Modulation of Excitability in Aplysia Tail Sensory Neurons by Tyrosine Kinases

Angela L. Purcell1 and Thomas J. Carew2

 1Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut 06520; and  2Department of Neurobiology and Behavior, University of California, Irvine, California 92697-4550


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Purcell, Angela L. and Thomas J. Carew. Modulation of Excitability in Aplysia Tail Sensory Neurons by Tyrosine Kinases. J. Neurophysiol. 85: 2398-2411, 2001. Tyrosine kinases have recently been shown to modulate synaptic plasticity and ion channel function. We show here that tyrosine kinases can also modulate both the baseline excitability state of Aplysia tail sensory neurons (SNs) as well as the excitability induced by the neuromodulator serotonin (5HT). First, we examined the effects of increasing and decreasing tyrosine kinase activity in the SNs. We found that tyrosine kinase inhibitors decrease baseline SN excitability in addition to attenuating the increase in excitability induced by 5HT. Conversely, functionally increasing cellular tyrosine kinase activity in the SNs by either inhibiting opposing tyrosine phosphatase activity or by direct injection of an active tyrosine kinase (Src) induces increases in SN excitability in the absence of 5HT. Second, we examined the interaction between protein kinase A (PKA), which is known to mediate 5HT-induced excitability changes in the SNs, and tyrosine kinases, in the enhancement of SN excitability. We found that the tyrosine kinases function downstream of PKA activation since tyrosine kinase inhibitors reduce excitability induced by activators of PKA. Finally, we examined the role of tyrosine kinases in other forms of 5HT-induced plasticity in the SNs. We found that while tyrosine kinase inhibitors attenuate excitability produced by 5HT, they have no effect on short-term facilitation (STF) of the SN-motor neuron (MN) synapse induced by 5HT. Thus tyrosine kinases modulate different forms of SN plasticity independently. Such differential modulation would have important consequences for activity-dependent plasticity in a variety of neural circuits.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tyrosine kinases are known to play important roles in developmental processes such as cellular proliferation and differentiation. Recently tyrosine kinases have also been implicated in processes thought to underlie learning and memory. For example, several tyrosine kinases, including Src and Fyn, are involved in the induction of hippocampal long-term potentiation (LTP) (Grant et al. 1992; Lu et al. 1998; O'Dell et al. 1991; Yu et al. 1997). Furthermore, disruption of tyrosine kinase activation produces behavioral deficits in the acquisition of some memory tasks (Grant et al. 1992; Whitechurch et al. 1997). In addition, homozygous mutant mice lacking the TrkB receptor tyrosine kinase exhibit an impairment in LTP (in CA1 of hippocampus) as well as learning deficits (Minichiello et al. 1999). Finally, mitogen-activated protein (MAP) kinase, activated downstream of receptor tyrosine kinases, is also required both for hippocampal LTP (English and Sweatt 1996, 1997) and for associative and spatial forms of learning (Atkins et al. 1998; Blum et al. 1999; Schafe et al. 1999).

Indirect evidence for tyrosine kinase function in synaptic plasticity also exists in Aplysia. Long-term facilitation (LTF) of the synapse between sensory neurons (SNs) and siphon motor neurons (MNs) accompanies long-term sensitization of the defensive gill and siphon-withdrawal reflex (Frost et al. 1985). Serotonin (5HT), a biogenic amine released during tail shock (Marinesco and Carew 2000), also produces LTF of these monosynaptic connections when repeatedly applied to either the intact CNS or cocultures of SNs and MNs (Clark and Kandel 1993; Emptage and Carew 1993; Montarolo et al. 1986; Zhang et al. 1997). Application of brain-derived neurotrophic factor (BDNF), which activates the TrkB receptor tyrosine kinase in vertebrates, to the intact CNS or SN-MN cocultures produces facilitation of the SN-MN synapse 24 h later (Giustetto et al. 1999; McKay and Carew 1996). Furthermore, treatment with TrkB-IgG protein blocks LTF induced by 5HT, suggesting that an endogenous ligand of the TrkB receptor tyrosine kinase is required for the induction of LTF (Giustetto et al. 1999). In addition, activation of MAP kinase, a downstream substrate of receptor tyrosine kinases, has been shown to be required for LTF induced by 5HT at the SN-MN synapse of Aplysia (Martin et al. 1997). Finally, the Drosophila mutant linotte, which displays impairments in odor-discrimination learning, has a disruption of a receptor tyrosine kinase (Dura et al. 1993, 1995).

In addition to their function in synaptic plasticity, tyrosine kinases have been shown to modulate ion channel activity (for review, see Siegelbaum 1994), thereby regulating neuronal firing patterns and excitability. For example, a number of different potassium channels have been shown to be regulated by tyrosine phosphorylation, including Kv1.2 (Huang et al. 1993; Lev et al. 1995), Kv1.3 (Bowlby et al. 1997; Fadool et al. 1997; Holmes et al. 1996a), Kv1.5 (Holmes et al. 1996b; Nitabach et al. 2001), and Kv2.1 (Sobko et al. 1998). Moreover, both sodium current (Hilborn et al. 1998; Ratcliffe et al. 2000) and calcium current (Hu et al. 1998) can be controlled by direct tyrosine phosphorylation of their respective channels. Tyrosine phosphorylation inhibits a nonselective cation current in pressure-sensitive cells in the leech (Catarsi et al. 1995), and in Aplysia, dephosphorylation of tyrosine residues by a protein kinase A (PKA)-activated tyrosine phosphatase switches the gating mode of a nonselective cation current in bag cell neurons (Wilson and Kaczmarek 1993). Finally, tyrosine phosphorylation has also been shown to regulate channel current through ionotropic receptors, such as the N-methyl-D-aspartate (NMDA) receptor (Yu et al. 1997) and the nicotinic ACH receptor (Huganir 1991).

