Journal of Applied Physiology Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 99: 1658-1667, 2005. First published July 21, 2005; doi:10.1152/japplphysiol.00334.2005
8750-7587/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
99/5/1658    most recent
00334.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, Y.-W.
Right arrow Articles by Su, C.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, Y.-W.
Right arrow Articles by Su, C.-K.

GABAB-receptor-mediated suppression of sympathetic outflow from the spinal cord of neonatal rats

Yi-Wen Cheng,1 Min-Chi Ku,1 Chiu-Ming Ho,2 Chok-Yung Chai,1 and Chun-Kuei Su1

1Institute of Biomedical Sciences, Academia Sinica, and 2Department of Anesthesiology, Taipei Veterans General Hospital and National Yang-Ming University, Taipei, Taiwan

Submitted 23 March 2005 ; accepted in final form 14 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Using a splanchnic nerve-spinal cord preparation in vitro that could spontaneously generate sympathetic nerve discharge (SND), we investigated the roles of intraspinal GABAB receptors in the regulation of SND. Despite an age-dependent difference in sensitivity, bath applications of baclofen (Bac; GABAB-receptor agonist) consistently reduced SND in a concentration-dependent manner. The drug specificity of Bac in activation of GABAB receptors was verified by application of its antagonist saclofen (Sac) or CGP-46381 (CGP). Sac or CGP alone did not change SND. However, in the presence of Sac or CGP, the effects of Bac on SND inhibition were reversibly attenuated. The splanchnic sympathetic preganglionic neuron (SPN) was recorded by blind whole cell, patch-clamp techniques. We examined Bac effects on electrical membrane properties of SPNs. Applications of Bac reduced excitatory synaptic events, induced membrane hyperpolarizations, and inhibited SPN firing. In the presence of 12 mM Mg2+ or 0.5 µM TTX to block Ca2+- or action potential-dependent synaptic transmissions, applications of Bac induced an outward baseline current that reversed at –29 ± 6 mV. Because the K+ equilibrium potential in our experimental conditions was –100 mV, the Bac-induced currents could not simply be attributed to an alteration of K+ conductance. On the other hand, applications of Bac to Cs+-loaded SPNs reduced Cd2+-sensitive and high-voltage-activated inward currents, indicating an inhibition of voltage-gated Ca2+ currents. Our results suggest that the activation of intraspinal GABAB receptors suppresses SND via a mixture of ion events that may link to a change in Ca2+ conductance.

intermediolateral cell column; hypertension; postnatal maturation; autonomic nervous system

ADMINISTRATIONS OF BACLOFEN (Bac), a GABAB-receptor agonist, have been suggested as useful treatments for certain neurogenic disorders, such as epilepsy (32, 69), muscular spasticity (19, 37, 68), pain (56), and hypertension (6, 29). Although the reason why Bac is effective in alleviating these disorders is not clear, it is possible that Bac can correct the anomaly of GABAB-receptor-mediated neurotransmission underlying these disorders. For instance, an overexpression of GABAB receptor in the nucleus tractus solitarius of the spontaneously hypertensive rat is involved in the diminution of baroreflex sensitivity and the development of chronic hypertension (20, 77). Clinically, intrathecal Bac therapy is a popular and safe treatment for chronic pain and severe spinal spasticity (50, 56). However, Bac-induced effects on sympathetic regulation at the spinal level seem to receive little attention.

In animal experiments, along the hierarchy of the neural axis, it has been shown that GABAB receptors exert multifarious effects on sympathetic regulation. Administrations of Bac may reduce catecholamine or acetylcholine release by acting on the peripheral GABAB receptors that are located presynaptically on the post- or preganglionic sympathetic nerve terminals (3, 34, 48, 51). Intrathecal administration of Bac to the thoracic spinal cord decreases blood pressure and heart rate (33). However, intraperitoneal or intracerebroventricular administration of Bac leads to a general excitation of sympathetic activity (49, 53) or elicits biphasic pressor and depressor responses (64). At the brain level, focal applications of Bac inhibit the presympathetic neurons in the rostral ventrolateral medulla (39). There, GABAB receptors exert tonic inhibitory effect on sympathoexcitation (4). Similarly, at the spinal level, GABAB receptors are tonically active to inhibit the cardiovascular sympathetic preganglionic neuron (SPN) (28, 30). Although these in vivo studies had indicated a general GABAB-receptor-mediated reduction of central sympathetic outflow, the underlying mechanisms were largely unknown.

GABAB receptors are generally considered as one of the presynaptic elements that can reduce synaptic strength (2, 15, 16). The inhibitory effects of GABAB receptors on neurotransmission could have been coupled with their modulations of voltage-gated channel activities. Activation of GABAB receptors attenuates high-voltage-activated Ca2+ currents in a variety of neurons (8, 13, 21, 27, 70). Bac also enhances voltage-dependent or the inwardly rectifying K+ currents (26, 38, 57, 63). With regard to the sympathetic control at the spinal levels, there is evidence indicating a presynaptic action of GABAB receptors, which may attenuate excitatory synaptic inputs to SPNs (47, 75). However, there was no evidence indicating that, intrinsic to SPNs, GABAB receptors would regulate their voltage-gated channel activities.

