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II. Institut Physiologisches, Universität Goettingen, 23 Humboldtallee, Germany; and Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Lalley, Peter M., Olivier Pierrefiche, Anne M. Bischoff, and Diethelm W. Richter. cAMP-dependent protein kinase modulates expiratory neurons in vivo. J. Neurophysiol. 77: 1119-1131, 1997. The adenosine 3
,5-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) second-messenger system influences neuronal excitability by modulating voltage-regulated and transmitter-activated channels. In this study we investigated the influence of the cAMP-PKA system on the excitability of expiratory (E) neurons in the caudal medulla of anesthetized, paralyzed, and artificially ventilated adult cats. We intracellularly injected the PKA inhibitors cAMP-dependent PKA inhibitor 5-22 amide (Walsh inhibitory peptide) and Rp-adenosine 3,5-cyclic monophosphothioate triethylamine (Rp-cAMPS), the PKA activator Sp-adenosine 3,5-cyclic monophosphothioate triethylamine (Sp-cAMPS), and the adenylyl cyclase activator forskolin and measured membrane potential, neuronal input resistance, and synaptic membrane currents. Inhibition of cAMP-PKA activity by Walsh inhibitory peptide or Rp-cAMPS injections hyperpolarized neurons, decreased input resistance, and depressed spontaneous bursts of action potentials. Action potential duration was shortened and afterhyperpolarizations were increased. Inhibitory synaptic currents increased significantly. Stimulation of cAMP-PKA activity by Sp-cAMPS or forskolin depolarized neurons and increased input resistance. Spontaneous inhibitory synaptic currents were reduced and excitatory synaptic currents were increased. Rp-cAMPS depressed stimulus-evoked excitatory postsynaptic potentials and currents, whereas Sp-cAMPS increased them. Sp-cAMPS also blocked postsynaptic inhibition of E neurons by 8-hydroxy-dipropylaminotetralin, a serotonin-1A (5-HT-1A) receptor agonist that depresses neuronal cAMP-PKA activity. To determine the predominant effect of G protein-mediated neuromodulation of E neurons, we injected guanosine-5-O-(3-thiotriphosphate) tetralithium salt (GTP-
-S), an activator of both stimulatory and inhibitory G proteins. GTP--S hyperpolarized E neurons, reduced input resistance, and increased action potential afterhyperpolarization. We conclude that the intracellular cAMP-PKA messenger system plays an important role in the activity-dependent modulation of excitability in E neurons of the caudal medulla. In addition, the cAMP-PKA pathway itself is downregulated during activation of 5-HT-1A receptors.
Second-messenger-mediated neuromodulation plays an important role in regulating membrane potential (Vm) and discharge properties of many types of neurons (Kaczmarek and Levitan 1987 Surgical preparation
Experiments were performed on 18 adult cats of either sex (2.5-4.5 kg). Care and use of animals were in accordance with the guiding principles of the German and American Physiological Societies. Animals were anesthetized with pentobarbital sodium (40 mg/kg ip initially, followed by 4-12 mg/h iv) to produce and maintain adequate levels of anesthesia. Supplemental doses of pentobarbital were given when systolic arterial pressure increased spontaneously to exceed 130-140 mmHg, and when discharges of phrenic nerve activity decreased in duration and increased in frequency to exceed 30 discharges per minute or showed a tonic discharge component. Additional anesthetic was also given if surgical procedures or pressure on a paw produced increases of heart rate and blood pressure or frequency of phrenic nerve discharges. Experiments were terminated by intravenous injection of pentobarbital sodium in sufficient quantities to produce irreversible cardiac arrest. Body temperature was maintained at 36-38°C by external heating. Atropine sulfate (0.2 mg/kg iv) was administered to reduce salivation and dexamethasone (0.3 mg/kg) was given to prevent brain edema. Neuromuscular paralysis was achieved by intravenous administration of gallamine triethiodide (4-8 mg/kg initially, followed by 4-8 mg/h). Animals were ventilated with oxygen-enriched air (65-75 vol % O2) through a tracheal cannula. End-tidal CO2 was maintained at 3.5-4.5% by adjusting ventilatory rate and tidal volume. A pneumothorax was performed bilaterally to increase stability of recording from E-2 neurons. Atelectasis was prevented by applying 1-2 cm H2O positive pressure to the expiratory air flow.
Recording and stimulation procedures
Phrenic nerve activity was amplified (×2,000-10,000), band-pass filtered (80-3,000 Hz), displayed on an oscilloscope, and registered on magnetic tape (frequency response 5 kHz DC) and chart recorder as raw and moving average (time constant = 10-100 ms) of the rectified discharge.
