.6.
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No potentiation of glutamate and GABA receptor-mediated currents activated by local uncaging
To assess whether the potentiation observed with 7-containing nAChRs also occurred with other ligand-gated ion channels, we elicted responses by the local uncaging of either glutamate (cGlu; 0.5 μM) or GABA (cGABA; 2 mM). Using a constant interval of 200 ms, the duration of the uncaging pulses was varied from 1 to 25 ms. As shown in Fig. 3, the amplitudes and kinetics of the second responses were not significantly different from the first for both cGlu (Fig. 3A) and cGABA (Fig. 3B) (n = 4 for each agonist), suggesting that the potentiation that we have observed appears to be selective for 7-containing nAChRs. Furthermore, the similar kinetics and amplitudes for both the first and second uncaging responses for both cGlu and cGABA demonstrate that equal amounts of agonist are released with similar time courses for both pulses. In addition, if the interval between pulses was varied (using the same range as for cCarb, 0.2–30 s; Fig. 1), the amplitudes and kinetics of the second responses were not significantly different from the first for both cGlu and cGABA (data not shown). This suggests that the potentiation that we have observed for the 7-containing nAChRs is not the result of differential amounts and/or kinetics of carbachol uncaging with the two different uncaging pulses.
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Representative traces of interneurones responding to photolysis of 500 μM cGlu (A) or 2 mM cGABA (B). Varying paired-pulse durations (1–25 ms, as indicated above traces) were delivered at a 200 ms interval (n 4 for each transmitter). Scale bars are 50 pA and 100 ms for cGlutamate, and 50 pA and 250 ms for cGABA.
Potentiation of 7 nAChR-mediated currents activated by pressure application of ACh
In order to test whether the potentiation that we have observed by the local photolysis of cCarb was observed using an alternative approach, 7-containing nAChRs were activated by the dual-pulse pressure applications of ACh (2 mM) at a 200 ms interval. When applying brief pressure pulses (3–5 ms), the amplitude of the second response was potentiated relative to the first, yielding a ratio of 3.8 ± 0.6 (n = 6; data not shown). This is in strong agreement with the degree of potentiation observed with cCarb photolysis using a 5 ms UV pulse duration. These data demonstrate that the potentiation of 7-containing nAChRs is observed with both carbachol and the natural agonist ACh, as well as local UV laser-based photolysis or pressure-applied activation techniques.
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Potentiation of 7 nAChR-mediated currents is unaffected by bath-applied agonist and holding potential
We considered the possibility that the potentiation of 7-containing nAChRs with dual cCarb uncaging pulses was due to the residual buildup of free agonist after the first uncaging pulse that remained during the second uncaging pulse. To test this, carbachol was bath-applied at 10 μM, a concentration at the base of the 7 nAChR dose–response curve (with an EC50 value, determined for 7 nAChRs expressed in Xenopus oocytes, of 285 μM; Khiroug et al. 2002) that did not induce desensitization during these experiments. Using an uncaging pulse duration of either 5 or 10 ms, with an interval of 200 ms between pulses, the bath application of carbachol had no significant effect on either the potentiation or the kinetics of either the first or second uncaging response (n = 4; data not shown). Although the residual agonist concentration may be higher than 10 μM, the bath-application of higher concentrations of carbachol led to significant desensitization, and thus could not be used.
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We also considered the possibility that the potentiation was dependent on holding potential. In six cells, where the holding potential was varied from –40 to –70 and –90 mV (using 15 ms duration pulses with an interval of 200 ms), there was no significant change in the potentiation ratio; these values were, respectively, 1.1 ± 0.2, 1.1 ± 0.1 and 1.2 ± 0.1.