Activities of SPNs are not driven only by the commands from the brain. An isolated spinal cord can generate substantial amounts of sympathetic activities (54, 58, 65, 71). Using a splanchnic nerve-spinal cord preparation, we have demonstrated that three or less thoracic spinal segments in vitro contain sufficient neural components to generate a sympathetic nerve discharge (SND) (62). The display of SND patterns in either tonic or bursting form is regulated by a spontaneous GABAergic activity acting on ionotropic GABAA receptors (60, 62). In the spinal cord of rats, metabotropic GABAB receptors are present (10, 14, 66). Thus it would be interesting to know whether the intraspinal GABAB receptors, being deprived from extraspinal synaptic inputs, were endogenously active to modulate sympathetic outflow.

In this study using an in vitro experimental model that had a better control of drug working concentrations, we aimed to determine GABAB-receptor-mediated effects on SND and the related ionic mechanisms underlying the regulation of sympathetic outflow from the thoracic spinal cord. We hypothesized that, similar to GABAA receptors, intraspinal GABAB receptors were endogenously active and would exert a tonic suppression of spinal SND. We also hypothesized that Bac-induced suppression of SND could be attributed to an attenuation of excitatory synaptic events to SPNs or a direct inhibition of SPN excitability due to an enhancement of K+ or a reduction of Ca2+ currents. Our data not only confirmed the presence of somatodendritic GABAB receptors on SPNs but also indicated complex ion events underlying Bac-induced SND suppression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
General procedures.   Neonatal Sprague-Dawley rats [postnatal (P) days 1–7] were used in this study, and all of the experimental protocols were approved by the Institutional Animal Care and Utilization Committee (protocol no. RRaIBMSC2003014). Methods in preparing a splanchnic nerve-thoracic spinal cord were modified from the procedures, as previously described (59, 62). Briefly, the nerve-cord preparation was fixed onto a recording chamber containing 8 ml 95% O2–5% CO2 equilibrated artificial cerebrospinal fluid (aCSF; in mM: 128 NaCl, 3 KCl, 1.5 CaCl2, 1.0 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, 30 D-glucose, and 3 ascorbate) and superfused at a rate of ~16 ml/min. A suction electrode placed on the proximal end of the splanchnic nerves was used to record compound action potentials, which were then amplified and filtered (WPI, DAM50; band pass: 0.1–1 kHz). All signals were stored on a pulse-code modulation tape recorder (Neuro-Corder, DR-890) for offline analysis. The amount of nerve discharge was measured by a time-based integrator (Gould, 13–4615-70; resetting every 5 s) to obtain total SND ( SND) or a leaky integrator (discharging time constant: 15 ms) to display the envelope of SND ({int} SND). The background level of neural recording was determined after a blockade of Ca2+-dependent neural activities by adding 24 mM Mg2+ to the bath solution. During experiments, the bath temperature was maintained at 24.5 ± 1°C.

Whole cell, patch-clamp recording.   The Bac-induced changes in electrophysiological properties of splanchnic SPNs were examined by using blind, whole cell, patch-clamp techniques. Patch pipettes were pulled from a borosilicate glass (AM-system, 6170) using a horizontal puller (P-97, Sutter Instrument, Navato, CA). The normal pipette solution contained (in mM) 145 potassium-gluconate, 5 NaCl, 10 EGTA, 1 CaCl2, 1 MgSO4, 4 ATP, and 10 HEPES (adjusted to pH 7.3 by 1 N KOH). When filled with normal pipette solution, the patch pipette had a resistance of 5–8 M{Omega}, with a liquid junction potential of ~8 mV. To examine Ca2+ currents in some experiments, 145 mM potassium-gluconate in the normal pipette solution was replaced with 135 mM cesium-gluconate and 10 mM TEA acetate to reduce K+ currents. Membrane currents or potentials were obtained with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) equipped with CV-203BU headstage, which was mounted on a three-dimensional micromanipulator. The patch pipette was advanced by steps (2 µm) into the tissue and positioned with a motion controller (PMC100, Newport), which also read the depth of pipette tip from the dorsal surface of the spinal cord. Signals were low-pass filtered at 5 kHz and processed by using a data-acquisition system (pClamp 6.0, Axon Instrument). The whole cell membrane capacitance (Cm) or recording access resistance was compensated or determined by the patch-clamp amplifier. In voltage-clamp mode, the waveform of current response to a 5-mV pulse was displayed on an oscilloscope to monitor a possible change in the access resistance. Only the results of experiments without an apparent alteration of access resistance were included in the analysis.

Antidromic stimulation of splanchnic SPN and measurements of electrical membrane properties.   To determine whether a spinal neuron was the SPN that had its axonal projection to the splanchnic nerves, the nerves were electrically stimulated by delivering square pulses (≤100 µA, 0.2 ms in duration, 0.5 Hz) to activate antidromic action potentials. A splanchnic SPN was then verified by using the following criteria: 1) the spinal neuron had an evoked spike with a fixed onset latency following the stimulation; and 2) the evoked spike was collided or disappeared when a spontaneous spike appeared within the propagation time of an antidromic action potential (Fig. 1). In practice, following a spontaneous spike, the antidromic onset latency was taken as a conservative estimate of the critical delay for electrical stimuli to activate antidromic spikes (40). In voltage-clamp mode with a holding potential of –60 mV (corrected for junction potential), we measured the electrical membrane properties, including whole cell membrane current (Im) and input resistance (Ri). The total resistance of whole cell, patch-clamp recording was determined by calculating the current responses elicited by a rectangular step pulse (–10 mV, 70 ms), whereas Ri was acquired by subtracting the recording access resistance from the total resistance.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Identification of a sympathetic preganglionic neuron (SPN) by collision tests. The neuron was recorded at a depth of 440 µm down the dorsal surface of the spinal cord. Arrow and dotted line indicate the time for delivering an electrical rectangular pulse to stimulate splanchnic nerves. Top: 5 traces are superimposed to demonstrate that a spike with constant onset latency (37.5 ms) is elicited with every stimulus. Bottom: 2 traces show that the spontaneous spikes appear around the time in delivering electrical pulse. Herein, the stimuli fail to elicit spikes.