In light of the evidence that tyrosine kinases and components of their signaling cascades can influence synaptic plasticity and electrical properties of cells, we were interested in exploring the role of tyrosine kinases in specific forms of plasticity in the Aplysia SNs and MNs. Here we show that manipulating the level of tyrosine kinase activity in the SNs modulates their excitability state in a bi-directional manner: blocking tyrosine kinase activity decreases SN excitability, while increasing kinase activity increases excitability. We also show that the tyrosine kinase pathway in the SNs can interact with the signaling cascade that mediates 5HT-induced increases in excitability. Finally, we demonstrate that tyrosine kinases can modulate different forms of short-term plasticity independently as tyrosine kinase inhibitors have no effect on 5HT-induced STF.

Some of the data presented in this paper have been reported previously in abstract form (Purcell and Carew 1999, 2000).


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INTRODUCTION
METHODS
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Preparation

Wild-caught adult Aplysia californica (obtained from Marinus, Long Beach, CA, or Marine Specimens Unlimited, Pacific Palisades, CA) were anesthetized by injection of isotonic MgCl2 (approximately 100 ml/100 g body wt). Pleural-pedal ganglia were removed from the animal and incubated for 30-45 s in 0.4% glutaraldehyde to reduce contraction of the connective tissue during 5HT application. Ganglia were pinned out in a silicone elastomer (Sylgard)-coated recording dish containing a 1:1 mixture of isotonic MgCl2 and artificial sea water (ASW) to prevent synaptic transmission during dissection. ASW consisted of (in mM) 460 NaCl, 55 MgCl2, 11 CaCl2, 10 KCl, and 10 Tris, pH 7.6. The ganglia were desheathed to expose the somatic clusters of the tail SNs in the pleural ganglion and the tail MNs in the pedal ganglion (Walters et al. 1983). During the experiment, the preparation was perfused at room temperature (20-22°C) at a rate of approximately 4 ml/min with ASW or ASW containing specific drugs and/or 5HT. In some cases where perfusion of drugs was cost prohibitive, experiments were performed in a static bath (see Experimental procedures).

Intracellular recordings

The preparation was illuminated from below with a dark-field condenser. Tail SNs and tail MNs were identified by their location in the somatic clusters of the pleural and pedal ganglia, respectively. Glass microelectrodes filled with a 3 M KCl solution for intracellular recording had resistances between 8 and 15 MOmega . Intracellular signals were amplified by Getting (model 5A, Iowa City, IA) and Axoclamp (model 2B, Axon Instruments, Foster City, CA) intracellular amplifiers. Data were recorded and analyzed by a MacLab data-acquisition system (AD Instruments, Mountain View, CA).

In all experiments, the resting membrane potential and the input resistance of each neuron were monitored to assess the health of the preparation. In synaptic experiments, the MN was hyperpolarized to -70 mV to prevent the cell from firing action potentials. The membrane potential of the SNs was not altered during any of the experiments. Excitability was determined by measuring the number of spikes elicited in the SN during a 300-ms depolarizing current pulse. Intracellular current injection was adjusted (typically from 0.5 to 2 nA) so that the SN fired two to three spikes in the baseline condition. Monosynaptic excitatory postsynaptic potentials (EPSPs) were evoked in the MNs by a 3-ms depolarizing current pulse in the SNs.

Experimental procedures

NEURONAL EXCITABILITY. Tests were administered with an interstimulus interval (ISI) of 2 min. After two stable baseline tests in ASW, the tyrosine kinase inhibitor genistein (Sigma, St. Louis, MO), its inactive isomer, genistin (Sigma), or vehicle (0.33% DMSO/0.43% ethanol) were perfused for 15 min prior to 5HT application, during which another two tests were administered (at 13 and 15 min). The preparation was then exposed to a solution containing 50 µM 5HT, and one of the drugs mentioned above for at least 16 min, with tests taken every 2 min. In other experiments, after stable baselines were established, two other tyrosine kinase inhibitors, herbimycin A (Gibco Life Technologies, Grand Island, NY) and lavendustin A (Calbiochem, La Jolla, CA), dissolved in DMSO were added directly to a 2-ml static bath from concentrated stock solutions achieving final bath concentrations of 5 and 50 µM, respectively. Fifteen minutes after drug addition, 220 µL of 5HT was added to the bath to a final concentration of 50 µM. A similar testing protocol to that described for the genistein experiments was used. In experiments using the tyrosine phosphatase inhibitor potassium bisperoxo(1, 10-phenanthroline)oxovanadate(V) (bpV) (Calbiochem), the preparation was exposed to the drug for 30 min in a static bath prior to 5HT application, with tests taken every 5 min beginning 15 min after exposure. 5HT was subsequently added to the static bath and tests resumed every 2 min for at least 10 min. In another series of experiments, the cAMP phosphodiesterase inhibitor Ro-20-1724 and adenylyl cyclase activator 7-deacetyl-7-(O-N-methyl piperazino)-gamma -butyryl-forskolin dihydrochloride (Calbiochem) were perfused alone or with genistein for 15 min prior to testing. Perfusion was stopped and testing performed in a static bath for 16 min before 5HT application. 5HT was added to the static bath (final bath concentration of 50 µM) and testing continued for an additional 10 min.

For the intracellular injection experiments, SNs were impaled with a beveled electrode containing either control buffer (25 mM HEPES, pH 7.4, 350 mM KCl, and 0.5% Fast Green) or control buffer with 100 U/ml of active human recombinant p60Src kinase (Upstate Biotechnology, Lake Placid, NY). Baselines were established in ASW as described in the preceding text. After two stable pretests, SNs were filled with the intracellular electrode solution by pressure ejection until the soma turned visibly green. Post tests were taken at 5 min intervals for an hour at which time 50 µM 5HT was applied to the preparation. Testing resumed for an additional 10 min at an ISI of 2 min. At the end of the experiment, an additional 1 or 2 nA above the testing current was injected into the cells to test for a ceiling effect. In a subset of experiments, 5 µM herbimycin A was added to the preparation 30 min before Src injection and remained in the bath for the duration of the experiment.

SHORT-TERM SYNAPTIC FACILITATION. A nondepressed baseline EPSP amplitude was established in ASW by eliciting single spikes in the SN with an ISI of 15 min. The drug or vehicle solution was applied directly to a static bath 25 min before 5HT application during which a third baseline was obtained. The short-term test was taken immediately after a 5-min 50 µM 5HT pulse in a static bath. In a subset of experiments, SN excitability was monitored simultaneously with changes in synaptic efficacy. In these experiments, a second SN was impaled and used to monitor excitability changes.

