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1School of Life Sciences, Department of Biology and Environmental Sciences, University of Sussex, Falmer, Brighton, United Kingdom; 2Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Tihany, Hungary; and 3Medical Research Council Laboratory for Molecular and Cell Biology, University College London, London, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Kupfermann 1974; Kupfermann and Weiss 1982; Marinesco et al. 2004; Teyke et al. 1990). An important identified modulatory neuron type that has a state-setting function in the feeding network are the cerebral giant cells (CGCs) of the snail Lymnaea (Yeoman et al. 1994a,b) and their homologs in other gastropods (Gillette and Davis 1977; Kupfermann and Weiss 1982; Weiss et al. 1981). Recently the CGCs have been linked to mechanisms of associative long-term memory (Kemenes et al. 2001; Kojima et al. 1997), indicating that they may be important for both nonassociative and associative plasticity of the feeding behavior. Despite the wealth of information on the function of the CGCs and their homologs in other systems, surprisingly little is known about the cellular mechanisms that underlie their role in behavioral plasticity. One testable hypothesis is that second-messenger-induced long-term plastic changes in specific ionic currents lead to persistent electrical changes in the CGC, which affect its input resistance, membrane potential, and firing properties, key determinants of its function as a modulatory neuron.
To better understand the contribution of particular current types to its electrical activity, we concentrated on the current that has the lowest threshold for activation in the CGC, the persistent sodium current (Staras et al. 2002). Similar types of persistent sodium currents have been shown to play important roles in the regulation of the membrane potential and firing properties of mammalian CNS neurons (Dibb-Hajj et al. 2002). The persistent sodium current of the CGC activates at a voltage (approximately –90 mV) where no other currents are known to be activated (Staras et al. 2002). This current can therefore have a role in determining the membrane potential and sub-threshold excitability of the CGCs and thus is a potentially important target for plastic changes underlying arousal or associative learning. Here we concentrated on the effects of cAMP on the persistent sodium current of the CGC because cAMP-dependent second-messenger cascades are known to be involved in many examples of long-term neuronal plasticity (Bailey et al. 1996; Dudai, 1987; Hu et al. 2001; Nguyen and Woo 2003; Nirenberg et al. 1983). We show that cAMP alters the electrical properties of the CGCs in the long term through modulating the persistent sodium current of its soma membrane. The electrical change that can be most directly linked to the cAMP-induced increase in the persistent sodium current is a prolonged depolarization of the soma membrane, which may have important functional implications for both the state-setting function of the CGCs and their proposed role in associative learning.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Conventional two electrode voltage- and current-clamp experiments were carried out on identified CGC neurons of the pond snail, Lymnaea stagnalis. We studied the persistent sodium current in both intact nervous systems where the CGCs can be examined in their normal neurochemical environment and in isolated neurons to determine if they were intrinsic to the cells. Animals were kept under a 12:12 light-dark cycle at 20 ± 2°C (mean ± SD) and fed lettuce three times a week. The CNS was dissected and pinned down in a silicone elastomer (Sylgard)-lined electrophysiology chamber in N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES)-buffered snail saline. The outer ganglionic sheath was removed using fine forceps, and the inner sheath was softened using a non-specific solid-protease (Sigma XIV, Sigma Chemical, Poole, UK). The paired CGCs are symmetrical neurons lying in the anterior lobes of the cerebral ganglia. For improved space clamp in intact CNS preparations, the neurons were axotomized by crushing all nerves leaving the cerebral ganglion, and recordings were made from the large (
90 µm) spherical cell bodies. The bath (volume: 1 ml) was perfused with saline solutions using a gravity feed system and a peristaltic pump. All experiments were performed at room temperature (20 ± 2°C).
Isolation of the CGCs for electrophysiological analysis in primary culture
Our cell culture procedure has been described in detail elsewhere (Straub and Benjamin 2001). Briefly, CGCs were individually isolated from the cerebral ganglia after the isolated nervous system had received an enzymatic treatments designed to soften the inner connective tissue. The enzyme treatment consisted of incubation in Protease type VIII (Sigma Chemical, Poole, UK) 1 mg/ml dissolved in the culture medium for 35 min. The cell bodies of the CGCs were exposed by mechanically disrupting the inner connective tissue and then removed, together with their main processes, by gentle suction with a fire-polished micropipette (tip diameter: 20–80 µm) prepared from 1.5-mm glass tubing (GC150T-10; Clark Electromedical Instruments, Reading, UK) that had been coated with Sigmacote (Sigma Chemical, Poole, UK). After isolation, neurons were transferred onto poly-L-lysine-coated culture dishes containing conditioned culture medium and cultured at 20°C for up to 3 days. We performed cultured cell recordings either in normal saline or in the culture medium. The sodium concentration in the culture medium was the same as in the normal saline and preliminary experiments revealed no difference in the persistent inward current amplitude between cultured cells recorded in these two different solutions.
cAMP injection
cAMP or 5'AMP was pressure injected at a concentration of 100 µM in the pipette, with 0.3% Fast Green dye (all from Sigma Chemical) in 0.05 M KCl. Fast Green was chosen as a marker dye because it has been used extensively in invertebrate neurophysiology to visualize the successful intracellular injection of material into single cells without affecting their cellular properties (e.g., Lewin and Walters 1999). The injection protocol consisted of 10 single pulses, 5-ms long each, at a pressure of 15 psi with 15-s intervals between pulses. Injection microelectrodes were pulled on a horizontal puller (P-87; Sutter Instrument, Novato, CA) from borosilicate glass (GC100F-10; Clark Electromedical Instruments, Reading, UK) with tip resistances of 28–35 M
when filled with cAMP + 0.05 M KCl or 8–10 M when filled with 4 M KAc. Depolarization controls were performed using KAc-filled microelectrodes to inject a current pattern that mimicked the direct voltage response induced by cAMP.