 
Chemicals.   Bac (GABAB receptor agonist), saclofen hydrochloride (Sac; GABAB receptor antagonist), and CGP-46381 (CGP; another GABAB receptor antagonist) were purchased from Tocris, whereas glycine (Gly; glycine receptor agonist) and TTX (fast Na+ channel blocker) were from Sigma. Concentrated solutions (in mM: 1 Bac, 10 CGP, 200 Gly, 10 Sac, or 0.25 TTX) were prepared by dissolving drugs in water and stored at –20°C. The final concentration of drugs in the bath solution was obtained by adding an aliquot of concentrated solutions directly to the bath chamber. A cumulative concentration-response test was conducted by sequentially raising drug concentrations in the bath solution to determine drug sensitivity.

Data analysis.   True neural signals were obtained by subtracting the background noise from the recorded signals. The height of SND under control conditions was taken as 100% activity. Drug effects on neural signals were then calculated as percent changes from control activity. For simple evaluation of the drug effects, a two-tailed Student's t-test was used. The data from testing the concentration-dependent effects of Bac were analyzed with the aid of Origin (version 4.1) and fit with a sigmoidal Boltzman function, y = A2 + (A1 – A2)/{1 + exp[(x – x0)/dx]}, where A1 and A2 are the upper and lower asymptotes, respectively, x0 is the half-maximal response, and dx is the width. In practice, the SND before Bac application (100% activity) was set as a fixed asymptote for A1. The effectiveness of Sac or CGP to antagonize Bac effects was evaluated first with ANOVA followed by post hoc multiple comparison, using adjusted t-test, with P values corrected by Bonferroni procedure. All values are presented as means ± SE. A statistical probability (P) value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Age-dependent Bac effects on the reduction of spinal SND.   Effects of GABAB-receptor activation on spinal SND were investigated by bath applications of Bac (final concentration: 0.1–9.6 µM). The spinal SND was reduced by Bac in a concentration-dependent manner (Fig. 2). Although SND was always reduced, Bac concentration required to achieve a similar extent of inhibition varied substantially between experiments. Our preliminary observations indicated that Bac sensitivity was higher in younger neonates. In seven of the nine experiments (78%) using neonatal rats of younger ages (1–3 days old), a reduction of SND to <70% of the control activity only required Bac at a concentration ≤ 0.4 µM. In all experiments using older neonates (5–7 days old; n = 4), a comparable SND inhibition could only be achieved by 1.6 µM Bac. Another series of experiments using neonates of different ages was, therefore, conducted to clarify a change in Bac sensitivity during early postnatal stages. Figure 3 shows an age-dependent shift of the concentration-response curves to the right. In older neonates, both effectiveness and potency of Bac on SND inhibition were attenuated. To minimize variations between experiments, the neonatal rats of similar ages were carefully selected for the same tests in the following experiments.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 2. Baclofen (Bac)-induced reduction of panels in A and B sympathetic nerve discharge (SND). Bottom panels in A and B: SND at faster sweep speed. A: saclofen hydrochloride (Sac) pretreatment reversibly attenuates the Bac-induced reduction of SND. SND, integrated SND resetting every 5 s. Dashed line in middle panel indicates the noise level of recording. A rat of an age of postnatal day 4 (P4) was used in this experiment. Bac (final concentration: 0.8–6.4 µM) reduced SND in a concentration-dependent manner. In the presence of 100 µM Sac (open bar on top), the effect of 3.2 µM Bac on SND inhibition was markedly reduced. After washout, 3.2 µM Bac still reduced SND. The noise level of the neural recording was determined by adding 24 mM Mg2+. B: a time control to demonstrate that the thoracic cord in vitro generates a stable SND.

 


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. Age-dependent attenuation of the potency and effectiveness of Bac on SND inhibition. Bac was cumulatively applied to the bath solution to achieve final concentrations of 0.1–1.6 µM for P1, 0.1–3.2 µM for P3, and 0.4–9.6 µM for P5 rats (n = 5 for each age group). In each group, the reduction of SND in response to Bac was well described by a sigmoidal curve (r = 0.9843, 0.9672, and 0.9704 for P1, P3, and P5 group, respectively). The concentrations of Bac required to achieve half-maximal responses of SND inhibition for the age group of P1, P3, and P5 was estimated as 0.16, 0.34, and 2.67 µM, respectively. The effectiveness of Bac as revealed by the maximal response of SND inhibition was attenuated in an age-dependent manner. Values are means ± SE.

