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The Journal of Neuroscience, November 15, 2001, 21(22):8707-8714

Cyclic Nucleotide-Gated Channels Contribute to the Cholinergic Plateau Potential in Hippocampal CA1 Pyramidal Neurons

J. Brent Kuzmiski and Brian A. MacVicar

Neuroscience Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plateau potentials are prolonged membrane depolarizations that are observed in hippocampal pyramidal neurons when spiking and Ca²⁺ entry occur in combination with muscarinic receptor activation. In this study, we used whole-cell voltage clamping to study the current underlying the plateau potential and to determine the cellular signaling pathways contributing to this current. When combined with muscarinic stimulation, depolarizing command potentials that evoked Ca²⁺ influx elicited a prolonged tail current (I_tail) that had an extrapolated reversal potential of 20 mV. I_tail was not observed when intracellular Ca²⁺ levels were chelated with 10 mM intracellular BAPTA, and I_tail was reversibly depressed in low external sodium. When I_tail was evoked at intervals >3 min, current amplitudes were stable for up to 1 hr. However, at shorter intervals, I_tail was refractory, with a time constant of recovery of 43.5 sec. The inhibitors of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and 6-anilino-5,8-quinolinequinone depressed I_tail and zaprinast, which blocks cGMP-specific phosphodiesterase, enhanced I_tail, suggesting that a component of I_tail was activated by cGMP. The inhibitors of cyclic nucleotide-gated (CNG) channels L-cis-diltiazem and 2',4'-dichlorobenzamil reversibly depressed I_tail. However, protein kinase G inhibition had no effect. Therefore, these results indicate that a component of I_tail is attributable to activation of CNG channels. We conclude that Ca²⁺ influx when combined with muscarinic receptor activation activates soluble guanylate cyclase and increases cGMP levels. The increased cGMP activates CNG channels and leads to prolonged depolarization. The cation conductance of the CNG channel contributes to the prolonged depolarization of the plateau potential.

Key words: seizure; acetylcholine; muscarinic receptors; cGMP; guanylate cyclase; hippocampus; epilepsy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We first reported that Ca²⁺ influx in combination with muscarinic (m1/m3) or metabotropic glutamate receptor (mGluR) stimulation generates prolonged depolarizations called plateau potentials (PP) in hippocampal pyramidal neurons (Fraser and MacVicar, 1996). We hypothesized that the PP was generated by a Ca²⁺-activated cation conductance (Crepel et al., 1994; Congar et al., 1997) because chelating intracellular Ca²⁺ prevented the Na⁺-mediated depolarization. Other reports have shown that plateau potentials occur in pyramidal neurons of other cortical regions (Klink and Alonso, 1997; Kawasaki et al., 1999). The PP is an attractive candidate for a major intrinsic conductance generating the prolonged depolarization observed during ictal phase of seizures (Dichter and Ayala, 1987; Fraser and MacVicar, 1996). Indirectly supporting this postulate is the observation that the PP is depressed by anticonvulsants, such as topiramate (Palmieri et al., 2000). However, the identity of the cation conductance generating the PP is still unknown.

We have investigated the contribution of cyclic nucleotide-gated (CNG) channels to the depolarizing current during the plateau potential. CNG channels are nonselective cation channels that in some configurations are also permeated by Ca²⁺ (Zagotta and Siegelbaum, 1996; Zufall et al., 1997). Several species of CNG channels have been cloned (Finn et al., 1996; Zagotta and Siegelbaum, 1996), and the olfactory CNG is expressed in the hippocampus (el-Husseini et al., 1995; Kingston et al., 1996; Bradley et al., 1997; Wei et al., 1998). The CNG channel is most highly expressed in the soma and proximal dendrites of pyramidal cells (Bradley et al., 1997). Cultured hippocampal neurons exhibit a cation current that is activated by a cGMP analog (Kingston et al., 1996; Bradley et al., 1997). Some (Kingston et al., 1996), but not all laboratories (Bradley et al., 1997) have reported the expression of the rod CNG channel in the hippocampus. The CNG channel is a potential candidate for the depolarizing current underlying the PP because cGMP metabolism is increased by muscarinic receptor or mGluR activation (Trivedi and Kramer, 1998; Wotta et al., 1998). Also, Ca²⁺ influx could potentially activate guanylate cyclase (GC) by stimulating formation of nitric oxide (NO) (Kingston et al., 1999).