Data analysis and statistics

Generally, two SNs per preparation were monitored for excitability changes. The responses of the two SNs were averaged to give a single value at each time point for the preparation. Due to the difference in drug application (perfusion vs. static bath), two sets of 5HT controls were performed (1 for each condition). No significant difference between the 5HT perfusion control and the 5HT static bath control was found at any of the time points [2-way ANOVA (treatment × time), F(1,4) = 0.707, NS]. Therefore the two groups were pooled for subsequent statistical analyses. EPSP amplitudes were measured as the peak voltage of the EPSP. The average of at least two pretests in ASW were taken as a baseline measure. Only EPSPs with pretests within 20% of the mean were used for further study (approximately 10% of all synapses were excluded by this criterion). All EPSP amplitudes were expressed as a percent of the baseline measure.

Spike amplitude and duration measurements were taken from SN recordings in which spikes were elicited by a 3-ms depolarizing stimulus. Spike amplitude was measured as the voltage change between the peak of the action potential and the average of the resting potential 15 ms before the current pulse. Spike duration was measured as the change in time between the peak of the action potential and the point at which the voltage was one-third of the spike amplitude. Input resistance measurements were obtained by injecting a 1-nA hyperpolarizing current pulse and recording the voltage at which the charge curve leveled out. For data analysis, both resting potential and input resistance were expressed as percentages of each cell's baseline value in normal ASW.

No significant effect of vehicle on 5HT-induced SN excitability was observed over any of the time points [2-way ANOVA (treatment × time), F(1,6) = 0.215, NS]. Therefore the vehicle control group was pooled with the 5HT control group for subsequent statistical analyses. In most cases, comparisons containing three or more between group variables were analyzed by an ANOVA followed by a Fisher's protected least significant difference (PLSD) post hoc analysis. In one case (Fig. 2B), a Dunnett's t-test was used as a post hoc analysis when multiple treatment groups were being compared with a control group. Simple comparisons between two groups were analyzed by an unpaired t-test. When comparing means within a group (for example, post-5HT treatment to pre-5HT treatment), a paired t-test was used. All probability values are two-tailed except for those reported for the three-electrode experiments. In this case, one-tailed t-tests were used because previous data predicted the expected direction of the effect. All data are displayed as the means ± SE.


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ABSTRACT
INTRODUCTION
METHODS
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Tyrosine kinase inhibitors attenuate 5HT-induced SN excitability

5HT induces several immediate changes in the tail SNs, including increases in both excitability and spike duration. These changes have been attributed mainly to activation of PKA and protein kinase C (PKC) (for review, see Byrne and Kandel 1996). In light of the recent evidence that growth factors can modulate plasticity induced by 5HT at Aplysia SN-MN synapses (Giustetto et al. 1999; McKay and Carew 1996; Zhang et al. 1997), we examined whether tyrosine kinases, the primary transducers of many growth factor signals, contributed to any of the short-term modulatory effects produced by 5HT.

In the intact pleural ganglion, excitability was measured in the tail SNs by determining the number of spikes elicited in the neuron in response to a 300-ms intracellular depolarizing pulse. The current was adjusted so that the neuron fired two to three spikes in the baseline condition. Genistein, a broad-spectrum tyrosine kinase inhibitor (Akiyama and Ogawara 1991; Akiyama et al. 1987), or its vehicle (0.33% DMSO, 0.43% ethanol) were applied to the preparation before and during the application of 50 µM 5HT. Representative results are shown in Fig. 1A. Summary data showing the time course of excitability changes are depicted in Fig. 1B. The increase in excitability induced by 5HT was stable over the time period tested (Fig. 1B). However, when 5HT was accompanied by genistein, 5HT-induced excitability was clearly attenuated. Application of 50 µM genistein reduced the number of spikes fired during 5HT to approximately half of that exhibited under control conditions (mean number of spikes ± SE at 8-min time point: genistein: 5.3 ± 1.0; vehicle: 9.1 ± 0.5). A twofold higher concentration of genistein (100 µM) further reduced the number of spikes elicited during 5HT application to a value less than baseline (mean number of spikes ± SE at 8-min time point: genistein: 1.6 ± 0.6).



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Fig. 1. The tyrosine kinase inhibitor genistein blocks serotonin (5HT)-induced sensory neuron (SN) excitability in a concentration-dependent manner. Sample traces (A) and summary data (B) are shown from experiments in which 5HT-induced SN excitability was measured in the absence or presence of the tyrosine kinase inhibitor genistein (Gen). Genistein (50 or 100 µM) or its vehicle (0.33% DMSO, 0.43% ethanol) was applied 15 min before 50 µM 5HT application (n = 5 for all groups). An additional group [artificial seawater (ASW); n = 10] received neither vehicle nor drug during the experiment. Representative traces (A) are shown for select time points during the course of the experiment (left to right): final pretest in ASW, 15 min after drug/vehicle application, and 8 min after 5HT addition. Data in B depict the mean (±SE) number of spikes over time for the different treatment groups.

In addition to its effect on 5HT-induced excitability, we observed that genistein alone produced a significant decrease in baseline excitability of the SNs [ANOVA F(3,16) = 7.53, P < 0.05]. This reduction was evident comparing the genistein group both to vehicle controls (Fig. 1B; mean number of spikes ± SE at 0-min time point: vehicle: 1.5 ± 0.2; 100 µM genistein: 0.6 ± 0.2; Fisher's PLSD, P < 0.05) and to inactive isomer controls (data not shown; mean number of spikes ± SE at 0-min time point: 100 µM inactive isomer genistin: 1.8 ± 0.2; Fisher's PLSD, P < 0.05). These results suggest that there is a baseline level of tyrosine kinase activity in the SNs in the absence of 5HT stimulation and that reducing this basal kinase activity produces a corresponding reduction in the excitability state of the SNs.