Saline and chemicals
The composition of normal saline was (in mM) 50 NaCl, 1.6 KCl, 3.5 CaCl2, 2.0 MgCl2, and 10 HEPES, dissolved in distilled water (pH 7.9 adjusted with 10 M NaOH). For Na+ replacement with Li+, the saline contained (in mM) 50 LiCl, 1.6 KCl, 3.5 CaCl2, 2.0 MgCl2, and 10 HEPES; pH 7.9 adjusted with 10 M LiOH. For studying sodium currents while blocking potassium and calcium currents, we used a nominally Ca-free saline, which contained (in mM) 50 tetraethylammonium chloride (TEA), 50 NaCl, 1.6 KCl, 0.02 CaCl2, 2.0 MgCl2, 10 HEPES, to which we added CdCl2 0.1–1.0, NiCl2 1.0, or 4-aminopyridine (4-AP) 4.0. The pH was adjusted to 7.9 by 10 M NaOH. For Na-replacement experiments (0 Na+) the saline contained (in mM) 50 N-methyl-D-glucamine, 1.6 KCl, 3.5 CaCl2, 2.0 MgCl2, 10 HEPES; pH 7.9 adjusted with concentrated HCl. Various pharmacological agents were used to investigate the properties of the persistent sodium current: tetrodotoxin (TTX, 10–50 µM), riluzole (50 µM) and phenytoin (100 µM). All the preceding chemicals were purchased from Sigma Chemical.
Recording methods
Conventional two-electrode voltage-clamp techniques were employed to measure voltage-activated currents. Microelectrodes were pulled from borosilicate glass (Harvard Apparatus, Edenbridge, UK) with tip resistances of 3–10 M for whole brain preparations and 7–20 M for cultured neurons (filled with 4 M KAc). For sodium current dissection experiments, we filled the current passing electrode with 2 M Cs2+ and 50 mM TEA. After impalement, a grounded metallic shield was placed between the electrodes to reduce the coupling capacitance. Axoclamp-2B microelectrode voltage-clamp amplifiers (Axon Instruments) were used in two-electrode voltage-clamp recording mode. Voltage and current signals were monitored online. For data acquisition and clamp protocols, the amplifiers were connected via a Digidata 1320 AD/DA converter (Axon Instruments) to a Pentium IV PC with pClamp 8 voltage-clamp software (Axon Instruments). Unfiltered signals were sampled at 10 or 20 kHz and stored digitally for off-line analysis. During acquisition, a standard leak-subtraction protocol was used before each voltage-clamp recording. This protocol consisted of three subsweeps, each being 1/3 of the amplitude of the test step waveform (P/3 protocol) and opposite in polarity to it. The leak was estimated in a voltage range (–110 mV) where no voltage-gated channels are active in the CGC soma membrane (Staras et al. 2002). To examine the influences of the elevation of the intracellular cAMP concentration on action potential generation, membrane potential and input resistance, we also made current-clamp recordings from CGCs impaled with two microelectrodes (filled with 4 M KAc, tip resistance: 8–15 M).
Data analysis
Analysis of electrophysiological traces was performed using Axon pClamp 8, MicroCal Origin and Microsoft Excel software. All traces used for analysis and figures were subjected to a low-pass Gaussian filter (300 or 400 Hz). Boltzmann sigmoidal curves were fitted to data for the generation of activation and inactivation functions of the persistent sodium current. Comparisons of the currents and electrical properties of the cells before and after pharmacological treatments or cAMP injection were made using t-tests or ANOVAs followed by Tukey’s HSD tests. Data are presented as means ± SE. Comparisons were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To characterize the basic properties of the CGC’s persistent sodium current in its full activation range (–90 mV to +30 mV), we used a nominally calcium-free saline with blockers of Ca and K currents added. We applied voltage steps or slow ramp protocols to the voltage-clamped CGC membrane from a holding potential of –110 mV. Examples of current traces from an experiment with the step activation protocol are presented in Fig 1A. A voltage step to –60 mV only activates a persistent inward current (Fig. 1A, top), whereas a step to –30 mV activates a transient as well as a persistent inward current (Fig. 1A, bottom). Both of these currents were identified previously as Na+ currents (INa(T), transient Na+ current; INa(P), persistent Na+ current) (Staras et al. 2002). We measured the time course of sodium current activation (time to half peak) in the –70 to +30-mV voltage range and found two distinct single-exponential functions for voltage dependence (Fig. 1B). The first function, which indicates slow activation at each voltage step, could be fitted to the data in the voltage range –70 to –30 mV and is therefore likely to characterize INa(P). The second function, which indicates fast activation at each voltage step, could be fitted to the data in the –30- to +30-mV voltage range and is therefore likely to characterize INa(T).