Potentiation of nAChR-mediated currents is regulated by [Ca2+]i level
Since the function of nAChRs, including 7-containing nAChRs in rat hippocampal interneurones (Khiroug et al. 2003), is regulated by intracellular Ca2+ ([Ca2+]i) levels, the effect of buffering [Ca2+]i to very low levels on the degree of potentiation was tested. Dialysing cells with the Ca2+ chelator BAPTA (20 mM) in the patch pipette solution significantly enhanced the degree of potentiation relative to control (Fig. 4). This increase in the extent of potentiation was significant for durations of 15 ms or less (for 200 ms intervals), and for intervals of 1 s or less (for 15 ms pulse durations) (Fig. 4B). The kinetics of uncaging responses in the presence of BAPTA were similar to those without BAPTA in that rise time and half-time of decay values slightly increased with the duration of the uncaging pulses, and that the rise times and decay of the second pulse were slower than the first (Table 1). Interestingly, for pulse durations of 15 ms or less, BAPTA dialysis significantly decreased the average amplitude of the first response compared to control, without significantly affecting the average amplitude of the second response (Table 1).
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A, representative traces illustrating paired-pulse potentiation evoked by a 5 ms pulse duration at a 200 ms pulse interval in a control cell (left) and in another cell dialysed with 20 mM BAPTA in the patch pipette (right). Scale bars are 50 pA and 200 ms. B, plot of Resp2/Resp1 ratio versus pulse duration using a 200 ms interval (left) or versus pulse interval using a 15 ms duration (right) for comparison of control () and BAPTA-dialysed cells (). Data are mean ± S.E.M. from 10 interneurones for BAPTA. Significant differences are indicated by an asterisk.
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Dependence of potentiation on CaN, PKC, and CaMKII
The modulatory effects of Ca2+ on the function of the 7-containing nAChRs may be direct, or indirect through Ca2+-dependent signal transduction cascades. For example, 7-containing nAChRs in chick ciliary ganglion neurones are known to be regulated by the Ca2+-dependent phosphatase calcineurin (CaN) and the Ca2+-dependent kinase CaMKII (Liu & Berg, 1999). To test whether the potentiation of the 7-containing nAChRs could be regulated by CaN, cells were dialysed with the CaN autoinhibitory peptide (CaN-AIP; 0.3 mM). Similar to the effects with BAPTA, dialysis with the CaN-AIP significantly enhanced the degree of potentiation relative to control (Fig. 5). The increase in potentiation was significant for durations of 15 ms or less for the 200 ms interval. Similar effects were observed upon dialysis with an inhibitory peptide of protein kinase C (PKC-I19–31; 0.2 mM), which also significantly enhanced the degree of potentiation relative to control for pulse durations of 15 ms or less (Fig. 5). The average current amplitude of the first response elicited for 5 ms duration pulses in the presence of either CaN-AIP or PKC-I19-31 was not significantly different from controls. Interestingly when we dialysed cells with the CaMKII inhibitor KN-93 (5 μM), there was no longer any significant potentiation for any pulse duration (Fig. 5). These data support the hypothesis that an intracellular Ca2+-dependent mechanism, perhaps through CaN, CaMKII, and/or PKC, regulates the degree of potentiation for 7-containing nAChRs in rat hippocampal interneurones in the slice.
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Plot of Resp2/Resp1 ratio versus pulse duration for control, or in the presence of either the calcineurin autoinhibitory peptide (CaN-AIP; 300 μM), or the PKC inhibitory peptide fragment 19-31 (PKC-I19-31; 200 μM), or a CaMKII inhibitor (KN-93; 5 μM). Data are means ± S.E.M. from 10 interneurones for CaN-AIP and PKC-I19-31, and 8 interneurones for KN-93.
Discussion
We have described a novel, transient potentiation of 7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices. Native nAChRs were activated with the local photolysis of caged carbachol (cCarb) using a paired-pulse protocol, where the duration of the UV laser pulses (5–25 ms) and the interval between pulses (200 ms to 30 s) were varied. The rapid kinetics of photolysis dramatically reduces the effects of desensitization on peak current amplitude, which is important since 7-containing nAChRs are known to desensitize rapidly (McGehee & Role, 1995; Khiroug et al. 2002; Quick & Lester, 2002). While maintaining a constant pulse duration (15 ms), interpulse intervals 1 s resulted in a strong (> 50%) decrease in the amplitude of the second response, which can be ascribed to desensitization. However, when the interpulse interval was 200 ms (at a duration of 5 ms), a > 3-fold increase in the amplitude of the second response was observed. Similar responses were also elicited with pressure-applied ACh, demonstrating that the natural agonist can induce this potentiation phenomenon, and also that it can be observed by activating the receptors via multiple methods. In addition, if a series of uncaging pulses (i.e. 4 at 5 ms durations and 200 ms intervals) was applied, the fourth uncaging response was still potentiated relative to the first.