We also observed that genistein altered the waveform of the SN action potential. Application of 100 µM genistein in the absence of 5HT decreased the amplitude of the action potential by approximately 22% and increased the duration of the action potential by 93% (data not shown). These effects were not observed with the inactive isomer or ASW alone. Consistent with these observations, Jonas et al. (1996) have shown that when tyrosine kinases are activated (as opposed to being blocked, as in these experiments), the exact opposite effects on cell excitability and spike waveform have been observed. In bag cell neurons of Aplysia application of insulin, which activates a tyrosine kinase receptor, increases excitability, increases the amplitude of the action potential and decreases the duration of the spike (Jonas et al. 1996). Further experiments will be directed at elucidating the ionic basis of these changes.

To examine the specificity of genistein's actions, we tested the effect of genistein's inactive isomer, genistin, as well as two additional tyrosine kinase inhibitors, herbimycin A and lavendustin A (Onoda et al. 1989; Uehara and Fukazawa 1991; Uehara et al. 1989) on 5HT-induced SN excitability (Fig. 2A). An overall ANOVA revealed a significant difference of treatment in the number of spikes elicited in 5HT [Fig. 2B; F(5,34) = 9.20, P < 0.05]. While genistein produced a significant concentration-dependent decrease in 5HT-induced excitability (50 µM, tD = 3.77 and 100 µM, tD = 7.47, P < 0.05), its inactive isomer, genistin (100 µM), had no significant effect (Fig. 2, A and B; tD = 1.37, NS), demonstrating that the effect of genistein was not due to nonspecific actions of the drug. In addition, both 5 µM herbimycin A and 50 µM lavendustin A significantly reduced the increase in excitability produced by 5HT (Fig. 2, A and B; tD = 4.67 and tD = 3.87, respectively, P < 0.05). Similar results were obtained if the change in number of spikes was analyzed to account for the shift in baseline (data not shown). These data show that tyrosine kinase activity modulates the increase in SN excitability produced by 5HT.



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Fig. 2. The effect of genistein on 5HT-induced excitability is specific. A: sample traces show that 2 other tyrosine kinase inhibitors, herbimycin A (Herb) and lavendustin A (Lav), also attenuate 5HT-induced SN excitability while genistin [Gen (-)], the inactive isomer of genistein, has no effect. Herbimycin A (5 µM), lavendustin A (50 µM), or genistin (100 µM) were applied to the preparations as described in Fig. 1. Traces (A) represent different time points during the course of the experiment (left to right): last pretest in ASW, 15 min after drug application, and 8 min after 50 µM 5HT application. Summary data (B) show the mean (±SE) number of spikes at 8 min after 5HT application for all drugs tested. No significant difference (NS) was found between preparations treated with 5HT alone and those treated with 5HT plus vehicle. Therefore these 2 groups were combined (pooled control) for subsequent comparison to treatment groups (see METHODS). *Significant difference (P < 0.05) from pooled control (n = 5 for all groups except for 5HT group where n = 10).

Inhibiting tyrosine phosphatase activity increases SN excitability

Since reducing tyrosine kinase activity attenuates SN excitability, the question arises whether the reverse is true: does increasing tyrosine kinase activity increase SN excitability? To explore this question, we unmasked the effects of endogenous tyrosine kinase activity by bath applying bpV, a membrane permeable tyrosine phosphatase inhibitor. Reducing the actions of opposing tyrosine phosphatase activity should lead to a net increase in the amount and/or duration of phosphorylation induced by underlying tyrosine kinase activity. We found that SN excitability was significantly increased in 100 µM bpV alone [Fig. 3, A and C1; ANOVA, F(2,11) = 5.13, Fisher's PLSD, P < 0.05]. The increase in excitability was stable over time (Fig. 3B). Subsequent addition of 5 µM 5HT produced a slight increase in excitability over that observed with bpV alone. However, the presence of the phosphatase inhibitor did not enhance the excitability produced by 5HT [Fig. 3C2; ANOVA, F(2,11) = 0.31, NS]. Moreover, bpV increased SN excitability in a concentration-dependent manner (Fig. 3C1) but had no effect on 5HT-induced excitability (Fig. 3C2).



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Fig. 3. Inhibition of tyrosine phosphatase activity produces increases in SN excitability. Endogenous protein tyrosine phosphorylation was increased by inhibiting phosphatase activity with the tyrosine phosphatase inhibitor potassium bisperoxo(1, 10-phenanthroline)oxovanadate(V) (bpV). The inhibitor (100 µM) or normal ASW was applied to the preparation 30 min before addition of 5 µM 5HT. A: representative traces from this series of experiments. B: the summary data for all time points. Traces in A, from left to right, correspond to the final pretest in ASW, 15 min in ASW/drug, and 8 min in 5HT. In the absence of 5HT, bpV increased excitability in a concentration-dependent manner (C1; at 15-min time point). *, significance (P < 0.05) from ASW control (n = 5 for ASW and 100 µM bpV groups and n = 4 for 10 µM bpV group). The concentration of the phosphatase inhibitor had no effect on 5HT-induced SN excitability (C2; at 38 min time point; no significant difference between groups). All graphs depict mean (±SE) number of spikes.

Importantly, these results suggest that there is a persistent level of tyrosine kinase activity in the baseline state (i.e., in the absence of 5HT-induced excitability) that can be modulated to produce changes in the cell's excitability. The fact that the phosphatase inhibitor had no effect on the excitability produced by 5HT suggests that the signaling pathways of tyrosine kinases and 5HT may converge on a common downstream substrate, for example, an ion channel (see DISCUSSION).

None of the tyrosine kinase inhibitors produced significant changes in the SN resting membrane potential [ANOVA, F(5,22) = 1.92, NS]. However, an overall ANOVA revealed a significant difference in input resistance among the inhibitors [F(5,23) = 5.76, P < 0.05]. A significant decrease in input resistance occurred in the presence of herbimycin A, lavendustin A, and the highest concentration of genistein (tD = 3.18, tD = 4.04, and tD = 2.86, respectively, P < 0.05), while the inactive isomer, genistin, had no significant effect (tD = 1.59). Consistent with this finding, application of bpV significantly increased the input resistance of the SNs compared with ASW controls (t8 = 4.60, P < 0.05) while having no significant effect on SN resting potential (t8 = 0.32, NS). These changes in resting membrane conductance could reflect changes in baseline ion channel activity due to modulation of tyrosine kinase activity. Further experiments will investigate the nature of the changes in input resistance.