Our strategy to delineate the roles for CNG channels in the PP required the use of whole-cell voltage clamping to quantify the tail current (I_tail) underlying the PP. We first ensured that we could voltage clamp the tail current, and then we examined the sensitivity of the current to pharmacological antagonists to the CNG channel. We report that blocking soluble GC (sGC) with 6-anilino-5,8-quinolinequinone (LY83583) (Leinders-Zufall and Zufall, 1995) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (Garthwaite et al., 1995) depressed the generation of I_tail. Antagonists to the CNG channel itself (2',4'-dichlorobenzamil or L-cis-diltiazem) (Zagotta and Siegelbaum, 1996; Wei et al., 1998) also reversibly inhibited I_tail; however, blocking the protein kinase activated by cGMP [protein kinase G (PKG)] and antagonists to NO synthase (NOS) had no effect on I_tail. Therefore, we conclude that Ca²⁺ influx in combination with muscarinic stimulation leads to cGMP formation that depolarizes pyramidal neurons by opening CNG channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hippocampal slice preparation. Hippocampal slices were prepared from Sprague Dawley rats, aged postnatal days 15-23. A block of tissue containing the hippocampus and surrounding structures was attached to a mounting tray with cyanoacrylate glue and immersed in chilled (0-4°C) modified oxygenated (95% O₂ and 5% CO₂) artificial CSF (aCSF) containing (in mM): 120 NaCl, 3.0 KCl, 1.3 MgSO₄, 2.0 CaCl₂, 1.5 KH₂PO₄, 26 NaHCO₃, and 10 D-glucose, pH 7.35. Horizontal slices (400 µm) were cut through the tissue block using a vibratome (VT100; Leica, Willowdale, Ontario, Canada). The slices were then transferred to a storage chamber with oxygenated aCSF and allowed to recover for at least 1 hr at room temperature.

Electrophysiology. Whole-cell voltage-clamp recordings (Hamill et al., 1981) from CA1 neurons within hippocampal slices were obtained using the "blind-patch" technique (Blanton et al., 1989). Slices were individually transferred to a recording chamber located on an upright microscope (Axioskop; Zeiss, Oberkochen, Germany) and submerged in rapidly flowing (1 ml/min) oxygenated aCSF. Bath temperature was maintained at 32-34°C with a Peltier unit and Cambion bipolar controller. The recording electrodes were pulled from 1.5 mm (outer diameter) borosilicate thin-walled glass capillaries (150F-4; World Precision Instruments, Sarasota, FL) in three stages on a Flaming-Brown micropipette puller (model P-87; Sutter Instruments, Novato, CA). Patch electrodes were filled with a solution containing (in mM): 115 Cs-methanesulphonate, 20 KCl, 10 Na-phosphocreatine, 10 HEPES, and 1.1 EGTA, pH 7.25. In some experiments, 10 mM BAPTA was substituted for the EGTA as described. When filled with intracellular solution, patch electrode resistance ranged from 4 to 8 M. All experiments were conducted in extracellular solution containing tetrodotoxin (TTX) (1.2 µM).

Membrane potentials and/or currents were monitored with either an Axoclamp 2A or Axopatch 200B amplifier (Axon Instruments, Foster City, CA), acquired through a Digidata 1200 series analog-to-digital interface onto a Pentium personal computer using Clampex 7.0 software (Axon Instruments). Data were sampled at a rate of 2-5 kHz and were low-pass filtered (four-pole Bessel) at 1-5 kHz. Series resistance was continuously monitored with hyperpolarizing voltage steps. Recordings with series resistance >20 M were rejected from analysis.

All chemicals were purchased from Sigma (St. Louis, MO), Molecular Probes (Eugene, OR), or Calbiochem (La Jolla, CA). Carbachol (Sigma), tetrodotoxin (Sigma), L-cis-diltiazem (Sigma), N^G-nitro-L-arginine (L-NNA) (Sigma), and N^G-nitro-L-arginine methyl ester, HCl (L-NAME) (Calbiochem) were dissolved in distilled H₂O and added to the aCSF from concentrated stocks. ODQ (Sigma), LY83583 (Sigma), 2',4'-dichlorobenzamil, HCl (Molecular Probes), and zaprinast (Calbiochem) were first made up as a stock in DMSO before being added to the aCSF. The final concentration of DMSO was always 0.1%; in control experiments, DMSO at these concentrations did not alter I_tail. BAPTA, tetracesium salt (Molecular Probes), KT5823 (Calbiochem), L-NNA, and L-NAME were dissolved directly into the patch-pipette solution.