 
Antagonizing Bac-induced SND suppression by Sac or CGP.   To evaluate the specificity of Bac in activating GABAB receptors, Bac effects on SND suppression were challenged by adding Sac or CGP. Due to the age-dependent sensitivity to Bac, nerve cord preparations from neonates of various ages were treated with different Bac concentrations to produce a SND suppression to ~50% of the control activity. The selected Bac concentrations would allow a reversible response in repetitive drug applications (Fig. 2). Applications of 100 µM Sac or CGP alone did not change SND (Fig. 4, B and C). However, in the presence of Sac or CGP, Bac effects on SND suppression were significantly reduced (Fig. 4, A–C). The specificity of CGP in antagonizing Bac effects was further verified by examining whether CGP could reduce Gly-induced SND suppression. Applications of 6 mM Gly reduced SND to 27 ± 4% of control activity (n = 5). This Gly-induced SND suppression was not affected by the pretreatment of 100 µM CGP (Fig. 4D).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Applications of CGP-46381 (CGP) or Sac antagonize Bac-induced SND inhibition. A: 6.4 µM Bac reversibly reduces SND, an effect that is abolished by 100 µM CGP. Five rats (P6–7) were used in this series of tests. B: 100 µM CGP alone does not affect SND, but the pretreatment of CGP reduces the effect of 3.2 µM Bac on SND inhibition (n = 5, P3–4). C: 100 µM Sac alone does not affect SND, but it reduces the effect of 4.8 µM Bac on SND inhibition (n = 6, P4–5). D: the reduction of SND induced by 6 mM glycine (Gly) is not affected by the pretreatment of 100 µM CGP (n = 5, P1–2). ns, Not significant. Values are means ± SE. **P < 0.01, ***P < 0.001.

 
Bac effects on the spontaneous firing and membrane potential of splanchnic SPNs.   SPNs were recorded by using blind whole cell, patch-clamp techniques to elucidate the causes of Bac-induced SND suppression. Electrical stimulations of the splanchnic nerves successfully elicited antidromical action potentials with fixed onset latencies of ~22–56 ms in 44 spinal neurons (Fig. 1). Under extracellular recording conditions, most splanchnic SPNs (66%) were spontaneously active, with the average firing rates of ~0.1–2 Hz. Under voltage-clamp conditions, we determined passive membrane properties, including Cm and Ri, which varied substantially in different SPNs (Cm: 0.5–4.2 pF; Ri: 180–650 M{Omega}). Under current-clamp conditions, applications of Bac always reduced SND and SPN firing (n = 5). However, the reduction of SPN firing might not always be accompanied with a membrane hyperpolarization (n = 2). Prominent membrane hyperpolarizations (–6.5 ± 2.1 mV) were observed in three SPNs, an example shown in Fig. 5. Figure 5 also shows that application of Bac reduces the occurrence of excitatory postsynaptic potentials.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5. Bac-induced membrane hyperpolarization, diminution of depolarizing synaptic potentials, and reduction of spontaneous firing in a SPN. Top: slow traces showing that Bac reversibly caused a membrane hyperpolarization and reduced SPN firing. Bottom (a, b, c): fast traces expanding from the segment of data as indicated by labels in the top panel. a, Control; b, after Bac application; c, after washout. Action potentials here were truncated to reveal better the depolarizing synaptic potentials. Depolarizing synaptic potentials reversibly disappeared after Bac application (b).

 
Bac-induced changes in baseline Im and whole cell Ri.   Under voltage-clamp conditions, SPNs were held at –60 mV. A time series plot was constructed by sampling Im and Ri with a frequency of every 10 s to examine Bac-induced changes along the time course (Fig. 6). Bac was applied to the nerve cord preparation when its synaptic transmission was intact or when it was interrupted by adding 12 mM Mg2+ or 0.5 µM TTX to aCSF. Figure 7 summarizes the changes of Im and Ri elicited by applications of 5 µM Bac. Bath applications of Bac to the cord with intact synaptic transmissions elicited small outward baseline Im (7.6 ± 1.7 pA, P < 0.01 by t-test, n = 5); this effect persisted in the presence of 12 mM Mg2+ (16.0 ± 6.1 pA, P < 0.05, n = 6) or 0.5 µM TTX (15.9 ± 6.2 pA, P < 0.01, n = 7). In the presence of 0.5 µM TTX and 100 µM Cd2+, applications of Bac also elicited outward baseline Im (11.3 ± 3.2 pA, n = 3). Thus applications of 5 µM Bac consistently produced hyperpolarizing outward baseline currents in a total of 21 SPNs. The Bac effects on Ri, however, varied between SPNs. In all of the trials in which 12 mM Mg2+ were used to reduce synaptic transmission, applications of Bac slightly increased Ri. Statistically, the consistent Bac effects led to a small but significant increase of Ri (Fig. 7; 18 ± 6%, P < 0.05 by t-test). In the trials when the synaptic transmission remained intact or was reduced by TTX, applications of Bac did not change Ri in a consistent manner. Figure 6 shows that the occurrence of outward baseline Im elicited by Bac is largely not accompanied by a simultaneous alteration of Ri, even though Bac may induce a transient inward Im with a concomitant rise of Ri.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6. Time series plot of Bac-induced changes of whole cell membrane current (Im) and input resistance (Ri) in two SPNs. Experiments were conducted in the presence of 0.5 µM TTX. Vh, Holding potential. A: bath application of Bac induced an outward baseline current (~23 pA), but it did not cause a concurrent change in Ri. a1 and a2: Original traces showing changes of Im in response to –10-mV step pulses. Each trace was acquired by averaging 12 current responses as labels (top a1, a2) indicated. Except for a vertical shift of the baseline current after Bac application, the step current responses of a1 and a2 were not different. B: Bac-induced transient and steady-state responses in Im and Ri. Steady-state membrane responses in A and B were similar. However, the time series plot shows a transient inward Im concomitant with a transient rise of Ri. Superimposed traces (b1, b2) show a reduction of step-current response, indicating a transient rise of Ri (b2, single trace without averaging, shaded arrows). Traces of b1 and b3 (average of 6 current responses) were not different.