Intracellular injections
Chemicals that influence PKA were injected intracellularly with current pulses (1-5 nA, 50-80 ms, 10 Hz) over a period of 1-3 min. The total charge applied to eject chemicals is expressed in nanocoulombs. Agents injected were the PKA inhibitors cAMPdependent PKA inhibitor 5-22 amide, also known as Walsh inhibitory peptide (Wiptide), and Rp-adenosine 3 Extracellular Ionophoresis
The 5-HT-1A receptor agonist 8-hydroxy-dipropylaminotetralin (8-OHDPAT), which postsynaptically hyperpolarizes E neurons and depresses discharges (Lalley et al. 1994 Measurements and analysis
Measurements of Vm, spontaneous EDPs or EDCs, IDPs or IDCs, stimulus-evoked s-EPSPs or s-IPSCs, and neuronal input resistance (Rn) were made on medullary E neurons that 1) exhibited maximum values of Vm that were more negative than Current-clamp recording from E neurons under control conditions reveals Vm fluctuations and discharge patterns such as those shown in Figs. 1-8 (see Ballantyne and Richter 1986
Depression of neuronal excitability by Wiptide and
Rp-cAMPS
Wiptide and Rp-cAMPS hyperpolarized Vm of E neurons, reduced or abolished action potential discharges, and increased spontaneous IDCs. Intracellular injection of Wiptide (30 or 100 µM, 30-315 nC), an inhibitory peptide that prevents phosphorylation of substrates by the catalytic subunit of PKA (Cheng et al. 1986
Enhancement of neuronal excitability by Sp-cAMPS
Sp-cAMPS (2 or 5 mM, 42-168 nC), an activator of PKA (Van Haastert et al. 1984
Increased excitability produced by forskolin
Forskolin injections (5 mM, 168-504 nC) produced increased excitability in all E neurons (n = 4). Vm was depolarized during all phases of the respiratory cycle (Fig. 6). The effects were qualitatively similar to those of Sp-cAMPS, but were of shorter duration (3-4 min). In two E neurons analyzed in voltage clamp, forskolin induced decreased IDCs and increased EDCs (Fig. 6).
Block of 5-HT-1A receptor-mediated inhibition by Sp-cAMPS
Ionophoretic application of 8-OHDPAT, a 5-HT-1A receptor agonist (Zifa and Fillion 1992
Depression of neuronal excitability by GTP- Intracellular injection of GTP-
We found that Wiptide and Rp-cAMPS depressed excitability of E neurons, whereas Sp-cAMPS and forskolin increased excitability. Sp-cAMPS also blocked depression of neuronal excitability produced by ionophoresis of the 5-HT-1A receptor agonist 8-OHDPAT. In addition, GTP- Specificity and selectivity of chemicals after intracellular ionophoresis
The effects of the chemicals administered by ionophoresis are presumed to be specific, because the quantities of chemicals injected into cells were small. Concentrations of drugs in the microelectrode solutions were, at the greatest, 0.003% of cations or anions in solution. The effective drug concentrations within the cytosol were therefore in the lower micromolar range. Because the quantities were small and the selectivity of the injected chemicals is high (Watling et al. 1995 Influence of pentobarbital anesthesia on the cAMP-PKA system and responses to intracellularly injected chemicals
In our experiments, substantial cAMP-PKA activity seemed to be preserved during pentobarbital anesthesia, because cAMP-PKA-active chemicals significantly increased or decreased neuron excitability. Because the effects were consistent with those reported in studies conducted on various unanesthetized preparations (Kaczmarek and Levitan 1987 Responses to injected chemicals during voltage clamp
We voltage clamped neurons to overcome the problem of voltage dependency of chemical effects on synaptic potentials and Rn. We are cognizant of the space-clamp problems associated with voltage clamping in such neurons. We realize that our measurements and analysis only apply to events occurring at the soma and proximal dendrites of neurons, and that they do not give information about responses in remote dendritic areas of respiratory neurons (Berger et al. 1984 cAMP-PKA system increases the excitability of E neurons
The present results demonstrate that the cAMP-PKA second-messenger system mediates an excitatory modulation of caudal medullary E neurons in vivo. Hyperpolarization of Vm and depression of action potential discharge occurred when PKA was inhibited. Activation of PKA, either with Sp-cAMPS or by stimulating adenylyl cyclase with forskolin to activate endogenous cAMP, depolarized neurons, increased spontaneous action potential burst discharges, and enhanced stimulus-evoked excitatory postsynaptic responses. The data are consistent with the in vitro observation that activation of the cAMP-PKA system increases the excitability of a wide variety of neurons. Our results do not conflict with findings of another study (Barraco et al. 1988 cAMP-PKA mediated increase of excitability in medullary E neurons may be related to decreased synaptic potassium and chloride currents
The cAMP-PKA system upregulates neuronal excitability through a variety of postsynaptic mechanisms. 1) Elevation of cAMP and activation of PKA decreases the current through voltage- and calcium-gated potassium channels (Akins and McClesky 1993 Downregulation of the cAMP-PKA system may occur during activation of 5-HT-1A receptors on E neurons
It is generally accepted that activation of 5-HT-1A receptors increases a G protein-dependent, inwardly rectifying potassium conductance (Andrade et al. 1986 cAMP-PKA system exerts tonic excitatory neuromodulation that can be overwhelmed by activation of various G proteins
The finding that Wiptide and Rp-cAMPS depressed excitability in E-2 neurons shows that the cAMP-PKA system is tonically active under normal in vivo conditions and suggests that there is persistent excitatory neuromodulation. There is, to our knowledge, no published evidence that directly links the actions of established neuromodulators of the respiratory network to specific second-messenger pathways. However, cAMP-PKA-mediated neuromodulation could be achieved by activating 5-HT-4 receptors (Torres et al. 1995 Functional implications
Many E neurons are bulbospinal and regulate expiratory motor output (Bainton and Kirkwood 1979
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Nicholls et al. 1992; Nicoll et al. 1990), including brain stem respiratory neurons (Arita et al. 1993; Barraco et al. 1988; Champagnat and Richter 1993). In expiratory (E) neurons of the caudal medulla, there is evidence that protein kinase C plays an important role in modulating excitability (Champagnat and Richter 1993; Haji et al. 1996).