Intracellular injection of active Src kinase increases SN excitability

The cytoplasmic Src tyrosine kinase has been observed to modulate different ion channels, including Kv1.3, Kv1.5, and the NMDA receptor (Fadool et al. 1997; Holmes et al. 1996a,b; Yu et al. 1997). To test whether this kinase may also affect the excitability of Aplysia SNs, we injected an active form of the human recombinant Src tyrosine kinase into the SNs. SNs injected with the Src kinase showed a steady increase in excitability over time (Fig. 4, A and B1). Src induced a significant increase in excitability at both 30 and 60 min after injection compared with the control solution [Fig. 4B2; 30 min: ANOVA, F(2,26) = 9.04, Fisher's PLSD, P < 0.05; 60 min: ANOVA, F(2,24) = 9.30, Fisher's PLSD, P < 0.05]. Subsequent addition of 5HT produced a significant increase in excitability over that observed at 60 min in both Src-injected and control-injected cells (Fig. 4B2; paired t-tests, control: t8 = 7.87, P < 0.05; Src: t13 = 5.44, P < 0.05). However, there was no significant difference between Src-injected cells and control-injected cells in the final levels of excitability induced by 50 µM 5HT [Fig. 4B2, ANOVA F(2,23) = 5.22, Fisher's PLSD, NS]. This finding is consistent with our earlier results showing that bpV application did not enhance 5HT-induced excitability.



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Fig. 4. Injection of active Src tyrosine kinase into SNs increases excitability. SNs were injected with either control buffer (25 mM HEPES, pH 7.4, 350 mM KCL, 0.5% Fast Green; FG) or control buffer containing 100 U/ml of active human recombinant p60 Src tyrosine kinase (Src). Excitability was monitored for 60 min at which time 50 µM 5HT was applied to the preparation. In a subset of these experiments, 5 µM herbimycin A was added to the bath 30 min before injection of p60 Src kinase and remained in the bath for the duration of the experiment (Src + herb). Representative traces from select time points during these experiments are shown (A; from left to right: pre test in ASW, 30-min postinjection, 60-min postinjection, and 6 min after 5HT application). Summary data depicting the time course of Src-induced excitability is shown in B1. Src-injected (n = 14) cells showed a significant increase in excitability compared with control-injected cells (n = 9) or Src-injected cells in the presence of herbimycin A (n = 6) at both 30 and 60 min after injection [B2;**, significance (P < 0.05) from control injection and Src injection + herbimycin groups]. However, no significant difference (NS) was observed between Src-injected cells (n = 14) and control-injected cells (n = 9) in the presence of 50 µM 5HT. Herbimycin A significantly attenuated 5HT-induced excitability as was observed previously [see Fig. 2; *, significance (P < 0.05) from both Src-injected and control-injected groups, n = 3].

It is possible that 5HT did not further increase excitability induced by Src injections because Src alone brought the SNs to a "ceiling" beyond which no further increase in excitability was possible. To examine the question, an additional 1-2 nA over the testing current was injected into Src-treated cells still in the presence of 50 µM 5HT. The additional current significantly increased the number of spikes elicited during the depolarizing pulse (5HT: 15.5 ± 1.1, n = 14; 5HT + current: 18.1 ± 1.5, n = 12; t11 = 3.51, P < 0.05; data not shown). These results show that a ceiling effect cannot account for the results shown in Fig. 4B2 and support the conclusion that the signaling cascades engaged by Src interact with those engaged by 5HT.

To confirm that the increase in excitability produced by Src was due to its tyrosine kinase activity, herbimycin A, a drug that irreversibly inactivates Src kinase, was applied to the preparation before and during Src injection. Herbimycin A completely blocked the enhancement of excitability induced by Src injection (Fig. 4, A and B1). In fact, the excitability of SNs injected with Src kinase in the presence of herbimycin A was not significantly different from that of SNs injected with control solution (Fig. 4B2, 30 and 60 min: Fisher's PLSD, NS). Consistent with our earlier results, herbimycin A significantly attenuated the increase in excitability induced by application of 50 µM 5HT [Fig. 4B2, ANOVA, F(2,23) = 5.22, PLSD, P < 0.01]. This series of experiments shows that injected constitutively active Src kinase causes an increase in Aplysia SN excitability through tyrosine phosphorylation. Furthermore, modulation by Src kinase can interact with 5HT-induced excitability (see DISCUSSION).

SN excitability induced by cAMP elevation is diminished by a tyrosine kinase inhibitor

We have shown that changes in the level of tyrosine kinase activity can alter both baseline SN excitability and excitability induced by 5HT. To further examine the possible site of action of tyrosine kinases in the signaling cascade, we examined the effects of genistein on excitability induced by elevation of cAMP which presumably leads to the activation of PKA, a kinase that has been implicated in 5HT-induced changes in the SNs (Baxter and Byrne 1990; Ghirardi et al. 1992; Goldsmith and Abrams 1992; Klein et al. 1986). Intracellular levels of cAMP were increased by applying 100 µM forskolin, an activator of adenylyl cyclase, and 500 µM Ro-20-1724, a phosphodiesterase inhibitor. Confirming a previous report (Goldsmith and Abrams 1992), we found that perfusion of these cAMP elevators produced an increase in excitability that was stable over time (Fig. 5A). Moreover, addition of 50 µM 5HT to the bath, which already contained the cAMP elevators, produced a further increase in excitability. Both the cAMP-induced increase in excitability and the further increase induced by 5HT were significantly reduced by blocking tyrosine kinase activity with genistein. Specifically, we observed a significant reduction in the amount of excitability induced by the cAMP elevators when 50 µM genistein was included in the bath (Fig. 5, A and B, 23-min time point, t8 = 3.30, P < 0.05). Subsequent addition of 5HT to the bath containing the cAMP elevators and genistein produced an increase in excitability over that with drugs alone. However, this increase was significantly less than that observed in preparations that were not exposed to genistein (Fig. 5B, 37-min time point, t8 = 3.26, P < 0.05).