 


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7. Overall effects of Bac-induced changes in Im and Ri among SPNs with intact or interrupted synaptic transmission. Indifferent to the status of synaptic transmissions, application of 5 µM Bac consistently elicited outward baseline currents. In the presence of 12 mM Mg2+ to reduce Ca2+-dependent synaptic transmission, application of Bac significantly increased Ri (18 ± 6%). Values are means ± SE. Significant changes induced by Bac: *P < 0.05, **P < 0.01.

 
Bac-induced changes of whole cell, current-voltage relationships.   Previous studies have indicated that all of the SPNs innervating the adrenal medulla express a Ba2+/Cs+-sensitive and hyperpolarization-activated inwardly rectifying conductance (74). To decipher whether splanchnic SPNs had similar currents that might be enhanced by Bac, the whole cell, current-voltage (I-V) relationship was determined by measuring the current responses elicited by voltage pulses from –100 to –40 mV, with a step increment of 10 mV. Experiments were conducted in the presence of 0.5 µM TTX, using normal pipette solution, and without the leak subtraction of passive current responses. Figure 8 shows that the whole cell I-V curve is linear, indicating that this SPN does not have inwardly rectifying conductances being activated by hyperpolarization. In this series of experiments (n = 4), an application of 5 µM Bac always induced outward baseline Im (26 ± 7 pA), but it might either decrease (–19 ± 4%, n = 3) or increase (+22%, n = 1) the slope of I-V curves. The I-V curves for control and Bac converged at –29 ± 6 mV (n = 4), a potential that was very different from K+ equilibrium potential (EK = –99.7 mV). In Cs+-loaded SPNs with external 0.5 µM TTX, applications of 5 µM Bac still caused an outward baseline Im (19 ± 4 pA, n = 5), which was reversed by application of 100 µM CGP.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 8. Linear current-voltage (I-V) curves of a SPN before and after Bac application. Tests were conducted in the presence of 0.5 µM TTX with a holding potential of –70 mV. A: responses of Im without leak current subtractions to voltage command (Vc) from –100 to –40 mV (rectangular pulses: 400 ms, 10-mV increment). An application of 5 µM Bac induced an outward baseline current of 19 pA (a shift of Im from –40 to –21 pA after Bac). After Bac, the magnitudes of pulse-evoked Im responses were reduced. B: I-V curves before and after Bac. Insets indicate curve fitting with a linear equation, showing correlation coefficient (r) that approximates 1. After Bac application, the I-V curve declined by 27% in slope and was shifted to the left, leading to a converging point of the two straight lines at –35.6 mV.

 
Bac reversibly reduces Cd2+-sensitive and high-voltage-activated inward currents in Cs+-loaded SPNs.   Bac effects on high-voltage-activated Ca2+ current were examined in Cs+-loaded SPNs. To acquire high-voltage-activated Ca2+ currents, the passive current responses elicited by the voltage pulses were subtracted by using a P/4 protocol, i.e., 4 prepulses with amplitude and reversed polarity were given before the full-size test pulse. The voltage pulses depolarizing Cs+-loaded SPNs less than or equal to –20 mV always induced inward currents. Without any treatment, the run-down of high-voltage-activated inward currents was apparent along the time course (decaying rate: –4.4 ± 1.1 %/min, n = 5). Therefore, we examined the changes induced by drug applications in a time series plot, which was acquired by determining the voltage-gated currents in response to successive rectangular depolarizing voltage pulses (–10 mV, 76 ms, 0.1 Hz). As shown in Fig. 9, application of 5 µM Bac promptly reduced high-voltage-activated inward currents, which were reversed by adding 100 µM CGP. This high-voltage-activated inward current was abolished by 100 µM Cd2+. Using a trend line to correct the time-dependent, run-down, quantitative analysis revealed that Bac applications significantly reduced the inward currents by –30 ± 4% (P < 0.005, n = 5). For comparison, another group of SPNs was recorded by using normal pipette solution, and tests were conducted in the presence of 0.5 µM TTX and 100 µM Cd2+ (n = 5). Under these conditions, high-voltage pulses elicited outward currents. A time series plot revealed that the outward currents were not affected by applications of Bac or CGP (Fig. 10).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 9. Time series plot of Bac and CGP effects on the Cd2+-sensitive and high-voltage-activated Ca2+ current (ICa) in a Cs+-loaded SPN. Experiments were conducted in the presence of 0.5 µM TTX. Bars on top indicate applications of Bac, CGP, or Cd2+. Data were collected 5 min after achieving whole cell, patch-clamp configurations to allow diffusion of pipette Cs+ into the neuron. Holding potential was –60 mV. Consecutive rectangular test pulses (76 ms) were given every 10 s to depolarize membrane from holding potential to –10 mV. Along the time course of tests, each circle represents the peak ICa elicited by the test pulse with leak current subtraction by a P/4 protocol. Raw ICa traces labeled by 1–7 in middle insets are the average responses of 6 consecutive test pulses and correspond to the numbered data points in the time series plot. The plot and raw ICa traces indicate that the high-voltage-activated ICa gradually run down along the time course (1–2), drop abruptly after Bac application (2–3), recover substantially after addition of CGP (4–5), and are virtually abolished by Cd2+ (6–7). The I-V curves in the bottom right inset were obtained by applying test pulses to depolarize membrane to potentials between –35 and 25 mV (400 ms, 15-mV increments) at the timing, as solid symbols and arrows indicated in the time series plot.