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) peaked at –26.4 ± 2.4 mV (n = 7) and reversed at 30.0 ± 7.1 mV. In the same experiments, the peak transient Na+ current (INa(T)) was observed at –27.9 ± 2.2 mV (n = 7), which is consistent with previous observations by Staras at al. (2002). The INa(P) amplitude measured at the maximum of the I-V relationship was –4.1 ± 0.3 nA, which constituted 8% of the peak INa(T) amplitude (–51.8 ± 4.1 nA, n = 7). A slow ramp at 40 mV/s (Fig. 1D) demonstrated INa(P) activation at around –90 mV and the peak at –28.5 ± 2.1 mV (n = 6) while INa(T) was completely inactivated. The INa(P) current-voltage relationships obtained with the ramp protocol were similar to those reconstructed from voltage steps and the INa(P) amplitudes were comparable.
The amplitude of INa(P) remained highly constant for the duration of even long voltage steps (800 ms, Fig. 1A), showing a decrease of only 5% at all voltage levels. This indicates ultra-slow inactivation of this current. However, the CGC INa(P), like similar currents in other systems (Dib-Hajj et al. 2002), may partially inactivate during the initial voltage step from the holding potential in a voltage-dependent manner. If so, a proportion of INa(P) will remain inactivated throughout the voltage step but will recover from inactivation following repolarization in a time-dependent manner. To generate the inactivation curve for INa(P), we used a command protocol stepping from a holding potential of –110 mV to conditioning step potentials from –80 to –10 mV (in 10-mV increments) followed by a short repolarization (10 ms) back to –110 mV and then by a test voltage step to –60 mV (n = 5, Fig. 2A). To test the time dependence of recovery from inactivation at the end of the voltage step, we used a protocol with fixed voltage conditioning and test pulses, –30 and –60 mV, respectively, and varied the interpulse interval from 10 to 70 ms (Fig. 2B). To plot the activation curve for INa(P), we fitted single exponentials to tail currents evoked by voltage steps back to –110 mV from between +30 and –90 mV (Fig. 2C, n = 7).
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The INa(P) inactivation curve (Fig. 2D) based on the type of experiment shown in Fig. 2A was best fitted with the Boltzmann equation I/Imax = 1/{1 +exp[(V – Vh)/k]}+ C, with Vh (half inactivation voltage) = –46 mV, k (slope factor) = 7.43, C, (constant indicating the proportion of current flowing through non-inactivated channels) = 0.11. The fact that the C value is >0 indicates that a proportion of the channels carrying INa(P) does not inactivate even at command potentials less negative than –30 mV.
Relative currents (mean I/Imax ± SEM) evoked by the second voltage step to –60 mV in the type of experiment shown in Fig. 2B are shown in Fig 2F. The current obtained after a 10-ms interval was 26% of Imax, whereas the current after a 70-ms prepulse was 95% of the steady-state current taken without a prepulse, demonstrating a fast recovery from voltage-dependent inactivation.
We also tested voltage-clamped CGCs (n = 10) for the presence of a resurgent Na+ current using conventional repolarization test protocols (Kiss 2003; Raman and Bean 1997) but found no evidence that it was present in the CGCs (data not shown).
INa(P) is TTX resistant
INa(P) activates at more negative potentials than any other current of the CGC (Staras et al. 2002), and this offers a unique opportunity to study the pharmacological properties of this current in a potential range without interference from other currents. We therefore tested the resistance of INa(P) to TTX in normal saline by measuring the steady-state currents evoked by depolarizing steps from the holding potential of –110 mV to step potentials between –85 and –55 mV. An example of the current evoked in the CGC in saline containing 50 µM TTX is shown in Fig. 3A and the averaged data for the potentials from –55 mV and below are shown in Fig. 3B. The persistent current showed a high degree of resistance to 50 µM TTX. INa(T) was also preserved in 50 µM TTX (data not shown), confirming previous observations (Staras et al. 2002). Riluzole and phenytoin, known blockers of the persistent Na+ current in some types of vertebrate neurons (e.g., cortical neurons of the rat brain, Chao and Alzheimer 1995; Urbani and Belluzzi 2000) had no effect on the INa(P) of the CGC (n = 9 and 4 experiments, respectively, data not shown).
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To address the issue of the potential contribution of a persistent Na+ conductance, such as INa(P), to the CGC membrane potential, we performed experiments in which the Na+ in the external solution was substituted by Li+. Li+ blocks the Na-Ca exchanger (Iwamoto et al. 1999; Sheu et al. 1986) but can substitute Na+ in voltage-gated Na+ channels (Sakmann et al. 2000; Smith et al. 1975). This allows the determination of the relative contribution of the Na-Ca exchanger and voltage-gated sodium conductances to the membrane potential. The substitution of Na+ by Li+ resulted in a hyperpolarization (by 6 mV) of the membrane potential (MP before, –58.6 ± 2.9 mV; MP after, –64.3 ± 3.5 mV, paired t-test, P < 0.05, n = 5, Fig. 4Ai and Bi). The CGC spikes also became significantly (paired t-test, P < 0.05, n = 5) narrower in Li+ (data not shown), which is thought to be indicative of the effect of lithium on Na-Ca exchangers in other systems, such as mammals (Janvier et al. 1997). The removal of Na+ (without replacement by Li+) resulted in a more significant hyperpolarization (by 30 mV) of the membrane (MP before, –57.8 ± 3.0, MP after, –85.8 ± 3.9, paired t-test, P < 0.01, n = 5, Fig. 4Aii and Bii). The size of the hyperpolarization in the absence of Na+ was significantly larger than in the presence of Li+ (Fig. 4B, *, unpaired t-test, P < 0.05). While the persistent current was preserved after Li+ substitution (Fig. 4Ci), it was absent in the absence of external Na+ (Fig. 4Cii). These experiments allowed us to establish that although the Na-Ca exchanger appears to make a contribution (5 mV) to the MP, a far bigger (25 mV) contribution appears to be due to a persistent Na+ conductance. That this conductance is likely to be INa(P) is supported by our calculations of the relationship between a 1-nA inward current flowing through the CGC membrane and the resulting change in membrane potential. The mean input resistance of the CGCs was 27 ± 3.2 M (n = 7), and thus the depolarizing voltage change in response to a 1-nA inward current is 30 mV. INa(P) increases by 1-nA in the –90 to –60 mV membrane potential range (see Fig. 1C) where no other persistent inward currents are activated and this also confirms that it can make a 30-mV contribution to the membrane potential.