Data analysis. Data were analyzed using Clampfit 8.0 (Axon Instruments). I_tail was quantified in each cell by calculating the area under the tail current. Area was calculated by summing the amplitudes of all individual data samples over time, relative to the baseline of prestimulus holding current. Samples were summed from the end of the depolarizing voltage command pulse over a 13 sec time period or to the point at which the current amplitude returned to prestimulus levels. Statistical comparisons were determined using either Student's t test or one-way ANOVA with Tukey's post hoc test (SPSS version 10.0; SPSS, Chicago, IL). In all cases, p < 0.05 was considered significant. Values are reported as mean ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results in this paper were obtained from 134 CA1 pyramidal neurons in the hippocampal slice preparation, using whole-cell patch-clamp techniques. Whole-cell recordings were made with patch pipettes containing a Cs⁺-based internal solution to reduce K⁺ currents and to improve space clamp (Colino and Halliwell, 1993). All experiments were performed in the presence of bath-applied TTX (1.2 µM) to block voltage-gated Na⁺ channels and to prevent Na⁺-dependent action potentials.

Cholinergic-dependent plateau potential and slow inward tail current

In the absence of carbachol, a nonhydrolyzable cholinergic agonist, positive current injection (800 msec, 0.1 nA) resulted in activation of Ca²⁺-dependent action potentials (n = 15) (Fig. 1A). After cessation of current injection, the membrane potential rapidly returned to resting levels. As shown previously with current-clamp recordings (Fraser and MacVicar, 1996), after a 5 min bath application of 20 µM carbachol, current injection evoked Ca²⁺ spikes that now elicited prolonged PPs (Fig. 1B). The average duration of the PP was 10.3 ± 1.2 sec (range of 3.7-18.4; n = 15). The PPs produced by carbachol were reversible (Fig. 1C).

View larger version (23K): [in this window] [in a new window]   Figure 1.   In the presence of carbachol, an Itail was observed under voltage clamp in the same cells that revealed a PP in current-clamp mode. A, Typical responses of a hippocampal CA1 pyramidal neuron under current-clamp conditions to hyperpolarizing and depolarizing current injection in control aCSF. Depolarizing current injection elicited robust Ca2+ spikes, and the membrane potential immediately returned to baseline levels after cessation of the current pulse. B, In the presence of 20 µM carbachol (CCH), Ca2+ spike firing evoked by identical stimuli resulted in a long-lasting PP. C, The PP was reversible after carbachol was washed from the slice. D, In the same cell, under voltage-clamp conditions in the absence of carbachol, an 800 msec depolarizing voltage pulse to 0 mV from a holding potential of 70 mV resulted in unclamped Ca2+ currents. At the offset of the pulse, Itail was not observed. E, In the presence of carbachol, a long-lasting inward Itail was induced at the offset of the depolarizing voltage pulse under voltage-clamp conditions. F, Itail was reversible after wash of carbachol. Recordings were made with a Cs+-based intracellular solution and 1.2 µM TTX in the external solution.

Long-lasting I_tails were observed under voltage clamp in the same pyramidal neurons, which had displayed a PP in carbachol under current-clamp conditions (n = 15/15) (Fig. 1). We examined I_tails at a holding potential of 70 mV after a voltage step to 0 mV for 800 msec before and after carbachol application. The inward I_tail was observed in a total of 77 pyramidal cells only after 20 µM carbachol was applied. Tail currents were quantified by measuring the area of the inward current over a 13 sec time period after the end of the voltage command pulse as described in Materials and Methods. Significant increases in I_tail areas were induced by carbachol (control area, 0.04 ± 0.02 nA · sec vs carbachol, 1.2 ± 0.2 nA · sec; p < 0.001; n = 15) (Fig. 1D,E); when carbachol was washed, I_tail diminished to control levels (0.1 ± 0.02 nA · sec; p < 0.001; n = 15) (Fig. 1F).