,5-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) system has been the most thoroughly studied second-messenger pathway. Activation of this system increases the excitability of many types of neurons (Kaczmarek and Levitan 1987). The influence of the cAMP-dependent protein kinase A pathway on respiratory neurons, however, remains unclear. Elevation of cAMP levels by extracellular application in in vitro and in vivo experiments was reported to either increase (Arita et al. 1993) or to decrease (Barraco et al. 1988) discharge frequency of different types of respiratory neurons. Indirect evidence from other studies suggests that in E neurons the cAMP-PKA system is a potentially important intracellular conveyer of neuromodulatory function. Discharge properties of caudal E neurons are strongly influenced by ionophoretic application of several putative neurotransmitters that have the potential to influence the cAMP-PKA system (Watling et al. 1995), including -aminobutyric acid (GABA) (acting on GABAB receptors; Haji and Takeda 1993), serotonin (5-HT) (5-HT-1A receptors; Lalley et al. 1994), adenosine (A1 receptors; Schmidt et al. 1995), dopamine, and norepinephrine (Fallert et al. 1979). In the medullary slice preparation of the rat, agonists for µ-opioid, 
2-adrenergic, and GABAB receptors alter frequency of respiratory neural discharges through Gi/o proteins, because the responses are pertussis toxin sensitive (Johnson et al. 1996). The effects of these agonists on discharge frequency could, potentially, be mediated by cAMP-dependent PKA. There are, however, no reported investigations that have directly determined whether the cAMP-PKA system controls excitability of E-2 neurons, nor have previous studies shown how other types of respiratory neurons are directly influenced by cAMP-dependent PKA.
-O-(3-thiotriphosphate) tetralithium salt (GTP--S) reveals neuromodulation as it does in other neurons (Andrade et al. 1986; Moises et al. 1994), and whether the PKA system influences responses to 5-HT-1A receptor activation. A preliminary account of this study has been published in abstract form (Pierrefiche et al. 1995).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
. Such electrodes allowed current flow without rectification.
; Lalley et al. 1995; Richter et al. 1996). Stray capacitances around the unshielded glass microelectrodes were compensated by the method of supercharging (Strickholm 1995a,b), which reduced settling time of the voltage artifact produced by brief current steps to 2-3 µS (Richter et al. 1996). During voltage clamp, holding potentials were in most tests set close to the Vm at which, during current clamp, the neurons were maximally hyperpolarized (
60 to 80 mV). In some experiments, holding potential was set to less negative levels (e.g., Fig. 6A). At all holding potentials, responses to cAMP-PKA-selective chemicals were consistent with the effects on Vm recorded in current-clamp or balanced bridge modes. Switching frequencies during voltage clamp ranged between 20 and 40 kHz and duty cycle was set at 25%. Electrode potentials, holding potentials, and Im were monitored on an oscilloscope, recorded on chart paper, and stored on magnetic tape.
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FIG. 6.
Activating the cAMP-PKA pathway with forskolin increases the excitability of E-2 neurons. Forskolin increases excitatory synaptic drive currents (EDCs), decreases IDCs, and depolarizes Vm. A1 and A2: recordings of Im. Vm was clamped to holding potential of 60 mV. B1 and B2: recordings of Vm. A1 and B1 were taken before injection (Control). A2 and B2 were taken 1 min after injection of forskolin (294 nC) from a 5 mM solution in 2 M potassium methylsulfate. Dashed horizontal lines in A: peak inward and outward currents (EDCs and IDCs) recorded under control conditions. Lines in B: peak inspiratory (bottom line) and expiratory (top line) Vm recorded under control conditions. A2, taken shortly after forskolin injection, shows that EDCs were increased and IDCs were decreased. B2 shows that forskolin depolarized Vm during the inspiratory and expiratory phases.