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Previous work has shown that persistent Na+ currents can be important targets for modulation by intracellular signaling cascades, including Protein Kinase C and A (PKC and PKA) (Astman et al. 1998; Carr et al. 2002; England et al. 1996; Franceschetti et al. 2000; Gold et al. 1996; Saab et al. 2003). It was important to assess the effects of the elevation of intracellular cAMP concentration on the persistent Na+ current of the CGCs in the absence of external synaptic or modulatory inputs because the TTX-R persistent sodium current of the DRG neurons, which shows close similarity to the INa(P) of the CGC, has been shown to be ligand modulated as well as voltage sensitive (Blum et al. 2002). So far INa(P) only has been investigated in CGCs in the intact cerebral ganglia, and before the cAMP experiments, it was therefore important to examine if this current could be evoked in the completely isolated CGC soma. To measure INa(P) in isolated CGCs (Fig. 5A), we used an activation protocol stepping from a holding potential of –110 mV to levels from –90 to –25 mV in 5-mV steps and measured the peak currents in different CGCs at 5 h and 1, 2, and 3 days after the isolation (n = 7, 9, 8, 7 cells respectively). We found that although on average the peak amplitude of the persistent sodium current in isolated CGCs was <10% of that in the whole brain, it could be reliably evoked in isolated CGCs throughout the 3-day observation period and showed no change between the four time points at which it was measured (data not shown, ANOVA, P > 0.05).
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24 h. Figure 5C shows typical current responses to the –110 to –55-mV voltage step in non-injected, 5'AMP-injected, depolarized and cAMP-injected CGCs. At 1.5-h post-injection, the amplitude of the persistent Na+ current in cAMP injected CGCs was significantly larger than in 5'AMP injected control and depolarization control CGCs (Fig. 5D, n = 5 in each group, ANOVA, P < 0.05, Tukey’s HSD test, P < 0.05) or non-injected CGCs (Fig. 5D, n = 7, Tukey’s HSD test, P < 0.05). A very similar result was seen already at 1 h after injection (data not shown, ANOVA, P < 0.05, Tukey’s HSD test, P < 0.05). At 10 min after injection with cAMP, the amplitude of the persistent current (–0.15 ± 0.02 nA, n = 5) was not yet significantly different from the baseline amplitude value measured in non-injected controls (–0.2 ± 0.05 nA, n = 7), or from 5'AMP injected or depolarization controls (n = 6 and 7, respectively, data not shown, ANOVA, P = 0.35). In two 1-day-old isolated CGCs, we managed to re-run the voltage-clamp protocol 2.5 h after cAMP injection and in one CGC at 24 h after cAMP injection. In each of these cases, the amplitude of the persistent sodium current was larger (–0.5, –0.3, and –0.5 nA, respectively) than the mean baseline amplitude that was measured in non-injected isolated CGCs (–0.2 nA).
cAMP affects the electrical properties of the CGC
The cAMP injection experiments in culture demonstrated that the intrinsic persistent sodium current of the CGC can be significantly boosted by cAMP around the average membrane potential level of the CGC in the intact CNS (–60 mV) (Staras et al. 2002). This was verified in experiments with cAMP injected into CGCs (n = 3) in intact cerebral ganglia, which also showed a large and prolonged increase in the amplitude of INa(P) (data not shown). To assess the potential effects of the increase in INa(P) on the electrical properties of the CGCs in its normal neurochemical environment, we injected cAMP or 5'AMP into CGCs in whole-brain preparations, and 1 h later we compared firing patterns, input resistance, and membrane potential values with uninjected controls. These experiments showed a switch from a tonic to a bursting firing mode (Fig. 6A), a decrease in input resistance (Fig. 6B), and a depolarization (Fig. 6C) resulting from the injection of the CGC with cAMP.
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) 1 h after injection the majority of CGCs in the cAMP-injected group (6 of 8) displayed a characteristic bursting behavior (example in Fig 6Ai). None of the control 5'AMP injected (n = 8, example in Fig. 6Aii) or non-injected CGCs (n = 6, example not shown) switched from the tonic firing to the bursting mode (
2 test, P < 0.05). The input resistance during the same 1-h post-injection period decreased significantly in the cAMP-injected CGCs (n = 8) compared to non-injected (n = 6) and 5'AMP-injected cells (n = 6, ANOVA, P < 0.05; Tukey’s HSD tests, P < 0.05, Fig. 6B). At the same time, the membrane potential also became significantly more depolarized in the cAMP-injected CGCs than in the 5'AMP-injected or non-injected cells (ANOVA, P < 0.05; Tukey’s HSD tests, P < 0.05, Fig. 6C).