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The extent of potentiation was also dependent on the duration of the uncaging pulses, where shorter duration pulses resulted in a greater degree of potentiation. Because the amplitude of the first response was much smaller for pulse durations less than 15 ms, it appears as if potentiation is contingent upon the submaximal activation of the 7 receptors. In addition, if the receptors are maximally activated, for example with durations of 15 ms or longer, little to no potentiation is observed; this is likely to be due to the possibility that potentiation under these conditions (i.e. maximal activation of the first response) is masked by receptor desensitization. Furthermore, the amplitude of the potentiated current is not larger than the first response to a maximal stimulus, suggesting that during potentiation, there is no recruitment of new channels. Therefore, it appears as if there is a fine balance between activation, potentiation, and desensitization of 7-containing nAChRs in rat hippocampal interneurones.
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It is well established that Ca2+ can activate and regulate a variety of signal transduction cascades, and plays a key role in the short- and long-term regulation of nAChRs (Quick & Lester, 2002). In addition, Ca2+ could be exerting an effect either directly or indirectly. Previously we showed that 7-containing nAChR recovery from desensitization was strongly dependent upon [Ca2+]i (Khiroug et al. 2003). In the present study, we found that both Ca2+ and Ca2+-dependent signal transduction cascades also affected potentiation. For example, the extent of potentiation was increased several-fold by dialysing cells with high concentrations of the Ca2+ chelator BAPTA (20 mM). Interestingly it appears as if the reduction in [Ca2+]i decreases the amplitude of the first response rather than significantly altering the amplitude of the second response. However as the duration of the uncaging pulses was decreased, the amplitudes of responses in cells dialysed with BAPTA were progressively lower than control. Although this is not a true dose–response relationship, this does suggest that the efficacy of the agonist to open the channels (i.e. the gating) might be affected by [Ca2+]i. In addition, it is possible that the reduced function of the channel on the first response due to BAPTA might be counteracted by the influx of Ca2+, so that the second response is far larger (and resulting in enhanced potentiation).
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Our data suggest that Ca2+ may be having an indirect effect on nAChR channel function since inhibiting either the Ca2+-dependent phosphatase calcineurin (CaN) or protein kinase C (PKC) significantly enhanced the extent of potentiation, while inhibiting CaMKII attenuated this potentiation. In chick ciliary ganglion neurones, the function of 7 nAChRs runs down with time in a Ca2+-dependent manner, and this rundown is enhanced by inhibition of CaN and prevented by block of CaMKII (Liu & Berg, 1999). Non-7 nAChRs have also been shown to be regulated by Ca2+-dependent processes. For example for 42 receptors expressed in Xenopus oocytes, the increase of [Ca2+]i and subsequent activation of PKC promotes the recovery from desensitization (Fenster et al. 1997, 1999; Quick & Lester, 2002). In rat chromaffin cells, however, the recovery of non-7 nAChRs from desensitization was slower when [Ca2+]i increases were prolonged (Khiroug et al. 1997, 1998). At present, it is unclear how inhibiting a Ca2+-dependent phosphatase (CaN) or kinase (PKC) exerts similar effects with respect to the nAChR potentiation described here, but it is likely that it occurs by altering the Ca2+-dependent phosphorylation state of the receptor.