,5-cyclic monophosphothioate triethylamine (Rp-cAMPS); the PKA activator Sp-adenosine 3,5-cyclic monophosphothioate triethylamine( S p - c A M P S ) ; f o r s k o l i n - 7 b - d e a c e t y l - 7 b - - ( m o r p h o l i n o ) b u t y r y l hydrochloride, a specific stimulator of adenylyl cyclase (Laurenza et al. 1989); and an irreversible, nonspecific activator of G proteins, GTP--S. Wiptide was dissolved in double-distilled water containing 1 mg/ml bovine serum albumin and added to the micropipette electrolyte solutions used for intracellular recording. Final concentrations of Wiptide were 30 µM in 3 M KCl (pH 6, injected with positive current pulses, n = 5 neurons) or 100 µM in 3 M potassium acetate (pH 7.5, injected with negative current pulses, n = 8). The results obtained with the different solutions and injection currents were similar. Although bovine serum albumin has been reported to increase low-voltage-activated calcium currents in neuroblastoma hybrid cells (Schmitt and Meves 1994), current injections from solutions containing equivalent concentrations of bovine serum albumin, but lacking Wiptide, were without effect. Rp-cAMPS (2 mM) and Sp-cAMPS (2 mM or 5 mM) were dissolved in 3 M potassium acetate or 2 M potassium methylsulfate, adjusted to pH 8-8.4, and injected with negative current pulses. GTP--S (5 mM) was dissolved in 2 M potassium methylsulfate and injected with negative current. Forskolin (5 mM) was also dissolved in 2 M potassium methylsulfate and injected with positive current.
), was ionophoresed before and after intracellular injection of Sp-cAMPS. Ionophoresis and recording were performed with parallel compound microelectrodes consisting of an intracellular micropipette and a three-barrel assembly for extracellular ionophoresis. The tip of the recording electrode extended 30-40 µm beyond the tips of the three-barrel assembly (Lalley et al. 1994). The intracellular pipettes contained Sp-cAMPS in potassium acetate as described above, whereas extracellular ionophoresis barrels contained 8-OHDPAT (40 mM) in double-distilled water, pH 4.5. 8-OHDPAT was retained with negative current (10 nA) and ejected with positive current during voltage- or current-clamp recording. 8-OHDPAT, Rp-cAMPS, Sp-cAMPS, forskolin, and Wiptide were purchased from Research Biochemicals International (Natick, MA). GTP--S was purchased from Sigma Chemical (St. Louis, MO).
55 mV under control (pretreatment) conditions and 2) showed minimal leak currents during voltage clamp at a holding potential that was close (±10 mV) to the Vm measured during the inspiratory phase under current clamp.
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
for a more detailed description). Vm fluctuates cyclically because of excitatory and inhibitory synaptic inputs and intrinsic membrane properties (Richter 1996). During the inspiratory phase when phrenic nerve activity is increasing, Vm is maximally hyperpolarized. At the end of inspiration, Vm begins to depolarize, but is slowed by synaptic inhibition during the postinspiratory phase as phrenic nerve discharge declines. If respiratory drive is sufficiently strong, depolarization will proceed until the threshold for discharge of action potentials is reached during late expiration, when phrenic nerve activity is absent. Voltage-clamp recording (Figs. 3 and 6) reveals cyclic variations of Im: net outward currents (IDCs) occur during the inspiratory phase and net inward currents (EDCs) develop during the expiratory phase.
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FIG. 1.
Inhibition of adenosine 3 ,5-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) by the cAMP-dependent PKA inhibitor 5-22 amide, also known as Walsh inhibitory peptide (Wiptide), depresses E-2 neuronal discharges. Wiptide injection into expiratory (E) neurons hyperpolarizes membrane potential (Vm), depresses action potential discharge, and alters the shape of action potentials. Traces in A-C: membrane potential (Vm, action potentials truncated), phrenic nerve activity (PN), and the moving average of PN discharge (PNa). Records in A1 were taken before Wiptide injection. Horizontal dashed line: control level of Vm during the inspiratory phase. Records in B1 and C were taken 1 and 3 min, respectively, after injection of Wiptide (30 µM in 3 M KCl) intracellularly with positive charge (53 nC). Note that the inspiratory phase Vm was hyperpolarized by Wiptide. Immediately after injection, action potentials were absent. After 1 min (B1), discharges of low intensity reappeared and increased in intensity until they recovered to control levels after 3 min (C). Vm, however, was still hyperpolarized during the inspiratory phase for an additional 7 min. B2 and B3: traces recorded at higher amplification and faster speed, superimposed to show the effects of Wiptide on the afterhyperpolarization and duration of action potentials. B2: action potentials aligned (top dashed reference line) to emphasize the increased amplitude of the fast afterhyperpolarization (bottom reference line). Traces at left were taken before Wiptide injection; traces at right after Wiptide injection. Traces in B3 show that Wiptide shortens action potential duration and illustrate the increased fast and slow afterhyperpolarizations.
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FIG. 2.