Similar to the INa(P) amplitudes measured in the voltage-clamp experiments, neither the input resistance nor the membrane potential values were different among the three groups measured at 10 min after injection (ANOVA, P > 0.05), indicating that the cAMP-induced changes in INa(P) and the electrical properties of the CGC emerged in parallel.
In a separate experiment, we tested if cAMP treatment also leads to an increase in the synaptic efficacy of the CGCs in the same time window where the increase in INa(P) and parallel changes in the electrical properties of this cell were found. We co-cultured CGCs (n = 5) with an identified monosynaptic follower cell, motoneuron B1 (Fig. 6Di). We compared the amplitude of post-synaptic excitatory postsynaptic potentials (EPSPs) evoked before and 1 h after the injection of cAMP into the CGC cell body (Fig. 6Dii) and found a significant increase in the CGC to B1 EPSP (paired t-test, P < 0.05, Fig. 6Diii).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Connor and Hockberger, 1984; Kononenko et al., 1983; Sudlow et al. 1993; Swandulla, 1987) in the Lymnaea CGCs cAMP injection into the soma directly stimulates an inward sodium current (INa, cAMP) (McCrohan and Gillette, 1988). The CGC INa,cAMP, which is voltage independent, lasts 5 s after cAMP injection (McCrohan and Gillette, 1988), whereas the effect of cAMP on the voltage-dependent INa(P) lasts for 24 h, indicating that cAMP is involved in both short- and long-term neuronal plasticity in the CGCs.
Several properties of the cAMP-responsive INa(P) of the CGC are similar to those of persistent Na+ currents that have been described previously both in vertebrate and invertebrate neurons (Clay, 2003; Crill, 1996; Kiss, 2003; Smith et al. 1975). At its peak voltage (–35 mV), INa(P) comprises only 5–10 % of the fast INa(T) in line with the values found in other neurons, yet it has considerable functional significance because it activates over a voltage range where no or few other voltage-gated channels are activated (Alzheimer et al. 1993; Crill 1996).
Of all the mammalian persistent sodium currents (for a review, see Crill 1996), the Lymnaea CGC INa(P) shows the greatest similarity to the TTX-resistant (TTX-R) low-threshold persistent sodium current carried by the mammalian Nav1.9 channel in small DRG neurons (Cummins et al. 1999; Dib-Hajj et al. 2002). Both the snail and mammalian persistent TTX-R sodium currents activate at approximately –80 mV, with slow and voltage-dependent activation kinetics, peak at approximately –30 mV and show a wide overlap between activation and steady-state inactivation, which might underlie the large, persistent currents that are observed near resting potential (between –70 and –40 mV) in both the DRG neurons and the CGC. A further similarity is that neither the persistent sodium current of the CGCs nor the persistent current sustained by the Nav1.9 channels of DRG neurons play a direct role in spike generation, and thus they both functionally differ from transient sodium currents. Computer simulation studies (Herzog et al. 2001) showed that the persistent TTX-R current exerts a strong influence on the resting potential of DRG neurons, shifting it from approximately –70 to approximately –50 mV. Our present work showed that the removal of INa(P) by using a Na-free saline leads to a 30-mV hyperpolarization of the CGC soma membrane in the intact CNS, of which only 6 mV can be attributed to the blocking of the Na-Ca exchanger. This indicated that like the TTX-R persistent current of the DRG neurons, activation of INa(P) can shift the resting membrane potential of the CGCs by 
20 mV, which was also confirmed by our calculations based on the resistance of the CGC soma membrane and the I-V relationship of INa(P) in the –80- to –50-mV range. Further support for a depolarizing influence of INa(P) on the CGC membrane came from our cAMP injection experiments that showed both a prolonged increase in INa(P) and a persistent soma membrane depolarization after injection with cAMP.
A clearly discernible INa(P) can be recorded in isolated neurons suggesting that INa(P) is intrinsic to the CGC soma membrane. However, INa(P) was found to be significantly smaller in amplitude in isolated CGCs compared to CGCs in the intact nervous system. This could be explained either by loss of functional TTX-R INa(P) channels due to the loss of the axon, as described for mammalian neurons (Dib-Hajj et al. 1998, 1999) or by the same number of individual channels each giving a smaller current response to the same voltage step in isolated neurons versus neurons in the intact CNS. Interestingly, the Nav 1.9 Na+ channels carrying the TTX-R persistent voltage-dependent Na+ current of the DRG neurons have been shown to be ligand activated (Blum et al. 2002). It is possible that in the intact Lymnaea CNS, the size of the voltage-activated CGC INa(P) is modulated by an external ligand, such as a neurohormone or neurotransmitter, the identity of which remains to be determined.
It was beyond the scope of this work to determine if INa(P) and INa(T) of the Lymnaea CGC are carried by the same or different populations of sodium channels. However, our work showed that even if the same channels conduct both currents, they must be in very distinct gating modes when conducting INa(P) versus INa(T). Apart from the most obvious difference in their inactivation kinetics (fast vs. ultra-slow), the two currents also have very different activation voltages and activation kinetics. Similarly to a number of other molluscan and mammalian voltage-activated sodium current types (e.g., Elliott and Elliott 1993; Gilly et al. 1997), the CGC INa(P) had slow activation kinetics. In addition, the steepness of the activation curve was smaller for INa(P) relative to that of the transient Na+ current INa(T), which was derived from previous work (Staras et al. 2002) resulting in a shift of the plateau of the INa(P) activation curve toward more positive voltages compared to INa(T). The monoexponential nature of the INa(P) tail current relaxation supports the idea that the channel carrying this current does not go through an open inactivated state (Pusch et al. 2000). Differences in the time-dependent expression of INa(T) and INa(P) in isolated CGCs (E. Nikitin, unpublished observations) also seem to support the notion that these sodium currents are carried by different channels but further work using patch-clamp methods will be required to decide whether or not this was the case. The lack of a resurgent current showed that the INa(P) of the Lymnaea CGC cannot be carried by repolarization-activated Na+ ion channels, as suggested for the INa(P) of the cerebellar Purkinje cells (Raman and Bean 2001).