Reversal potential and sodium dependence of I_tail

Previously, we reported that activation of PPs depends on increased intracellular [Ca²⁺] ([Ca²⁺]_i) and that the depolarization is attributable to TTX-insensitive Na⁺ influx through a nonselective cation channel (Fraser and MacVicar, 1996). We investigated the role for TTX-insensitive Na⁺ influx in I_tail by replacing extracellular [Na⁺] ([Na⁺]_o) with equimolar N-methyl-D-glucamine. Reducing [Na⁺]_o from 152 to 26 mM significantly reduced the area of I_tail (normal aCSF, 1.8 ± 0.3 nA · sec vs low sodium, 0.3 ± 0.1 nA · sec; p < 0.002; n = 6) (Fig. 2A,B); I_tail recovered after replacement of the NaCl (1.5 ± 0.3 nA · sec compared with control; p = 0.63; n = 6) (Fig. 2C). The results of these experiments are summarized in the plot of I_tail areas in Figure 2D.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Itail was dependent on Na+ influx independent of TTX-sensitive channels, varied linearly with voltage, and was blocked by intracellular BAPTA. A, In 20 µM carbachol, Itail was observed after a depolarizing voltage step (800 msec) to 0 mV from a holding potential of 70 mV. B, Reducing [Na+]o from 152 to 26 mM depressed Itail. External NaCl was substituted with equimolar N-methyl-D-glucamine. C, Restoring external NaCl to 152 mM completely reversed the depression of Itail induced by low [Na+]o. D, Summary of the effects of reducing [Na+]o and after washout on the area of Itail. Mean ± SE areas of Itail are plotted (n = 6). *p < 0.002 compared with control data. E, The reversal potential of Itail was assessed by ramping the voltage from 50 to 100 mV in 100 msec in the presence of carbachol. Ramps obtained during Itail were compared with ramps obtained from identical voltages without an evoked Itail. F, Subtraction of the ramp currents without Itail (ramp 1) from the ramp currents during Itail (ramp 2) from 65 to 100 mV revealed that Itail varied linearly with membrane potential in the voltage range tested. The reversal potential in this cell determined by linear extrapolation (dotted line) was 20.1 mV. G, Superimposed recordings in carbachol from different CA1 pyramidal neurons from the same slice loaded with intracellular pipette solution containing either 1.1 mM EGTA or 10 mM BAPTA (indicated by arrows). Chelating [Ca2+]i with BAPTA depressed Itail. H, Summary of the mean ± SE Itail areas from pyramidal neurons recorded with either (in the micropipette) 1.1 mM EGTA (n = 10) or 10 mM BAPTA (n = 9). **p < 0.001, EGTA compared with BAPTA.

We next examined the reversal potential for I_tail to see whether a nonselective cation channel underlies the depolarization. A current attributable to a nonselective cation channel should reverse at approximately 20 to 0 mV. The I-V relationship of I_tail was obtained by ramping the holding potential from 50 to 100 mV (in 100 msec) when I_tail was evoked after a 800 msec depolarizing prepulse to 0 mV in the presence of carbachol (20 µM) (protocol shown in Fig. 2E). Ramp currents obtained during the I_tail (Fig. 2F2) were compared with ramp currents obtained from identical holding potentials without a prepulse to evoke I_tail (Fig. 2F1). An example I-V plot is shown in Figure 2F. Subtraction of the control ramp current from the ramp current during the I_tail revealed an inward current that showed an extrapolated reversal potential in this cell of approximately 20 mV (Fig. 2F). The inward currents revealed by the ramp subtractions were fitted by a computed linear regression (r² = 0.98-1), which were then used to extrapolate the reversal potential. The average reversal potential estimated by linear extrapolation was 23.6 ± 8.5 mV (n = 7).

Ca²⁺ dependence of I_tail

In the previous report, PPs were abolished after intracellular perfusion with 10 mM BAPTA, a Ca²⁺ chelator (Fraser and MacVicar, 1996). In this study, when we included 10 mM BAPTA in the intracellular solution to prevent Ca²⁺ increases, the amplitude of I_tail (in the presence of 20 µM carbachol) was greatly depressed (Fig. 2G,H). The mean area of I_tail after 15-20 min intracellular perfusion with BAPTA was 0.3 ± 0.08 nA · sec (n = 9). As a control, we recorded I_tail in other cells in the same slices using pipettes filled with control intracellular solution that contained 1.1 mM EGTA. In the control cells, I_tail was evoked in pyramidal neurons in carbachol, and the area of I_tail was significantly increased compared with BAPTA-filled cells (1.4 ± 0.2 nA · sec; n = 10; p < 0.001). These data suggest that, similar to the PP, activation of I_tail is dependent on elevations in [Ca²⁺]_i.

I_tail is refractory

Before we could examine the actions of pharmacological agents on I_tail, we had to ensure that I_tail could be reliably evoked at a specific intervals and that it was stable over extended periods of time. We found that, if I_tail was evoked with intervals <3 min, the area of the current was significantly reduced (Fig. 3A). The normalized area of I_tail recorded at various intervals is plotted in Figure 3B, showing the gradual recovery with longer intervals. A single exponential curve was fit to the normalized current areas at intervals of 7.5, 15, 30, 45, 60, 90, and 180 sec after an initial I_tail was evoked. The time constant of recovery of I_tail to 63% was 43.5 sec (n = 9), calculated from the fit curve. I_tail was fully recovered at 180 sec. To examine rundown over an extended time, I_tail was activated every 180 sec for up to 1 hr (Fig. 3C). After 51 min, the area of the I_tail was 99.8 ± 22.9% of the first I_tail evoked (n = 6) (Fig. 3C). Plateau potentials in subiculum have also been shown to be refractory when elicited in short intervals (Kawasaki et al., 1999).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Itail was refractory when evoked at short intervals but was stable for up to 1 hr when evoked at >3 min intervals. A, Itail was evoked in the presence of 20 µM carbachol with a depolarizing voltage step to 0 mV from a holding potential of 70 mV (left). Another depolarizing voltage step to 0 mV 15 sec after the initial Itail was evoked resulted in an Itail with a reduced area (middle). A depolarizing voltage step to 0 mV 180 sec after an initial Itail was evoked resulted in a Itail with a similar area (right). B, Plot of the normalized mean ± SE Itail areas at intervals of 7.5, 15, 30, 45, 60, 90, and 180 sec with respect to the initial evoked Itail, showing that activation of Itail had a refractory period (n = 9). A single exponential curve was fit (solid line), and the time constant of recovery was calculated to be 43.5 sec (indicated by dotted lines). C, The normalized mean ± SE Itail areas evoked every 3 min were plotted against time for 51 min (n = 6).