 


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 10. Time series plot of high-voltage-activated outward currents (Iout) in a SPN. Bars on top indicate applications of Bac and CGP. Experiments were conducted by using normal pipette solution and in the presence of external 0.5 µM TTX and 100 µM Cd2+. Holding potential was –60 mV. Consecutive test pulses (76 ms) to depolarize membrane from holding potential to 30 mV were given every 10 s. Raw Iout traces in the bottom insets are responses with leak current subtraction by a P/4 protocol and correspond to numbered data points in the time series plot. Each circle in the time series plot represents the peak Iout elicited by the test pulse. Along the time course of tests, Iout was not changed by application of Bac or CGP.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
We have demonstrated that Bac activates intraspinal GABAB receptors and inhibits the spinally generated SND. Surprisingly, Bac sensitivity in SND suppression is age dependent, suggesting a functional change of intraspinal GABAB receptors in sympathetic regulation at early postnatal stages. The inhibition of SND could be attributed to both the diminution of excitatory synaptic potentials to SPNs and the elicitation of outward baseline currents that directly hyperpolarized SPNs themselves. The appearance of this hyperpolarizing current in SPNs seems to depend on a mixture of ion events that may not lead to a concomitant change in passive membrane properties. Another new finding is that activation of somatodendritic GABAB receptors on SPNs reduces high-voltage-activated Ca2+ currents. Our findings indicate that intraspinal GABAB-receptor activities suppress central sympathetic outflow through mechanisms both antecedent and intrinsic to SPNs.

Age-dependent sensitivity of Bac: a clinical implication.   The age difference of Bac sensitivity in SND inhibition suggests that GABAB receptors undergo postnatal changes. Molecular biological cloning of GABAB receptors has demonstrated the presence of different clones coding for GABAB-receptor subunits, namely, GABABR1a/b and GABABR2. The combination of these two clones to form a heterodimer (GABABR1 + GABABR2) is required to obtain full biological activity (11, 21, 72, 76). Developmentally, the expression of GABABR1a/b splice variants is undergoing a postnatal change, showing a shift from GABABR1a in neonatal to GABABR1b in adult stages (22). The functions of GABAB receptors also undergo postnatal development (7, 22, 41, 43, 67, 78). Intriguingly, the autonomic nervous system is not mature at birth (5, 25, 31). Thus, in terms of the sympathetic control at the spinal level, alterations of Bac sensitivity may occur, because the reconstitution of the receptor subunits can, in turn, affect the binding affinity of Bac (43) or even their coupling with G protein (1). Although we have not resolved the exact mechanisms, our results strongly suggest that a prominent change of spinal GABAB receptor plays a role in modulating the sympathetic outflow during postnatal maturation.

Our findings have some clinical implications. In our experiments, bath application of Bac to an in vitro spinal cord preparation simulates the regimen that uses intrathecal Bac to alleviate autonomic dysfunction (6). We observed that Bac in activating GABAB receptor consistently reduces SND. This observation clearly suggests an overall inhibition of the sympathetic outflow at the spinal level during intrathecal administration of Bac. However, the ability of Bac to inhibit SND largely depends on the age of neonates. The age-dependent Bac sensitivity in SND inhibition could not be due to the thickness of tissue as animals age. In one of our laboratory's previous studies, bath applications of N6-cyclopentyladenosine (CPA; adenosine A1-receptor agonist) also reduced SND (52). We did not find any age-dependent CPA effect as Bac induced. Besides, compared with CPA, Bac has a lower molecular weight (of Bac and CPA: 213.7 and 335.4, respectively), which presumably makes Bac a better permeate than CPA and allows Bac to sink into the tissue faster than CPA. Moreover, as shown in Fig. 2, the reduction of SND reached a plateau 3–5 min after Bac applications. Thus, given 15 min for each concentration test here, the age difference in Bac sensitivity is unlikely due to the lack of equilibrated time required for drug diffusion. Similar to our findings in age-dependent sympathetic suppression, Bac as an anticonvulsant has a distinct age-related difference in its effectiveness (69). The evidence suggests a necessity to carefully assess the dose that is required for a Bac regimen on younger individuals.