The most significant finding of the present work was that a brief elevation of the level of intracellular cAMP leads to a prolonged increase of INa(P). This observation lends further support to the notion that the INa(P) of the Lymnaea CGC shows close similarities to the persistent TTX-R sodium current of the the small DRG neurons. The persistent sodium current of the small DRG neurons increases in response to activation of the cAMP/PKA pathway (England et al. 1996; Gold et al. 1996), which is thought to increase injury-induced neuronal excitability (Hu et al. 2001), an important form of neuronal plasticity. PKA was also shown to increase spontaneous activity in DRG neurons (Hu et al. 2001). The same authors suggested that the persistent TTX-R sodium current may have contributed to this increase, but they did not test this hypothesis. In the present work, we showed both that cAMP increases INa(P) and changes the electrical properties of the CGC soma membrane, some of which (e.g., membrane depolarization) may be directly linked to an increase in INa(P) (see lithium experiment). cAMP injection also induced bursting activity in the CGC. Previous work has shown that cAMP can induce bursting pacemaker activity in the usually silent MGCs of Aplysia (Drake and Treistman 1981), and it was suggested by the same authors that this effect may be mediated by the enhancement of a voltage-dependent sodium current, which still remains to be identified in the Aplysia MGCs. As well as affecting the membrane potential, the TTX-R persistent sodium current is also thought to make an important contribution to the electrogenic properties of the small DRG neurons (Cummins et al. 1999; Herzog et al. 2001). The burst-inducing effect of cAMP in the Lymnaea CGC might be mediated through similar effects of INa(P) on CGC membrane excitability.
Recent work in our laboratory has shown that single-trial food-reward conditioning in Lymnaea leads to the rapid activation of the cAMP-dependent protein kinase PKA (Kemenes, unpublished observations) and phosphorylation of the cAMP responsive element binding protein CREB (Ribeiro et al. 2003) in the cerebral ganglia, key sites for neuronal plasticity induced by behavioral classical conditioning (Straub et al. 2004). These observations indicate that in neurons of the cerebral ganglia cAMP levels can be elevated under physiological conditions by classical conditioning. A possible link between a cAMP-induced increase in INa(P) and long-term associative memory has been provided by the recent observation that as well as activating PKA and CREB, single-trial food-reward conditioning also leads to a subsequent significant increase in the peak amplitude of INa(P) (Nikitin et al. 2004).
As well as a cAMP-induced increase in INa(P), we also found a cAMP-mediated enhancement of the CGC to B1 EPSPs in cell culture (Fig. 6D). This confirmed previous observations made by Nakamura et al. (1999) that were based on PKA injection into the CGCs in the intact Lymnaea CNS and demonstrated that similarly to other model systems, cAMP treatment leads to synaptic plasticity in the CGCs. An emerging notion in the learning and memory field is that changes in both intrinsic ionic currents and synaptic efficacy are important for neuronal and behavioral plasticity (Daoudal and Debanne 2003; Marder et al. 1996; Zhang and Linden 2003). Our experiments provided direct evidence that the activation of the cAMP/PKA pathway leads to parallel changes in the electrical properties of the soma membrane and the efficacy of the synaptic connections of an identified modulatory neuron type, thought to be involved in both non-associative (feeding arousal) and associative (food-reward and aversive learning) behavioral plasticity.
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Address for reprint requests: G. Kemenes, School of Life Sciences, Dept. of Biology and Environmental Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK (E-mail: G.Kemenes{at}sussex.ac.uk)
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|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Alzheimer C, Schwindt PC, and Crill WE. Modal gating of Na+ channels as a mechanism of persistent Na+ current in pyramidal neurons from rat and cat sensorimotor cortex. J Neurosci 13: 660–673, 1993.[Abstract]
Astman N, Gutnick MJ, and Fleidervish IA. Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices. J Neurophysiol 80: 1547–1551, 1998.
Bailey CH, Bartsch D, and Kandel ER. Toward a molecular definition of long-term memory storage. Proc Natl Acad Sci USA 93: 13445–13452, 1996.
Blum R, Kafitz KW, and Konnerth A. Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature 419: 687–693, 2002.[CrossRef][Medline]
Brezina V, Orekhova IV, and Weiss KR. Neuromuscular modulation in Aplysia. II. Modulation of the neuromuscular transform in behavior. J Neurophysiol 90: 2613–2628, 2003.
Carr DB, Cooper DC, Ulrich SL, Spruston N, and Surmeier DJ. Serotonin receptor activation inhibits sodium current and dendritic excitability in prefrontal cortex via a protein kinase C-dependent mechanism. J Neurosci 22: 6846–6855, 2002.
Chao TI, Alzheimer C. Effects of phenytoin on the persistent Na+ current of mammalian CNS neurons. Neuroreport. 11: 1778–1780, 1995.