I_tail requires activation of guanylate cyclase independent of PKG activity

To explore the possibility that carbachol and/or Ca²⁺ act via a cGMP-dependent pathway (Trivedi and Kramer, 1998; Wotta et al., 1998), we tested inhibitors of steps in cGMP signaling. First, we evaluated the effects of inhibitors of sGC. LY83583 (20 µM), an inhibitor of guanylate cyclase and cGMP channels, reduced the area of I_tail (control, 1.7 ± 0.4 nA · sec vs LY83583, 0.3 ± 0.07 nA · sec; p < 0.02; n = 5) (Fig. 4A,B). In several neurons, the later portion of I_tail was blocked in contrast to the complete lack of residual I_tail when BAPTA was included in the pipette or [Na⁺]_o was reduced (Fig. 2). ODQ (20 µM), another sGC inhibitor with no reported action on the cGMP channel (Garthwaite et al., 1995), inhibited the activation of I_tail (area in ODQ, 0.5 ± 0.1 nA · sec compared with area of control, 1.5 ± 0.2 nA · sec; p < 0.02; n = 5). The results of the experiments with the guanylate cyclase inhibitors are summarized in Figure 4C. Conversely, we examined the effects of inhibiting cGMP-specific phosphodiesterase by bath application of zaprinast (20 µM) to see whether the area of I_tail was enhanced when cGMP levels were potentially increased. We used a submaximal concentration of carbachol (5 µM) to elicit I_tail. To quantify the actions of zaprinast, we first evoked I_tail twice in control solution, and then zaprinast was bath applied for >8 min and I_tail was again evoked. All values for I_tail, including the washout values, were normalized to the first control. The normalized area of the I_tail increased from 1.1 ± 0.1 in control to 2.4 ± 0.6 in zaprinast (n = 10; p < 0.05). The enhancement of I_tail was reversible as the area decreased to 1.5 ± 0.3 (not significantly different compared with control) when zaprinast was washed out. In the example shown in Figure 4D-F, bath application of zaprinast reversibly enhanced the area of I_tail. These results indicate that I_tail was dependent on increased cGMP levels that could elicit I_tail by either activating CNG channels directly in CA1 pyramidal neurons or by activating cGMP-dependent protein kinases (PKG).

View larger version (18K): [in this window] [in a new window]   Figure 4.   Itail required sGC activity but was independent of PKG activity. A, In the presence of 20 µM carbachol (CCH), Itail could be evoked with a depolarizing voltage step to 0 mV from a holding potential of 70 mV. B, After bath application of 20 µM LY83583, Itail was depressed. C, Summary of the mean ± SE Itail areas in the presence of the sGC inhibitors LY83583 or ODQ or a PKG inhibitor compared with Itail controls. Bath application of either 20 µM LY83583 (n = 5) or 20 µM ODQ (n = 5) significantly depressed Itail area. *p < 0.02, compared with control Itail areas. Intracellular perfusion of the PKG inhibitor KT5823 (10 µM) for >30 min did not alter Itail area (n = 5, KT5823; n = 5, control). Itail areas from cells perfused with KT5823 were compared with control areas of Itail obtained from pyramidal neurons in the same slices. D, Itails of reduced areas were evoked with a depolarizing voltage step to 0 mV from a holding potential of 70 mV with perfusion of a submaximal dose of carbachol (5 µM). E, Bath application of zaprinast enhanced the evoked Itail. F, The increase in Itail area by zaprinast was reversible after wash.

Although we found previously that activation of protein kinases and phosphorylation was unnecessary for PP genesis (Fraser et al., 2001), we determined the involvement of PKG, which can be activated by cGMP. Intracellular perfusion of the selective PKG inhibitor KT5823 (10 µM) (Lei et al., 2000) in the patch pipette for 30 min failed to inhibit I_tail (Fig. 4C). In the same slices in which control intracellular solution was used, there was not a significant difference in the area of I_tail (control, 1.8 ± 0.2 nA · sec vs KT5823, 1.6 ± 0.1 nA · sec; p=0.36; n = 5). Therefore, we conclude that a guanylate cyclasecGMP pathway, independent of PKG, is required for activation of I_tail.