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Fig. 5. Genistein inhibits SN excitability induced by cAMP elevators. SN excitability was induced by combined bath application of forskolin (100 µM) and cAMP phosphodiesterase inhibitor Ro-20-1724 (500 µM). These cAMP elevators were either applied alone or with 50 µM genistein 31 min before addition of 50 µM 5HT. A: the summary data for these experiments. Genistein significantly inhibited the increase in excitability induced by the protein kinase A (PKA) activators as well as the increase produced by subsequent 5HT application [B; *, significance (P < 0.05) from respective control groups; n = 5 for both groups]. B: the data here correspond to the 23- and the 37-min time points shown in the summary graph (A).

These results show that genistein can attenuate the enhancement of excitability produced by elevation of cAMP as well as that produced by 5HT. Since elevation of cAMP levels leads to the activation of PKA and since 5HT is thought to mediate its effects through elevation of PKA activity, these results suggest that tyrosine kinase activity influences SN excitability at a site that is downstream of PKA's actions. Subsequent addition of 5HT produces a comparable further increase in excitability to that induced by cAMP elevation in both the presence or absence of genistein (Fig. 5A). This result suggests that inhibiting tyrosine kinase activity reduces baseline SN excitability, thereby attenuating the effects of 5HT modulation. Therefore the 5HT cascade may be functionally independent of tyrosine kinase activity.

Tyrosine kinase activity is not required for short-term synaptic facilitation

We have shown that manipulating the degree of tyrosine kinase activity can modulate (both increase and decrease) the level of excitability in SNs. We next examined whether tyrosine kinases play a role in other rapidly induced modulatory effects of 5HT. It is well established that a brief (5 min) pulse of 5HT produces an enhancement of synaptic efficacy between tail SNs and tail MNs that lasts for approximately 15 min (Mauelshagen et al. 1996; Walters et al. 1983). To examine the effect of blocking tyrosine kinase activity on short-term facilitation (STF), 100 µM genistein or its vehicle were applied to the preparation 25 min prior to a 5-min 5HT pulse. As shown in Fig. 6, the drug alone had no significant effect on synaptic transmission (Fig. 6, A and B; t18 = 1.15, NS). In addition, genistein had no effect on the input resistance of the MN (t16 = 0.06, NS). Subsequent 5HT exposure produced significant STF in both the vehicle control group (t18 = 5.50, P < 0.05) and the group exposed to genistein (t18 = 2.60, P < 0.05). Moreover, there was no significant difference in the amount of STF observed between the genistein and vehicle groups (t18 = 0.46, NS). In a subset of these experiments, excitability was simultaneously monitored in a second SN (Fig. 7, A1 and B1). Although genistein did not block STF produced by 5HT, consistent with our previous results, it completely blocked the 5HT-induced increase in excitability (Fig. 7, A2 and B2). Thus there was a significant difference in the number of spikes fired during 5HT between the vehicle and genistein groups (t4 = 4.67, P < 0.05). Moreover, 5HT produced a significant increase in excitability compared with vehicle alone (t4 = 5.74, P < 0.05), while no difference was observed between genistein + 5HT and genistein alone (t4 = 0.50, NS). Finally, significant STF was observed in both the vehicle and genistein groups (vehicle; t4 = 7.15 and genistein; t4 = 3.93, P < 0.05), and no difference was found in the amount of facilitation between groups (t4 = 0.08, NS). Therefore excitability and STF induced by 5HT in the SNs exhibit independent modulation: while tyrosine kinase activity clearly alters the level of SN excitability, it does not appear to be involved in short-term modulation of synaptic efficacy.



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Fig. 6. Tyrosine kinase inhibition does not affect STF. Sample traces (A) and summary data (B) from experiments in which STF was induced by a 5 min application of 50 µM 5HT to stable SN-motor neuron (MN) synapses (see METHODS). Genistein (100 µM) or its vehicle was applied 25 min before 5HT application, during which another pretest was obtained. The short-term test was taken immediately after 5HT offset. Excitatory postsynaptic potential (EPSP) amplitudes are plotted as a percentage of the average baseline EPSP. The pre in B denotes the pretest obtained in the vehicle or 100 µM genistein alone. Significant short-term facilitation (STF) was produced in preparations treated with genistein (n = 9) as well as those treated with vehicle (n = 11). No significant difference was observed in the amount of facilitation between vehicle- or genistein-treated preparations (see RESULTS). Note that although the EPSP amplitude was decreased in the presence of genistein in the example shown in A, on average the drug had no significant effect on baseline synaptic transmission (see B). - - -, baseline EPSP amplitude.



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Fig. 7. Tyrosine kinase inhibitors differentially affect 2 forms of 5HT-induced short-term plasticity. In a subset of experiments, a 2nd SN was monitored for changes in excitability in the same preparations as synaptic efficacy of SN-MN connections was being tested. Genistein (100 µM) or its vehicle was applied to preparations as described in Fig. 6. Both SN excitability and STF were tested after a 5-min application of 50 µM 5HT. Representative traces from these experiments are shown in A, 1 and 2. Traces are shown (from left to right) for the pretest in ASW, 15 min in vehicle/drug, and immediately after 5HT offset. - - -, baseline EPSP amplitude. B1: the mean (±SE) number of spikes for the 2nd SN. In vehicle-treated preparations, 5HT induced a significant increase in excitability compared with that in vehicle alone [*, significance (P < 0.05)]. In preparations treated with genistein, the number of spikes elicited in the presence of 5HT was not significantly different (NS) than that in genistein alone. There was a significant difference in the number of spikes elicited between vehicle- and genistein-treated preparations in the presence of 5HT (P < 0.05, n = 3 for both groups). B2: mean (±SE) percent baseline EPSP amplitude. Although genistein blocked 5HT-induced SN excitability, it had no effect on 5HT-induced STF in the same preparations. Significant STF was observed in both vehicle and genistein-treated preparations. No significant difference was observed in the amount of facilitation between the 2 groups (n = 3 for both groups).