Clay JR. On the persistent sodium current in squid giant axons. J Neurophysiol 89: 640–644, 2003.
Connor JA and Hockberger P. A novel membrane sodium current induced by injection of cyclic nucleotides into gastropod neurones. J Physiol 354: 139–162, 1984.
Crill WE. Persistent sodium current in mammalian central neurons. Annu Rev Physiol 58: 349–362, 1996.[CrossRef][Web of Science][Medline]
Cummins TR, Dib-Hajj SD, Black JA, Akopian AN, Wood JN, and Waxman SG. A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J Neurosci 19: RC43, 1999.
Daoudal G and Debanne D. Long-term plasticity of intrinsic excitability: learning rules and mechanisms. Leam Mem 10: 456–465, 2003.[CrossRef]
Dib-Hajj SD, Fjell J, Cummins TR, Zheng Z, Fried K, LaMotte R, Black JA, and Waxman SG. Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic pain. Pain 83: 591–600, 1999.[CrossRef][Web of Science][Medline]
Dib-Hajj SD, Tyrrell L, Black JA, and Waxman SG. NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down- regulated after axotomy. Proc Natl Acad Sci U S A 95: 8963–8968, 1998.
Dib-Hajj S, Black JA, Cummins TR, and Waxman SG. NaN/Nav1.9: a sodium channel with unique properties. Trends Neurosci 25: 253–259, 2002.[CrossRef][Web of Science][Medline]
Drake PF and Treistman SN. Mechanisms of action of cyclic nucleotides on a bursting pacemaker and silent neuron in Aplysia. Brain Res 218: 243–254, 1981.[CrossRef][Web of Science][Medline]
Dudai Y. The cAMP cascade in the nervous system: molecular sites of action and possible relevance to neuronal plasticity. CRC Crit Rev Biochem 22: 221–281, 1987.[Web of Science][Medline]
Elliott AA and Elliott JR. Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia. J Physiol 463: 39–56, 1993.
England S, Bevan S, and Docherty RJ. PGE2 modulates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurons via the cyclic AMP- protein kinase A cascade. J Physiol 495: 429–440, 1996.
Franceschetti S, Taverna S, Sancini G, Panzica F, Lombardi R, and Avanzini G. Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurons. J Physiol 528 Pt 2: 291–304, 2000.
Gillette R, and Davis WJ. The role of the metacerebral giant neurone in the feeding behaviour of Pleurobranchaea. J Comp Physiol 116: 129–159, 1977.[CrossRef]
Gilly WF, Gillette R, and McFariane M. Fast and slow activation kinetics of voltage- gated sodium channels in molluscan neurons. J Neurophysiol 77: 2373–2384, 1997.
Gold MS, Reichling DB, Shuster MJ, and Levine JD. Hyperalgesic agents increase a tetrodotoxin-resistant Na+ current in nociceptors. Proc Natl Acad Sci USA 93: 1108–1112, 1996.
Herzog RI, Cummins TR, and Waxman SG. Persistent TTX-resistant Na+ current affects resting potential and response to depolarization in simulated spinal sensory neurons. J Neurophysiol 86: 1351–1364, 2001.
Hu SJ, Song XJ, Greenquist KW, Zhang JM, and LaMotte RH. Protein kinase A modulates spontaneous activity in chronically compressed dorsal root ganglion neurons in the rat. Pain 94: 39–46, 2001.[CrossRef][Web of Science][Medline]
Iwamoto T, Uehara A, Nakamura TY, Imanaga I, and Shigekawa M. Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+). J Biol Chem 274: 23094–23102, 1999.
Janvier NC, Harrison SM, and Boyett MR. The role of inward Na(+)–Ca(2+) exchange current in the ferret ventricular action potential. J Physiol 498: 611–25, 1997.
Kemenes I, Kemenes G, O’Shea M, and Benjamin PR. Conditioning-induced changes in the electrical activity of a molluscan modulatory neuron can account for the expression of a long-term memory trace. Soc Neurosci Abstr 27: 644.6, 2001.
Kiss T. Evidence for a persistent Na-conductance in identified command neurones of the snail, Helix pomatia. Brain Res 989: 16–25, 2003.[CrossRef][Web of Science][Medline]
Kojima S, Nanakamura H, Nagayama S, Fujito Y, and Ito E. Enhancement of an inhibitory input to the feeding central pattern generator in Lymnaea stagnalis during conditioned taste-aversion learning. Neurosci Lett 230: 179–182, 1997.[CrossRef][Web of Science][Medline]
Kononenko NI, Kostyuk PG, and Shcherbatko AD. The effect of intracellular cAMP injections on stationary membrane conductance and voltage- and time- dependent ionic currents in identified snail neurons. Brain Res 268: 321–338, 1983.[CrossRef][Web of Science][Medline]
Kupfermann I. Feeding behavior in Aplysia: a simple system for the study of motivation. Behav Biol 10: 1–26, 1974.[CrossRef][Web of Science][Medline]
Kupfermann I and Weiss KR. Activity of an identified serotonergic neuron in free moving Aplysia correlates with behavioral arousal. Brain Res 241: 334–337, 1982.[CrossRef][Web of Science][Medline]
Lewin MR and Walters ET. Cyclic GMP pathway is critical for inducing long-term sensitization of nociceptive sensory neurons. Nat Neurosci 2: 18–23, 1999.[CrossRef][Web of Science][Medline]
Marder E, Abbott LF, Turrigiano GG, Liu Z, and Golowasch J. Memory from the dynamics of intrinsic membrane currents. PNAS 93: 13481–13486, 1996.