I_tail is mediated by cGMP-gated cation channels

To test the hypothesis that I_tail is mediated by cGMP-gated cation channels, we used bath applications of two CNG channel blockers, 2',4'-dichlorobenzamil and L-cis-diltiazem (Koch and Kaupp, 1985; Nicol et al., 1987). The first CNG channel blocker that we used was the amiloride derivative 2',4'-dichlorobenzamil (100 µM). In the example shown in Figure 5A-C, application of 2',4'-dichlorobenzamil reversibly reduced the area of the I_tail. The results of these experiments are summarized in the plot of the I_tail area in Figure 5D. Significant changes in I_tail area were induced by 2',4'-dichlorobenzamil (control, 2.1 ± 0.2 nA · sec vs 2',4'-dichlorobenzamil, 0.3 ± 0.06 nA · sec; p < 0.001; n = 7); this reduction in area of I_tail was reversible (1.6 ± 0.2 nA · sec; p = 0.2 compared with control; n = 7).

View larger version (23K): [in this window] [in a new window]   Figure 5.   Antagonists of cyclic nucleotide-gated channels depressed Itail. A, In the presence of 20 µM carbachol (CCH), Itail was evoked with depolarizing voltage steps to 0 mV from a holding potential of 70 mV. B, Bath application of 100 µM 2',4'-dichlorobenzamil (DCB) depressed generation of Itail. C, The inhibition of Itail was reversible after wash of 2',4'-dichlorobenzamil. D, Summary plot of the mean ± SE areas of Itail before, after, and wash of 2',4'-dichlorobenzamil. Application of 2',4'-dichlorobenzamil reversibly depressed Itail (n = 7; *p < 0.001) compared with Itail control. E, Summary of the effects of bath application of L-cis-diltiazem on mean ± SE Itail area. Itail was depressed by L-cis-diltiazem (n = 7; **p < 0.01 compared with control). In three neurons that were stable in the wash for >30 min, Itail partially recovered from the depression induced by L-cis-diltiazem.

We also examined the effects of L-cis-diltiazem on the carbachol-activated I_tail. L-cis-diltiazem is the inactive isomer of a Ca²⁺ channel blocker that has been reported to inhibit CNG channels. The site of action of L-cis-diltiazem is suggested to be located on the cytoplasmic side of the channel (Koch and Kaupp, 1985; Stern et al., 1986; Haynes, 1992; McLatchie and Matthews, 1992, 1994). However, at physiological pH, ~50% of the L-cis-diltiazem is unprotonated and can cross the membrane. Therefore, L-cis-diltiazem was bath applied. Figure 5E shows that L-cis-diltiazem (100 µM) inhibited activation of I_tail. The area of I_tail was reduced from 1.8 ± 0.4 nA · sec in control to 0.4 ± 0.05 nA · sec after application of L-cis-diltiazem (p < 0.01; n = 7). A subset of these cells (n = 3) were stable for >30 min in wash in carbachol containing aCSF, and I_tail recovered to 0.9 ± 0.1 nA · sec in these cells. Similar to the effects of sGC inhibition, there was still a residual I_tail observed in several neurons after inhibition of CNG channels with 2',4'-dichlorobenzamil (Fig. 5) or L-cis-diltiazem.

Effect of nitric oxide synthase inhibitors on I_tail

We tested the possibility that NO causes the increased guanylate cyclase activity required for activation of I_tail by applying two inhibitors of NOS. Slices were bathed for >1 hr in aCSF containing the NOS inhibitors L-NAME (1 mM) or L-NNA (100 µM). I_tails evoked in these slices did not differ from those evoked in control slices [control, 1.6 ± 0.5 nA · sec vs L-NAME, 1.4 ± 0.3 nA · sec (p = 1.00; n = 5) or vs L-NNA, 1.3 ± 0.2 nA · sec (p=0.98; n = 5)]. In addition, neither L-NAME (1 mM) (control, 1.6 ± 0.5 nA · sec vs L-NAME, 1.6 ± 0.4 nA · sec; p=1.00; n = 10) nor L-NNA (500 µM) (control, 1.6 ± 0.5 nA · sec vs L-NNA, 1.6 ± 0.3 nA · sec; p=1.00; n = 10) had any significant effect on I_tail when included in the intracellular recording electrode (Fig. 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results in this paper indicate that CNG channels contribute to the prolonged depolarization during the cholinergic plateau potential. We used whole-cell voltage-clamp recordings to quantify the I_tail after voltage steps to activate Ca²⁺ currents in CA1 pyramidal neurons. A significant I_tail was only observed when carbachol was perfused. The reversal of I_tail at 20 mV indicated that the current was likely attributable a nonselective cation channel. The inward current was carried principally by Na⁺ and was blocked by including high concentrations of the Ca²⁺ chelator BAPTA in the pipette. The current did not exhibit significant rundown over 1 hr when evoked at intervals of >3 min. At shorter intervals, I_tail was refractory. Inhibiting sGC with ODQ or LY83835 depressed the current, and, conversely, inhibiting phosphodiesterase, which degrades cGMP, enhanced I_tail. Two separate antagonists of the CNG channel, L-cis-diltiazem and 2',4'-dichlorobenzamil, depressed I_tail. Inhibition of the kinase activated by cGMP (PKG) using protocols shown to be effective in other studies (Lei et al., 2000) had no effect. These results indicate that Ca²⁺ influx activates sGC, leading to increased cGMP levels. The increased cGMP activates CNG channels, causing a depolarization attributable to the nonselective cation conductance. Surprisingly, we could find no evidence that the activation of sGC was attributable to increased NO because high concentrations of NOS inhibitors had no effect on I_tail.