Activation of somatodendritic GABAB receptors elicited hyperpolarizing outward baseline currents without consistent changes in Ri.   There were discrepant observations with regard to a direct postsynaptic GABAB-receptor activity in SPNs. In CA3 hippocampal regions, it seems that only the presynaptic GABAB receptors function in early postnatal stages (24). Similar findings in the SPNs of immature rats (12–20 days old) also have reported that Bac only attenuates excitatory synaptic transmission to SPNs without affecting Ri, suggesting a lack of somatodendritic GABAB-receptor-mediated effects on SPNs (75). However, a recent study by Whyment et al. (73) indicates that applications of 50 µM Bac induce a dramatic decrease of Ri (~34%), which persists in the presence of TTX and is caused by an increase of K+ conductance (73). Under conditions with intact or interrupted synaptic transmissions, given a holding potential at –60 mV, we found that Bac consistently elicited hyperpolarizing outward baseline currents with inconsistent changes in Ri among SPNs. This inconsistency in Bac-induced changes of Ri has also been observed in the neurons located in the rostral ventrolateral medulla (39) or some of the SPNs in the thoracic spinal cord (See Fig. 5 in Ref. 73). Compared with observations from other studies, our results clearly indicate a heterogeneous response in different SPNs that may or may not reveal a discernible postsynaptic GABAB-receptor-mediated effect on Ri. We believe that the discrepancy of observations may be due to a complex ion mechanism underlying the hyperpolarizing outward baseline current. This might explain why activation of the somatodendritic GABAB receptors on SPNs did not elicit a concurrent change in Ri.

Compared with the inconsistent effects of Bac on Ri when the tests were conducted in the presence of 0.5 µM TTX, Bac consistently increased Ri when 12 mM Mg2+ was used to reduce Ca2+-dependent synaptic transmission. This observation implies that an activation of Ca2+-dependent channel activities by Bac, which can be blocked by the presence of high Mg2+, may contribute to a decrease of Ri in some Bac tests. On the other hand, Bac-induced outward baseline currents persisted in the presence of external TTX and Cd2+ or when most K+ currents were removed by intracellular Cs+ loading. These findings exclude the possible involvement of TTX-sensitive Na+, high-voltage-activated Ca2+, or leak K+ conductance in causing the hyperpolarizing outward baseline current.

Reduction of Cd2+-sensitive and high-voltage-activated Ca2+ currents by GABAB-receptor activation plays the key role in SND inhibition.   Activation of GABAB receptors in various neurons has been reported to activate inwardly rectifying K+ currents (23, 57), facilitate L-type but attenuate N-type Ca2+ currents (13, 36, 55), reduce low-voltage-activated Ca2+ currents (45), or cause an indirect suppression of Ca2+-dependent K+ currents (12, 46). All of these ion events may occur at either presynaptic nerve terminals or postsynaptic somatodendritic area to alter the neuronal excitability. In our studies, we did not find a discernible inwardly rectifying conductance when splanchnic SPNs were hyperpolarized between –80 and –100 mV (Fig. 8). In the SPNs that innervate the adrenal medulla, there is a hyperpolarization-activated inwardly rectifying current, which is sensitive to external Ba2+ or intracellular Cs+ and is reduced in K+-free aCSF (74). It was not clear, in our experimental conditions, why we did not find a likely current in splanchnic SPNs. Under physiological conditions, the functional operation of inwardly rectifying K+ conductances falls in a potential range between resting level and EK, lending a more hyperpolarized resting level than those neurons without such conductances (44). Compared with the resting potential of phrenic motoneurons at approximately –65 mV (61), the SPNs in our studies here, which were examined under the same experimental conditions as for phrenic motoneurons, had a fairly depolarized resting potential at approximately –50 mV. The resting potentials of those SPNs examined in thin slice preparations are –48.2 ± 1 mV (17). Thus the lack of hyperpolarization-activated inward rectification and the fairly depolarized resting potential did not indicate an existence of inwardly rectifying K+ conductance in splanchnic SPNs. In our studies, application of Bac still induced hyperpolarizing outward baseline current in Cs+-loaded splanchnic SPNs. Moreover, the I-V curves of control and after Bac were linear and did not converge toward EK. Taken together, our results did not support the notion that Bac reduced SND through an intrinsic mechanism of SPNs attributing to the inwardly rectifying K+ conductance. The possibility that Bac reduces SND by an enhancement of inwardly rectifying K+ conductances on the pre-SPN spinal neurons, however, cannot be excluded.

Applications of Bac to Cs+-loaded SPNs reduced the Cd2+-sensitive and high-voltage-activated inward currents. This result indicates a downregulation of high-voltage-activated Ca2+ channel activities by GABAB receptors. In contrast, with TTX and Cd2+ to reduce voltage-gated Na+ and Ca2+ currents, activation of GABAB receptors did not affect the high-voltage-activated outward current, thus excluding the involvement of delayed-rectified K+ current (Fig. 10). Bac effects on high-voltage-activated Ca2+ current may not be unique to SPNs. In three of three Cs+-loaded spinal neurons that did not respond to antidromic stimulation of splanchnic nerves, applications of 5 µM Bac reduced high-voltage-activated Ca2+ currents (data not shown). Thus it is likely that Bac exerts its effect on SND inhibition via a universal reduction of Ca2+ influx at multiple levels, including SPN, non-SPN, and even pre-SPN spinal neurons. Although how the diminution of high-voltage-activated Ca2+ influx into somatodendritic area of some spinal neurons could reduce SND was unknown, an attenuation of Ca2+ influx into the presynaptic nerve terminals apposing SPNs would decrease excitatory postsynaptic potentials, as we observed in Fig. 5. Previous studies have shown that SPNs receive prominent intraspinal GABAergic, glycinergic, and glutamatergic inputs (9, 18, 35, 42, 60). The release of these amino acid neurotransmitters might, therefore, be regulated by presynaptic GABAB receptors.