    DISCUSSION
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Several studies have shown that the increase in membrane excitability induced by 5HT in Aplysia SNs is mediated by PKA (for review, see Byrne and Kandel 1996). Pharmacological manipulations which increase cAMP levels or direct injection of cAMP into SNs produce an increase in excitability similar to that produced by 5HT application (Baxter and Byrne 1990; Goldsmith and Abrams 1992; Hochner and Kandel 1992; Klein et al. 1986). Moreover, inhibitors of PKA block or attenuate 5HT-induced increases in SN excitability (Goldsmith and Abrams 1992; Hochner and Kandel 1992). On a mechanistic level, 5HT and agents that activate PKA both modulate a specific type of potassium current, the "S" (serotonin) current (IKS) (Baxter and Byrne 1989; Klein et al. 1982; Pollock et al. 1985). Single-channel recordings revealed an S-type potassium channel is modulated by 5HT, cAMP (Siegelbaum et al. 1982), and PKA (Shuster et al. 1985). In addition, both 5HT and cAMP reduce a calcium-activated potassium current (IK,Ca) in SNs (Walsh and Byrne 1989). In light of this evidence, the current model for 5HT-induced changes in SN excitability suggests that 5HT increases the level of cAMP, thereby activating PKA (Byrne and Kandel 1996), leading to the suppression of both IKS and IK,Ca and the enhancement of SN excitability (Baxter et al. 1999).

In the present study, we provide evidence for a novel mechanism by which the excitability state of the SNs can be modulated. Table 1 summarizes the results of manipulations of tyrosine kinase activity on SN excitability. We find that inhibition of tyrosine kinase activity reduces baseline excitability as well as the increase in excitability produced by both 5HT and PKA. Conversely, we find that, in the absence of 5HT, functionally increasing the effects of cellular tyrosine kinase activity by application of a tyrosine phosphatase inhibitor or by direct injection of the active Src tyrosine kinase increases SN excitability. Moreover, the tyrosine kinase signaling cascade appears to interact with downstream components of 5HT modulation as their combined effects are not simply additive. These results show that the excitability state of the SNs is modulated by tyrosine kinases and this modulation can influence the subsequent effects of 5HT in the SNs.


                              
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Table 1. Summary of effects of tyrosine kinase manipulations on SN excitability

Changes in tyrosine kinase activity modulate the excitability state of the SNs

Our data show that increasing or decreasing the level of tyrosine kinase activity produces corresponding changes in the excitability state of the SNs. An interesting implication of these results is that there is persistent tyrosine kinase activity within the SNs since application of a tyrosine phosphatase inhibitor produces an increase in excitability in the absence of 5HT. This finding suggests that tyrosine phosphatases tightly regulate the level of substrate phosphorylation on tyrosine residues. When the opposing phosphatase activity is removed by an inhibitor, the persistently active tyrosine kinases now become the dominant enzyme and are able to more effectively phosphorylate their substrates. Furthermore treatment with a tyrosine kinase inhibitor in the absence of 5HT decreases baseline SN excitability, suggesting that the baseline phosphorylation levels can be downregulated as well.

Several examples of modulation by persistent tyrosine phosphorylation have been reported previously. The voltage-dependent potassium channel Kv1.3 found in mammalian brain exhibits a basal level of tyrosine phosphorylation (Holmes et al. 1996a). Moreover, when cells expressing this channel are treated with a tyrosine phosphatase inhibitor, increases in tyrosine phosphorylation of the channel occur in a time- and dose-dependent manner, with a corresponding suppression of current through the channel. In leech pressure-sensitive neurons, a cation channel is inhibited by basal tyrosine phosphorylation as phosphatase treatment of inside-out patches containing the channel increases channel activity (Aniksztejn et al. 1997). In Aplysia bag cell neurons, the level of tyrosine phosphorylation of a cation channel or a closely associated channel protein determines the gating mode of the channel as well as its behavior to subsequent second-messenger signals (Wilson and Kaczmarek 1993). Collectively, these studies suggest several candidate loci for tyrosine regulation that can regulate basal ion channel activity and thus the firing properties and excitability state of the neurons.

Two possible models of tyrosine kinase modulation

We have shown that blocking tyrosine kinase activity can attenuate excitability induced by 5HT and increasing tyrosine kinase activity increases SN excitability, which occludes further increases induced by 5HT. These data suggest that tyrosine kinases function downstream of the 5HT receptor and either operate in series with the 5HT cascade or converge on a common substrate. We have also shown that a tyrosine kinase inhibitor can attenuate excitability induced by elevation of cAMP levels, suggesting that the tyrosine kinase functions downstream of PKA activation as well. These findings suggest two possible models that describe how tyrosine kinases may modulate SN excitability in conjunction with the 5HT cascade (Fig. 8). The first model proposes that tyrosine kinases function in series with the 5HT cascade and operate downstream of PKA (Fig. 8A). This scenario requires that PKA would eventually lead to the activation of a tyrosine kinase. Evidence for this type of signaling cascade has recently been reported in mouse adipocytes (Fredriksson et al. 2000). Norepinephrine induces the expression of vascular endothelial growth factor through a cascade involving beta -adrenoreceptors, PKA activation, and the Src tyrosine kinase. Activation of a tyrosine kinase downstream of PKA has also been observed in primary cultures of striatal neurons (Vincent et al. 1998). The tyrosine kinase inhibitor genistein attenuates forskolin-induced MAP kinase activation in these neurons. However, this type of signaling cascade is not yet well understood and it is not yet clear how PKA leads to the activation of tyrosine kinases.



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Fig. 8. Two models of tyrosine kinase function in 5HT-induced SN excitability. The results of this study predict 2 possible mechanisms of tyrosine kinase function in the modulation of SN excitability. The 1st model (A) proposes that tyrosine kinases act in series with the 5HT cascade. The tyrosine kinase would act downstream of PKA activation and possibly modulate channels directly. The 2nd model (B) suggests that tyrosine kinases are not directly activated by 5HT but rather intersect the 5HT cascade downstream or at the level of PKA. Tyrosine kinases, activated by molecules, such as cytokines and growth factors, would phosphorylate substrates, possibly including ion channels and thus change baseline ion channel activity. The cell's response to subsequent exposure to neuromodulators (i.e., 5HT) would be modified due to the altered ion channel activity. TYRK, tyrosine kinases.