Inhibition of PKA by Wiptide delays the onset of expiratory discharges and reduces neuronal input resistance (Rn). Wiptide injection (100 µM, 120 nC) into E-2 neurons shortens discharge duration following Vm hyperpolarization, reduces Rn, and increases spontaneous inhibitory synaptic drive currents (IDCs) during inspiration and postinspiration. A1 and A2: effects of Wiptide on Vm and Rn. Dashed reference line: control inspiratory phase Vm. In A1 and B, the small, regularly spaced downward deflections of Vm were produced by 0.3-nA, 60-ms current pulses to measure Rn. After Wiptide injection, Vm was strongly hyperpolarized. In addition, threshold for action potentials was reached later in the expiratory phase. Rn was also reduced during the inspiratory and expiratory phases. The amplified and expanded records in B are segments from A1 and A2 showing the effect of Wiptide on Rn. Top trace: control segment. Bottom traces: records taken after Wiptide was injected. Because of decreased Rn, the constant current pulses fail to elicit hyperpolarizing potentials in the bottom trace.
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FIG. 3.
Wiptide inhibition of PKA activity enhances inhibitory synaptic currents, while reducing and delaying excitatory synaptic currents. Records of membrane currents (Im, top traces), Vm (middle traces), and phrenic nerve activity (bottom traces) were taken from the same E neuron during alternating periods of voltage- and current-clamp recording. During voltage-clamp recording, holding potential was 70 mV, corresponding to the most negative Vm of the inspiratory phase recorded during current clamp. Dashed lines: maximal outward inhibitory currents (top line), maximum inward excitatory currents (middle line), and maximum negative Vm values (bottom line) recorded under control cconditions. Im and Vm displays at right: superimposed segments taken from the left and middle. Wiptide injection (150 nC) increased synaptically mediated inhibitory currents and hyperpolarized Vm during the inspiratiory phase, and delayed onset of inward currents and threshold for discharge during the expiratory phase. There was also a small reduction of peak inward Im.
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FIG. 4.
Rp-adenosine 3 ,5-cyclic monophosphothioate triethylamine (Rp-cAMPS) block of PKA augments synaptic inhibition. Injection of Rp-cAMPS hyperpolarizes Vm and depresses evoked excitatory postsynaptic potentials and action potentials. Records show Vm of an E neuron, phrenic nerve activity (bottom trace), and the moving average of phrenic nerve action potential frequency (middle trace). Records were taken before (control, left) and after injection of Rp-cAMPS (180 nC from a 2 mM solution). Right: superimposed segments taken from the left and middle. Rp-cAMPS hyperpolarized Vm during inspiratory and postinspiratory phases and reduced the rate of depolarization during the postinspiratory phase, evidenced by the plateau of Vm. In addition, Rp-cAMPS abolished action potentials during the postinspiratory phase evoked by single shocks delivered to the ipsilateral brain stem in the region of the pre-Bötzinger complex. In the control records, 2 evoked action potentials are evident, 1 during postinspiration when discharge is otherwise absent, the other during expiration just after the neuron began to discharge spontaneously. Responses to stimulation are absent during the postinspiratory period after Rp-cAMPS injection, as shown in the middle and right.
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FIG. 5.
Activation of PKA by Sp-adenosine 3 ,5-cyclic monophosphothioate triethylamine (Sp-cAMPS) increases excitability. A1 and A2: depolarization and increased Rn after injection of Sp-cAMPS (42 nC from a 2 mM solution) into an E neuron. Dashed horizontal line in A1: control Vm during the inspiratory phase. Regularly spaced negative deflections of Vm are electrotonic potentials evoked by constant current pulses. B1 and B2: superimposed traces of excitatory postsynaptic currents evoked by stimulating the ipsilateral medulla in the pre-Bötzinger region. Holding potential was 75 mV. Four traces are superimposed in each record. Dashed lines: peak amplitude of evoked excitatory postsynaptic currents during control, which was increased after injection of Sp-cAMPS.
-S depressed the excitability of E neurons.
View this table:
TABLE 1.
Effects of Wiptide on MP, EDCs, IDCs, and Rn
; Knighton et al. 1991), depressed action potential discharges in 10 of 13 E neurons. In 3 of the 10 depressed neurons, discharges were initially abolished. This was followed by reduced discharges that subsequently returned to control levels. Figure 1B1 illustrates the transition from no discharge to low-intensity firing in one of the three neurons. In seven neurons, discharges were greatly reduced but not abolished by Wiptide. Three E neurons were unresponsive. The effects of Wiptide administration on E neurons are summarized in Table 1.
), significantly increased IDPs during the inspiratory and postinspiratory phases. In three E-2 neurons, this resulted in the appearance of a plateau potential during postinspiration (Fig. 4). Rp-cAMPS, like Wiptide, did not significantly change the peak amplitudes of spontaneous EDPs, but delayed the onset of action potential discharges. In addition, stimulus-evoked excitatory postsynaptic potentials, evoked by stimulating the ipsilateral medulla, were appreciably depressed by Rp-cAMPS during the inspiratory and postinspiratory phases (Fig. 4). In three neurons in which measurements were made, Rp-cAMPS reduced Rn during all phases of the respiratory cycle (Table 2).