View larger version (13K): [in this window] [in a new window]   Figure 6.   Itail was independent of nitric oxide production. A, The effects of the nitric oxide inhibitors L-NAME and L-NNA are summarized in the histogram. Itail area was not depressed by either bath application for >1 hr (1 mM) (n = 5) or intracellular perfusion (1 mM) (n = 10) of L-NAME. Inhibition of nitric oxide synthase by bath application (n = 5) or intracellular perfusion (n = 10) of L-NNA did not affect Itail area.

CNG channels are a family of related proteins that consist, in the native form, of  subunits, which form homomeric pores, and  subunits, which modify channel properties and sensitivity to antagonists when coexpressed with  subunits (Zagotta and Siegelbaum, 1996; Wei et al., 1998; Bonigk et al., 1999). Distinct CNG channels were first described in retinal rod and cone cells and in olfactory cells and are classified as CNC1 (rod CNG channel), CNC2 (cone CNG channel), and CNC3 (olfactory CNG channels). There is substantial evidence, however, that CNG channels are more widely expressed and that they play roles in synaptic function in other brain regions, such as the hippocampus (Wei et al., 1998; Kingston et al., 1999). The olfactory CNG channel (CNC3) is expressed in hippocampal pyramidal neurons, and this channel is highly permeable to Ca²⁺ (el-Husseini et al., 1995; Kingston et al., 1996; Bradley et al., 1997; Wei et al., 1998). The rod CNG channel was found in the hippocampus by some (Kingston et al., 1996) but not all investigators (Bradley et al., 1997). Although application of membrane-permeable forms of cGMP [8-bromo-cGMP (8-Br-cGMP)] induced an inward current in cultured hippocampal neurons (Leinders-Zufall et al., 1995; Bradley et al., 1997), the functions of CNG channels in the hippocampus are unknown. Increased cGMP is apparently involved in the induction of LTP because guanylate cyclase inhibition and Rp-8-Br-cGMPs, a cGMP-dependent protein kinase antagonist, blocked induction of LTP in CA1 (Zhuo et al., 1994; Arancio et al., 1995). In addition, transgenic mice lacking the olfactory CNG  subunit exhibited an attenuation of LTP (Parent et al., 1998).

Our results indicate that activation of CNG channels contribute to the depolarization during the plateau potential. Our results do not differentiate between olfactory or rod type of CNG channels. There are differences between the two CNG channels that may be used to determine channel type in future experiments. Olfactory CNG channels are blocked by LY83583 (Leinders-Zufall and Zufall, 1995). We observed depression by LY83583; however, LY83583 also inhibits sGC. We also observed inhibition of I_tail by ODQ, another inhibitor of sGC. Therefore, our results do not differentiate between these two possible actions of LY83583, and we cannot attribute the actions of LY83583 to either enzyme inhibition or channel block. This would be best resolved in future experiments by directly activating CNG currents in pyramidal neurons in hippocampal slices by cGMP itself and then testing the effects of LY83583. Inhibition of sGC or antagonists of CNG channels often did not totally block I_tail. In contrast, there was no residual I_tail in BAPTA or low [Na⁺]_o. It is possible that there are multiple components of I_tail that we have not resolved.