In addition, we found that 5HT application was able to further increase excitability over that induced by cAMP elevators in the presence or absence of genistein, suggesting that tyrosine kinases may function independently of the 5HT cascade. This result contrasts with two previous studies (Baxter and Byrne 1990; Hochner and Kandel 1992), which report that either bath application of cAMP analogues or direct injection of Sp-cAMPS into the SNs occluded further changes in excitability produced by 5HT. Several experimental factors could account for the differences in these observations, such as different pharmacological agents and type of preparation. For example, the experiments in both the study by Baxter and Byrne (1990) and by Hochner and Kandel (1992) were performed in excised SNs and used cAMP analogues to elevate cAMP levels, whereas we used the intact nervous system and forskolin/Ro-20-1724 to increase cAMP levels in this study. Further experiments will resolve this apparent difference.

A more likely model that is consistent with our results is that tyrosine kinases are not activated downstream of 5HT but rather they phosphorylate substrates that are modulated downstream of 5HT and PKA (Fig. 8B). Changes in persistent tyrosine phosphorylation through changes in the balance between tyrosine kinases and tyrosine phosphatases would adjust the baseline ion channel activity and modify the cell's response to subsequent exposure to neuromodulators, such as 5HT. As mentioned previously, there are several ion channels that have basal tyrosine phosphorylation. It is conceivable that a channel, such as the S channel, is modulated by both tyrosine kinases and PKA. Alternatively, tyrosine kinases may modulate PKA directly. In Aplysia, the catalytic subunit of PKA can have one of two different amino termini (Beushausen et al. 1992). Only catalytic subunits containing the second amino terminus can be phosphorylated in vitro by Src kinase. Furthermore it has been shown that the substrate selection of PKA depends on which amino terminus the catalytic subunit possesses (Panchal et al. 1994). An intriguing possibility is that tyrosine phosphorylation could regulate the dynamics of PKA substrate phosphorylation. Future experiments will help distinguish between these two possible models.

STF is not modulated by tyrosine kinases

Although inhibiting tyrosine kinase activity clearly reduced SN excitability, it had no effect on STF. This result suggests that substrates for tyrosine kinases are not required for the enhancement of transmitter release in the short term. It also demonstrates that two different types of short-term plasticity that are induced by the same neuromodulator (5HT) can be differentially regulated. Therefore stimuli that induce changes in tyrosine kinase activity (i.e., growth factors, cytokines) may affect the firing properties of the SNs and their subsequent responses to neuromodulators without changing the synaptic efficacy of the SNs onto their follower cells. In addition to modulatory plasticity induced by 5HT, the SNs also exhibit intrinsic (homosynaptic) plasticity that is activity dependent. Low rates of firing give rise to homosynaptic depression (Armitage and Siegelbaum 1998; Castellucci et al. 1970), while high rates produce synaptic facilitation (e.g., post tetanic potentiation) (Bao et al. 1997; Schaffhausen et al. 2001; Walters and Byrne 1984). Thus tyrosine kinase modulation of excitability (but not STF) in the SNs could provide a means of differential regulation of homosynaptic plasticity, leaving heterosynaptic plasticity (e.g., induced by 5HT) unaltered. This confers a high degree of flexibility in the induction of synaptic plasticity at the SN-MN synapse.

Tyrosine kinase activity provides a mechanism for gain control of cellular plasticity

There are many different external signals that can trigger changes in cellular tyrosine kinase activity. Several cytokines and growth factors utilize tyrosine kinases to initiate gene expression after injury or during periods of development. Our results suggest that changes in the persistent level of tyrosine kinase activity can have a major impact on Aplysia SN excitability. Therefore alterations in the amount of tyrosine phosphorylation within a neuron can provide a cellular context which can affect the cell's response to subsequent signaling events.

This type of modulation of plasticity may be operative during development as well. Growth factors more prevalent during particular periods of development could activate tyrosine kinase cascades unique to that growth period. The increased SN excitability due to high tyrosine phosphorylation levels may, in turn, facilitate the occurrence of specific types of activity-dependent plasticity (Hawkins et al. 1983; Sutton and Carew 2000; Walters and Byrne 1983, 1984).

If particular forms of ion channel function or plasticity are regulated by tyrosine kinases, this could provide a mechanism for modulation within a particular developmental window. An example of this type of modulation of plasticity in a specific developmental window is observed in vertebrates with the change of subunit composition of the NMDA receptor. During development, the majority of NMDA receptors contain NR2B subunits that cause the receptor to have a longer open time and allow a greater calcium influx. As the animal matures, the relative composition of the NMDA receptor changes, with proportionally greater amounts of NR2A subunits; thus the channel open time is decreased considerably (Carmignoto and Vicini 1992; Sheng et al. 1994). Consistent with this observation, the threshold for LTP is lower in developing animals expressing NMDA receptors containing NR2B subunits (Kirkwood et al. 1995). Furthermore, in adult transgenic animals that only have NR2B-containing NMDA receptors, the threshold for LTP is lower and they perform better than normal adults in learning tasks (Tang et al. 1999).

In conclusion, tyrosine phosphorylation can, in principle, adjust the excitatory state of a neuron, which in turn could contribute to a variety of forms of plasticity both intrinsic to the cell and with its synaptic partners. Thus elucidation of the role of tyrosine kinase activity in different types of cellular plasticity will help to gain a more complete understanding of how differential regulation of specific second-messenger cascades may facilitate information processing in the nervous system.


    ACKNOWLEDGMENTS

The authors thank J. H. Byrne, C. M. Sherff, M. A. Sutton, and U. Mueller for helpful comments on an earlier version of the manuscript.

This work was supported by a predoctoral National Science Foundation fellowship to A. L. Purcell and National Institute of Mental Health Grant RO1 MH-14-1083 to T. J. Carew.


    FOOTNOTES

Address for reprint requests: T. J. Carew (E-mail: tcarew{at}uci.edu).

Received 7 December 2000; accepted in final form 14 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society