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TABLE 2.
Effects of Rp-cAMPS on E neurons
), increased neuronal excitability in 13 of 14 E-2 neurons. Inspiratory IDPs were significantly reduced (4.77 ± 1.02 mV), expiratory EDPs were significantly augmented (2.31 ± 0.81 mV), and neurons discharged longer bursts of action potentials. In three neurons, action potential afterhyperpolarization decreased in amplitude and duration (not shown). In addition, Sp-cAMPS increased Rn throughout the respiratory cycle (n = 3). An example of the effects on Vm and Rn is presented in Fig. 5, A1 and A2.
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TABLE 3.
Effects of Sp-cAMPS on E neurons
), depresses excitability of medullary respiratory neurons through postsynaptic inhibition (Lalley et al. 1994). The underlying mechanism could be a 5-HT-1A receptor-mediated suppression of the cAMP-PKA pathway (Hoyer et al. 1994). To test whether such a transduction mechanism might link activation of 5-HT-1A receptors to depression of E neurons, we determined whether activation of PKA by Sp-cAMPS would circumvent the depressant effects of 8-OHDPAT on 5 E neurons.
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FIG. 7.
Activation of PKA blocks depression of excitability mediated by activation of serotonin-1A (5-HT-1A) receptors. Sp-cAMPS blocks depression produced by ionophoresis of 8-hydroxy-dipropylaminotetralin (8-OHDPAT), a selective 5-HT-1A receptor agonist. A-D: current-clamp records of Vm taken from an E neuron, phrenic nerve activity, and the moving average of phrenic nerve action potential frequency. Dashed line: control inspiratory phase Vm. Before injection of Sp-cAMPS, ionophoresis of 8-OHDPAT (70 nA, 40 mM) hyperpolarized Vm and abolished discharges of action potentials (A and B). Injection of Sp-cAMPS (336 nC from a 2 mM solution) depolarized Vm (C) and blocked the effects of 8-OHDPAT ionophoresis.
-S
-S (5 mM), a nonselective and irreversible activator of G proteins (Andrade et al. 1986; Nicholls et al. 1992), depressed the excitability of four E neurons. Depression was characterized by hyperpolarization of Vm throughout the respiratory cycle, depression of action potential discharge, and reduction of Rn (Fig. 8).
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FIG. 8.
Guanosine-5 -O-(3-thiotriphosphate) tetralithium salt (GTP--S) injection hyperpolarizes Vm and decreases excitability of E-2 neurons. A1-A3: hyperpolarization of Vm, depression of action potential discharge, reduction of Rn, and increased spike afterhyperpolarization following injection of GTP--S. Dashed reference line in A1-A3: peak inspiratory phase Vm recorded under control conditions. Regularly spaced negative deflections of Vm are electrotonic potentials evoked by constant current pulses to detect changes of Rn. Records in A2 were taken shortly after injection of GTP--S (30 nC) from a 5 mM solution in 2 M potassium acetate. Constant current pulses were not applied when record was taken. Note that GTP--S increased inhibitory synaptic drive potentials (IDPs), as evidenced by the hyperpolarization of Vm during the inspiratory phase. A3, taken 5 min after injection, shows further increase of IDPs, abolition of action potentials, and decreased Rn, evidenced by the reduced amplitudes of electrotonic potentials. B1-B3: expanded records from the same E-2 neuron to illustrate the effect of GTP--S on spike afterhyperpolarization. Records in B2 were taken shortly after injection. Note the large increase in afterhyperpolarization. The superimposed records in B3 further illustrate that GTP-2-S increases spike afterhyperpolarization amplitude and duration.
-S produced a progressive depression and eventual abolition of action potential discharges and a marked reduction in Rn (Fig. 8, A1-A3). An additional effect seen was a marked increase of the fast afterhyperpolarization of the action potential (Fig. 8, B1-B3). In other E neurons, GTP--S produced a more modest but significant hyperpolarization of Vm. Action potential discharges were reduced in intensity, but not abolished, and action potential afterhyperpolarizations remained unchanged.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
-S depressed the excitability of E neurons. Before interpreting these findings, certain issues need to be addressed. These concern 1) whether the chemicals employed to alter activity of the cAMP-PKA system were specific; 2) whether the effects of the chemicals were restricted to cells into which they were injected; 3) whether pentobarbital anesthesia influences the cAMP-PKA system and consequently modifies responses to injected chemicals; and 4) whether the responses to chemicals recorded during voltage clamp are accurate indexes of effects on neuron excitability.
), we presume that they specifically altered the activity of the cAMP-PKA system.