Marinesco S, Kolkman KE, and Carew TJ. Serotenergic modulation in Aplysia. I. Distributed serotonergic network persistently activated by sensitizing stimuli. J Neurophysiol 92: 2468–2486, 2004.
McCrohan CR and Gillette R. Cyclic AMP-stimulated sodium current in identified feeding neurons of Lymnaea stagnalis. Brain Res 438: 115–123, 1988.[CrossRef][Web of Science][Medline]
Nakamura H, Kobayashi S, Kojima S, Urano A, and Ito E. PKA-dependent regulation of synaptic enhancement between a buccal motor neuron and its regulatory interneuron in Lymnaea stagnalis. Zool Sci. 16: 387–394, 1999.[CrossRef][Web of Science]
Nguyen PV and Woo NH. Regulation of hippocampal synaptic plasticity by cyclic AMP-dependent protein kinases. Prog Neurobiol 71: 401–437, 2003.[CrossRef][Web of Science][Medline]
Nikitin E, Kiss T, Kemenes I, O’Shea M, Benjamin PR, and Kemenes G. The effect of classical reward conditioning on the persistent sodium current of a modulatory neuron contributing to associative plasticity in a molluscan feeding network. FENS Abstr. 2: A043.18. 112, 2004.
Nirenberg M, Wilson S, Higashida H, Rotter A, Krueger K, Busis N, Ray R, Kenimer JG, and Adler M. Modulation of synapse formation by cyclic adenosine monophosphate. Science 222: 794–799, 1983.
Pusch M, Bertorello L, and Conti F. Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels. Biophys J 78: 211–226, 2000.[Web of Science][Medline]
Raman IM and Bean BP. Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons. J Neurosci 17: 4517–4526, 1997.
Raman IM and Bean BP. Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms. Biophys J 80: 729–737, 2001.[Web of Science][Medline]
Ribeiro MJ, Serfozo Z, Papp A, Kemenes I, O’Shea M, Yin JCP, Benjamin PR, and Kemenes G. Cyclic AMP response element-binding (CREB)-like proteins in a molluscan brain: cellular localization and learning-induced phosporylation. Eur J Neurosci 18: 1223–1234, 2003[CrossRef][Web of Science][Medline]
Saab CY, Cummins TR, and Waxman SG. GTP gamma S increases Nav1.8 current in small-diameter dorsal root ganglia neurons. Exp Brain Res 152: 415–419, 2003.[CrossRef][Web of Science][Medline]
Sakmann BF, Spindler AJ, Bryant SM, Linz KW, and Noble D. Distribution of a persistent sodium current across the ventricular wall in guinea pigs. Circ Res 87: 910–914, 2000.
Sheu SS, Sharma VK, and Uglesity A. Na+–Ca2+ exchange contributes to increase of cytosolic Ca2+ concentration during depolarization in heart muscle. Am J Physiol Cell Physiol 250: C651–656, 1986.
Smith TG, Jr., Barker JL, and Gainer H. Requirements for bursting pacemaker potential activity in molluscan neurones. Nature 253: 450–452, 1975.[CrossRef][Medline]
Staras K, Gyori J, and Kemenes G. Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding. Eur J Neurosci 15: 109–119, 2002.[CrossRef][Web of Science][Medline]
Straub VA and Benjamin PR. Extrinsic modulation and motor pattern generation in a feeding network: a cellular study. J Neurosci 21: 1767–1778, 2001.
Straub VA, Styles BJ, Irelan, JS, O’Shea M, and Benjamin PR. Central localization of plasticity involved in appetitive conditioning in Lymnaea. Learn Mem 11: 787–793, 2004.
Sudlow LC, Huang RC, Green DJ, and Gillette R. cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch. J. Neurosci 13: 5188–5193, 1993.[Abstract]
Swandulla D. Cationic membrane conductances induced by intracellularly elevated cAMP and Ca2+: measurements with ion-selective microelectrodes. Can J Physiol Pharmacol 65: 898–903, 1987.[Web of Science][Medline]
Teyke T, Weiss KR, and Kupfermann I. Appetitive feeding behavior of Aplysia: behavioral and neural analysis of directed head turning. J Neurosci 10: 3922–3934, 1990.[Abstract]
Urbani A, Belluzzi O. Riluzole inhibits the persistent sodium current in mammalian CNS neurons. Eur j Neurosci 12: 3567–3574.
Weiss KR, Koch UTJ, Mandelbaum DE, and Kupfermann I. Neural and molecular mechanisms of food induced arousal in Aplysia californica. Adv Physiol Sci 23: 305–344, 1981.
Yeoman MS, Pieneman AW, Ferguson GP, Ter Maat A, and Benjamin PR. Modulatory role for the serotonergic cerebral giant cells in the feeding system of the snail, Lymnaea. I. Fine wire recording in the intact animal and pharmacology. J Neurophysiol 72: 1357–1371, 1994a.
Yeoman MS, Kemenes G, Benjamin PR, and Elliott CJH. Modulatory role for the serotonergic cerebral giant cells in the feeding system of the snail, Lymnaea. II. Photoinactivation. J Neurophysiol 72: 1372–1382, 1994b.
Zhang W and Linden DJ. The other side of the engram: experience-driven changes in neuronal intrinsic excitability. Nat Rev Neurosci 4: 885–900, 2003.[CrossRef][Web of Science][Medline]
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