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We considered several possibilities to explain the molecular mechanism responsible for potentiation. The first is that residual free agonist from the first uncaging pulse remains at the time of the second pulse, thus increasing the actual agonist concentration during the second pulse, and thus activating more channels. This does not seem likely for several reasons. First, we did not see any potentiation with responses due to the uncaging of either GABA or glutamate, as might be expected if the buildup of residual agonist was responsible for potentiation. Second, the bath-application of a low dose of carbachol had no effect on potentiation; if the residual buildup of carbachol was involved in the potentiation, this bath-application of carbachol may have had an effect. Lastly, the estimated concentration required to produce a 3-fold increase in current amplitude did not appear to be attainable. The maximal effective dose of free carbachol possible using 0.5 mM cCarb (with a quantal yield of 0.8) (Milburn et al. 1989) would be 0.4 mM, a concentration which would be expected to activate responses that were 60% of maximum (based on carbachol dose–response curves for rat 7 receptors expressed in Xenopus oocytes) (Khiroug et al. 2002). Since the amplitude of the 5 ms uncaging pulses was 25% of the longest duration (i.e. 25 ms) pulse (which we will assume here achieved a carbachol concentration of 0.4 mM), we estimate that the amplitude of these brief (i.e. 5 ms) pulses activates currents to 15% of maximum (i.e. from the dose–response curve) (Khiroug et al. 2002). The approximate carbachol concentration corresponding to 15% of maximum is 120 μM. We estimate that in order to achieve a 3-fold potentiation for the second 5 ms pulse, the carbachol concentration would have to reach 300 μM. Based on these calculations, this is not possible, particularly in light of the fact that the free agonist concentration after the first pulse (which we estimated may have reached 120 μM) should have decayed to well below 20 μM (the foot of the dose–response curve) by the start of the second pulse (as the baseline current generally had returned to baseline levels). Therefore it does not seem likely that residual free agonist can explain the potentiation of the 7 nAChRs.
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Another consideration was whether some of the receptors had bound agonist molecules (e.g. singly liganded channels) after exposure of the first response, even if the free agonist concentration has decayed to below 20 μM. In this case, the channels might be ‘primed’ to respond more robustly to the second pulse of agonist due to increased efficacy, resulting in an apparent potentiation of the response amplitude. However this did not appear to be the case since the bath application of a low dose of carbachol (10 μM), a dose that did not induce desensitization during our experiments but which might be able to prime some channels, had no effect on either the amplitude or potentiation of 7 receptors. In addition, neither residual agonist nor priming are consistent with the fact that Ca2+ and Ca2+-dependent intracellular signal transduction cascades significantly affected the extent of potentiation. In addition, such a priming mechanism does not explain the significantly slower decay of the second (i.e. potentiated) response.
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Although clear evidence indicates that nAChRs can directly regulate synaptic plasticity in the hippocampus (Hunter et al. 1994; Fujii & Sumikawa, 2001; Ji et al. 2001; McGehee, 2002), as well as cognitive processes in humans and animal models (Levin, 2002), the cellular mechanisms involved are not fully understood. In the brain and in particular in the hippocampus, 7-containing nAChRs are located both at postsynaptic sites on interneurones where they mediate fast cholinergic excitatory synaptic transmission (Alkondon et al. 1998; Frazier et al. 1998), and on presynaptic terminals where their activation facilitates glutamatergic release (Gray et al. 1996). However, these nAChRs might also be located and function extrasynaptically in the hippocampus, and work by non-synaptic, diffuse, so called ‘volume transmission’ (Umbriaco et al. 1995).
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The medial septum/diagonal band (MS/DB) complex, via the septohippocampal pathway, is thought to be critical for the generation and/or maintenance of the hippocampal theta rhythm in vivo (Buzsaki, 2002). In the rat hippocampus, interneurones receive cholinergic input from the medial septum–diagonal band complex (MSDB) of the basal forebrain (Frotscher & Léránth, 1985; Woolf, 1991), as well as from intrinsic cholinergic interneurones (Matthews et al. 1987; Cobb et al. 1999). Septal cholinergic neurones and GABAergic interneurones fire in rhythmic bursts to entrain hippocampal interneurones (Jones et al. 1999; Buzsaki, 2002). In addition, intrinsic cholinergic interneurones can pattern network activity, via nAChRs, in the hippocampus, and nAChR activation increases theta activity in freely moving rabbits (Jones et al. 1999). The time dependence of the potentiation that we have observed in the present study is physiologically consistent with the timing of the various forms of rhythmic activity (e.g. theta rhythms; Buzsaki, 2002) and synchrony in the hippocampus directly linked with plasticity and cognitive processes. Thus this newly described phenomenon of paired-pulse potentiation could be participating in regulating rhythmic patterns of electrical activity in the hippocampus.
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