; Fiorillo and Williams 1996). The small quantities of the other more permeable chemicals would have been considerably diluted in the extracellular fluid. Measurements of Vm and Rn support the conclusion that the critical site of action was in the soma and proximal dendrites of cells into which the chemicals were injected. In most neurons so far studied, suppression of the cAMP-PKA system depresses neuronal excitability. If Rp-cAMPS, for example, had diffused from cells and depressed E neurons presynaptically, the neuronal inhibition would have been linked to depressed release of an excitatory neurotransmitter, probably glutamate (Bianchi et al. 1995; Richter 1996), and Rn would have decreased (Collingridge and Lester 1989), contrary to what we observed in this study.
; Van Haastert et al. 1984; Watling et al. 1995), we presume that anesthesia did not appreciably influence the responses to injected chemicals. The manner in which barbiturates influence the cAMP-PKA system is not yet resolved. Barbiturates stimulate cAMP production in lymphoma cells (Gonzales 1995), but depress it in membrane preparations from rat brain (Da
ura et al. 1989). If the latter study is indicative of how barbiturates influence cAMP in neurons, the cAMP-PKA system may be even more active in E-2 neurons under physiological conditions when anesthetics are absent.
; Kreuter et al. 1977). However, in all experiments the changes evoked by second-messenger-selective chemicals on Im during voltage clamp were consistent with the measurable effects on Vm, and are supported by results obtained from nonrespiratory neurons (Kaczmarek and Levitan 1987; Van Haastert et al. 1984; Watling et al. 1995). In some cells in the present study, action potentials were completely blocked during voltage clamp (Fig. 3), or the cells did not discharge action potentials spontaneously (Fig. 6). In these situations, as well as in tests where action potentials were present, the changes in Im evoked by injected chemicals were compatible with the observed alterations of Vm. For example, in Fig. 3, Wiptide hyperpolarized Vm during the inspiratory phase and delayed the time to discharge of action potentials during the expiratory phase. During voltage clamp, the IDCs that support inspiratory phase hyperpolarization of Vm increased and the time to peak of EDCs was delayed. In Fig. 6, inward current increased during the expiratory phases whereas outward current decreased during inspiratory phases after injection of forkolin. Correspondingly, forskolin depolarized Vm during all phases. Therefore we believe that the somata of neurons were adequately voltage clamped to the degree that their morphology and the experimental conditions allow. We assume that the Im responses to chemicals injected into the region where the voltage was adequately controlled by voltage clamp are accurate indicators of their effects on neuronal excitability in the proximal regions of the neuron.
) that showed that cAMP analogues injected into respiratory regions of the medulla depressed discharges of respiratory neurons. In that investigation, cAMP analogues would have influenced relatively large numbers of neurons pre- and postsynaptically. Accordingly, inhibition may have been achieved through cAMP-mediated activation of inhibitory interneurons leading to enhanced release of inhibitory neurotransmitter at synapses on respiratory neurons. Such effects have been demonstrated previously at synapses in the hippocampus (Capogna et al. 1995; Sciancalepore and Cherubini 1995).
; Gereau and Conn 1994; Goldsmith and Abrams 1992; Grega and Macdonald 1987; Rudy 1988; Swope et al. 1992; Wright and Zhong 1995) and increases current flow through calcium and sodium channels (Colwell and Levine 1995; Ewald and Levitan 1987; Grega and Macdonald 1987; Liu and Lasater 1994; Nicholls et al. 1992; Sudlow and Gillette 1995; Swope et al. 1992). 2) The cAMP-PKA system opposes the postsynaptic actions of inhibitory neurotransmitters. It desensitizes GABAA receptors (Blackstone et al. 1994; Browning et al. 1990; Raymond et al. 1993; Schwartz et al. 1991), reduces GABAA- and glycine-gated chloride currents (Agopyan et al. 1993; Schwartz et al. 1991), and depresses GABAB-gated potassium currents (Bowery 1993). 3) In addition, the cAMP-PKA system enhances glutamate-regulated neuronal excitation by augmenting inward cation currents through N-methyl-D-aspartate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor channels (Blackstone et al. 1994; Cerne et al. 1993; Greengard et al. 1991). 4) Furthermore, metabotropic glutamate receptors that increase cAMP levels, such as the mGluR1a subtype (Schoepp and Conn 1993), may increase neuronal excitability by increasing intracellular Ca2+ concentration, decreasing calcium-mediated and voltage-dependent potassium currents and increasing other sorts of cation currents (Greene et al. 1994).
; Richter et al. 1992). In the present study, spontaneous IDCs and hyperpolarization of neurons during the inspiratory and postinspiratory phases were significantly increased when PKA was inhibited and depressed when PKA was activated. It seems reasonable, therefore, to assume that the cAMP-PKA system downregulates chloride and potassium currents that are synaptically activated during the inspiratory and postinspiratory phases. In addition, action potential duration and fast action potential afterhyperpolarization were altered by the cAMP-PKA-selective agents, possibly through modulation of calcium- or voltage-dependent potassium currents (Rudy 1988). There is evidence that such currents contribute to repolarization and afterhyperpolarization of action potentials in E neurons (Champagnat and Richter 1994; Haji et al. 1996; Richter et al. 1993). In the present investigation we observed increased Rn during the late expiratory phase following injections of Sp-cAMPS, which could signal cAMP-mediated inhibition of inwardly rectifying potassium conductances (Akasu and Shoji 1994). We do not, however, have direct evidence that the injected chemicals alter potassium and chloride currents. In addition, our findings do not explain why these currents are more sensitive to cAMP-PKA modulation than are depolarizing cationic currents.