There is substantial Ca²⁺ permeation of some forms of CNG channels, particularly the olfactory form (Frings et al., 1995; Leinders-Zufall et al., 1997; Dzeja et al., 1999). The likeliest CNG channel underlying this current in the hippocampus is the olfactory CNG channel (Kingston et al., 1996; Bradley et al., 1997). This implies that Ca²⁺ influx will occur in pyramidal cells when I_tail and CNG channels are activated, which could have profound effects on cell signaling. We have confirmed here that the plateau potential is prevented by BAPTA, which will chelate and attenuate rises in [Ca²⁺]_i. Therefore, Ca²⁺ influx through CNG channels would be expected to increase the Ca²⁺ load in pyramidal neurons, thereby leading to further activation of the plateau potential. The degree of Ca²⁺ permeability of CNG channels is also determined by the  subunits that are coexpressed with  (Dzeja et al., 1999). If the CNG channel configuration in hippocampal slices (Bradley et al., 1997) is identical to that found in hippocampal cell culture (Kingston et al., 1996), then there should be considerable Ca²⁺ permeability.

Our results point to several key components in the pathway leading to activation of the CNG channel and the plateau potential, as illustrated in Figure 7. Key steps are the Ca²⁺ influx leading to increased [Ca²⁺]_i and the concurrent activation of muscarinic receptors. We propose that the concurrent stimulation of muscarinic receptors and increased [Ca²⁺]_i leads to activation of sGC and increased cGMP levels. The increased levels of cGMP induces opening of CNG channels, causing the depolarizing current underlying the plateau potential. Both LY83583 and ODQ are potent inhibitors of sGC, and both depressed I_tail. The two CNG channel antagonists depressed I_tail, consistent with our conclusion that a component of the inward current is attributable to the CNG cation current. The extrapolated reversal potential for I_tail was close to an estimated reversal potential for a mixed cation conductance. We also propose that zaprinast-sensitive phosphodiesterases contribute to the termination of the CNG current because I_tail was enhanced in zaprinast. Protein phosphatase activation also contributes to the generation of the plateau potential (Fraser et al., 2001) in a manner similar to the modulation of Ca²⁺-activated K⁺ currents by muscarinic receptors (Pedarzani et al., 1998). The transduction pathway between increased Ca²⁺ and activation of sGC is still to be determined. When we started these experiments, we hypothesized that Ca²⁺-dependent activation of NOS could increase NO generation, causing sGC activation (Wotta et al., 1998). Alternatively, muscarinic receptors have been linked to activation of endothelial NOS (Han et al., 1998). However, our results do not support any role for NOS in activation of sGC. Quite high concentrations of two different NOS inhibitors had no effect on I_tail. Therefore, the pathway leading to sGC activation is still to be determined. There are alternative explanations for our observations, such as modification of [Ca²⁺]_i mechanisms by cGMP. Conclusive evidence for this model will come from more direct studies in hippocampal neurons on the CNG channel itself.

View larger version (29K): [in this window] [in a new window]   Figure 7.   Proposed model for the activation of Itail and generation of plateau potentials. We propose that stimulation of muscarinic receptors (m1/m3) coupled to G-proteins in combination with Ca2+ influx through high-voltage-activated Ca2+ channels (HVA) can activate sGC, leading to an increase in intracellular cGMP and opening of CNG channels. The mechanism by which Ca2+ activates sCG does not apparently require nitric oxide. Influx of Na+ and Ca2+ through CNG channel openings mediates Itail and the prolonged depolarization during the plateau potential. Activation of protein phosphatase (PP) is required for plateau potential generation (Fraser et al., 2001), which may serve to increase the sensitivity of CNG channels to cyclic nucleotides. As part of Itail termination, cGMP-specific phosphodiesterase (PDE) can metabolize cGMP to 5'-GMP.

These findings could be relevant to the generation of seizures and epilepsy. A generalized seizure in the whole animal involves a prolonged depolarization, which is termed the tonic component of the ictal seizure (Dichter and Ayala, 1987). Often this is followed by repetitive depolarizations called the clonic phase. There is no doubt that recurrent collaterals are important in synchronizing neuronal activity in networks leading to seizures (MacVicar and Dudek, 1980; Traub et al., 1989; Jefferys, 1998), and it is likely that multiple currents contribute to seizure generation. However, the conversion of neuronal networks from bursting to prolonged tonic depolarizations could entail the enhancement of a prolonged inward current. The CNG current underlying I_tail is an excellent candidate for an intrinsic current that could be a key contributor to the ictal tonic depolarization.

	FOOTNOTES

Received May 10, 2001; revised Aug. 2, 2001; accepted May 31, 2001.

This work was supported by grants from Canadian Institutes of Health Research (CIHR). J.B.K. was supported by a studentship from the Savoy Foundation, and B.A.M. held Senior Scientist awards from CIHR and Alberta Heritage Foundation for Medical Research. We thank Dr. P. Schnetkamp for helpful comments.

Correspondence should be addressed to Dr. Brian A. MacVicar, Neuroscience Research Group, Department of Physiology and Biophysics, 3330 Hospital Drive N.W., Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada. E-mail: macvicar{at}ucalgary.ca.

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