: Anwyl 1990; Bobker and Williams 1990). The transduction mechanism seems to vary in different types of neurons. In some neurons, the cAMP-PKA system is downregulated when 5-HT-1A receptor activation mobilizes Gi protein, leading to depression of adenylyl cyclase (Hoyer et al. 1994). In other neurons, 5-HT-1A receptor activation may mobilize G proteins that directly alter potassium conductances (Andrade et al. 1986). In medullary E neurons, 5-HT-1A receptor activation hyperpolarizes Vm, depresses excitability, and decreases Rn (Lalley et al. 1994), probably by increasing membrane conductance to potassium ions (Anwyl 1990; Richter et al. 1996). The effects of 5-HT-1A receptor activation could be mediated by downregulation of adenylyl cyclase. This is suggested by 1) the similar effects of 8-OHDPAT and the chemicals that impair cAMP-PKA activity, and 2) by the block of 8-OHDPAT effects after injection of Sp-cAMPS. Activation of PKA by Sp-cAMPS may have circumvented the 5-HT-1A receptor-mediated reduction of endogenous cAMP. Nonetheless, there may be additional transduction mechanisms subsequent to 5-HT-1A receptor activation including a direct, G protein-mediated increase in potassium channel current.
) or metabotropic glutamate receptors (Cerne et al. 1993; Schoepp and Conn 1993). The latter may serve to augment a primary tonic excitatory drive mediated by activation of N-methyl-D-aspartate and AMPA/kainate receptors (Bianchi et al. 1995; Pierrefiche et al. 1991; Richter 1996; Richter et al. 1992).
-S is an analogue that indiscriminately activates all G proteins. Nonetheless, the net effect on E neurons was a depression of excitability (Fig. 7), even if Gs protein and therefore cAMP activity, was elevated by GTP--S. Thus E neurons and other types of respiratory neurons may be depressed through other G protein-activated mechanisms that potentially alter multiple second-messenger systems and ion channel proteins (Shepherd 1994). Activation of 5-HT-1A (Fig. 7), GABAB, 2-adrenergic, A1, and µ- and 
-opiate receptors could serve this purpose (Andrade et al. 1986; Bowery 1993; Johnson et al. 1996; Moises et al. 1994; Nicoll 1988; Schmidt et al. 1995; Taiwo et al. 1992). In E neurons, such mechanisms may serve to augment the powerful periodic inhibition evoked by GABA and glycine during the inspiratory and postinspiratory phases. There is probably dynamic interplay between G protein-dependent neuromodulators that are tonically excitatory and those that downgrade excitability, such as 5-HT, catecholamines, opioids, and GABA.
; Ballantyne and Richter 1986; Long and Duffin 1984). Such neurons are synaptically excited by glutamate, which activates N-methyl-D-aspartate and AMPA/kainate receptors (Pierrefiche et al. 1991). Because many studies have shown that the cAMP-PKA pathway modulates current flow through potassium channels, chloride channels, and glutamate-gated cation channels of various types of neurons, there is the possibility that similar events take place in E neurons. We postulate that the primary excitatory actions of ionotropic glutamate receptor activation (Bianchi et al. 1995; Pierrefiche et al. 1991) are modulated by the cAMP-dependent PKA: along with the protein kinase C pathway (Champagnat and Richter 1993; Haji et al. 1996), it influences respiratory motor output. Potentially, these second-messenger systems might alter the intensity and burst duration of respiratory neuron discharges and thus modify respiratory drive and pattern. In addition, responses of E-2 neurons to hypoxia and ischemia (Richter et al. 1991) and the degree of neuron damage (Haddad and Jiang 1993) might be influenced by the cAMP-PKA pathway and other second-messenger systems. During hypoxia, levels of GABA, 5-HT, and adenosine increase (Haddad and Jiang 1993), leading to increased activation of functionally identified metabotropic receptors (GABAB, 5-HT-1A, and A1 receptors). Because these receptors mediate downregulation of cAMP-dependent PKA activity and decrease neuronal excitability, they may potentially subserve an important neuroprotective function in E neurons.
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ACKNOWLEDGEMENTS |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB 406, C2) and National Heart, Lung, and Blood Institute Grant HL-29563.
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FOOTNOTES |
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Address for reprint requests: P. M. Lalley, Dept. of Physiology, University of Wisconsin, Madison, WI, 53706.
Received 12 March 1996; accepted in final form 29 October 1996.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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URA, T.,
KUROKAWA, T.,
YAMASHITTA, A.,
ISHIBASHI, S.
Effective inhibition by pentobarbital of forskolin-stimulated adenylate cyclase activity in rat brain.
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