The source of tonic GABAB-receptor-mediated activity under in vivo conditions is extrinsic to the spinal cord.   Under the present in vitro conditions, we did not find significant effects of Sac or CGP. The lack of antagonistic effect suggests an absence of endogenous GABAB-receptor-mediated activity within the spinal cord. This observation is not consistent with those obtained from other in vivo studies that support the notion of a tonic GABAB-receptor-mediated activity at the spinal level (28, 30). The existence of spinal GABAergic activity is evident, because our laboratory has previously demonstrated that bicuculline or picrotoxin, by eliminating endogenous GABAA-receptor-mediated activities, induces a sympathetic output in bursting forms (60, 62). Thus the discrepancy of observations between our in vitro and other's in vivo studies cannot be attributed to the lack of endogenous GABAergic activity under the present in vitro conditions. It has been reported that the excitation of SPNs elicited from the dorsal root stimulation is suppressed by Bac (47). Therefore, the spontaneous GABAB-receptor activity may arise from the local spinal reflex under in vivo conditions. In contrast to the actions of intraspinal GABAA receptors that mainly affect the patterns, rather than the amounts of SND (60), activation of intraspinal GABAB receptors causes a general reduction of sympathetic outflow. Thus intraspinal GABAB receptors act as a simple target subserving the commands from the extraspinal GABAergic sources for sympathetic regulation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by the National Science Council of the Republic of China (NSC 90–2320-B-001–043 and NSC 92–2320-B-001–025).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We are grateful to Dr. C.-Y. Tang for insightful discussion, S.-L. Phoon for technical assistance, C. C.-J. Hsieh for editorial service, and M. Seah for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C.-K. Su, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (e-mail: csu{at}ibms.sinica.edu.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Al-Dahan MI and Thalmann RH. Effects of guanosine 5'-O-(3-thiophosphate) and calcium on {gamma}-aminobutyric acidB binding as a function of postnatal development. J Neurochem 52: 313–316, 1989.[CrossRef][ISI][Medline]
  2. Alford S, Christenson J, and Grillner S. Presynaptic GABAA and GABAB receptor-mediated phasic modulation in axons of spinal motor interneurons. Eur J Neurosci 3: 107–117, 1991.[CrossRef][ISI][Medline]
  3. Alkadhi KA, Salgado D, Davis CA, and Udofia EI. Effects of {gamma}-aminobutyric acid on the compound action potential of the rat superior cervical ganglion. Arch Int Pharmacodyn Ther 322: 66–79, 1993.[ISI][Medline]
  4. Avanzino GL, Ruggeri P, Blanchi D, Cogo CE, Ermirio R, and Weaver LC. GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists. Am J Physiol Heart Circ Physiol 266: H1722–H1728, 1994.[Abstract/Free Full Text]
  5. Bartolome J, Mills E, Lau C, and Slotkin TA. Maturation of sympathetic neurotransmission in the rat heart. V. Development of baroreceptor control of sympathetic tone. J Pharmacol Exp Ther 215: 596–600, 1980.[Free Full Text]
  6. Becker R, Benes L, Sure U, Hellwig D, and Bertalanffy H. Intrathecal baclofen alleviates autonomic dysfunction in severe brain injury. J Clin Neurosci 7: 316–319, 2000.[CrossRef][ISI][Medline]
  7. Ben-Ari Y, Tseeb V, Raggozzino D, Khazipov R, and Garoarsa JL. {gamma}-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life. Prog Brain Res 102: 261–273, 1994.[ISI][Medline]
  8. Bertrand S, Nouel D, Morin F, Nagy F, and Lacaille JC. Gabapentin actions on Kir3 currents and N-type Ca2+ channels via GABAB receptors in hippocampal pyramidal cells. Synapse 50: 95–109, 2003.[CrossRef][ISI][Medline]
  9. Bogan N, Mennone A, and Cabot JB. Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons. Brain Res 505: 257–270, 1989.[CrossRef][ISI][Medline]
  10. Bowery NG, Hudson AL, and Price GW. GABAA and GABAB receptor site distribution in the rat central nervous system. Neuroscience 20: 365–383, 1987.[CrossRef][ISI][Medline]
  11. Brauner-Osborne H and Krogsgaard-Larsen P. Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells. Br J Pharmacol 128: 1370–1374, 1999.[CrossRef][ISI][Medline]
  12. Cardozo DL and Bean BP. Voltage-dependent calcium channels in rat midbrain dopamine neurons: modulation by dopamine and GABAB receptors. J Neurophysiol 74: 1137–1148, 1995.[Abstract/Free Full Text]
  13. Carter TJ and Mynlieff M. {gamma}-Aminobutyric acid type B receptors facilitate L-type and attenuate N-type Ca2+ currents in isolated hippocampal neurons. J Neurosci Res 76: 323–333, 2004.[CrossRef][ISI][Medline]
  14. Castro AR, Pinto M, Lima D, and Tavares I. Nociceptive spinal neurons expressing NK1 and GABAB receptors are located in lamina I. Brain Res 1003: 77–85, 2004.[CrossRef][ISI][Medline]
  15. Chen G and van den Pol AN. Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons. J Neurosci 18: 1913–1922, 1998.[Abstract/Free Full Text]
  16. Delgado-Lezama R, Aguilar J, and Cueva-Rolon R. Synaptic strength between motoneurons and terminals of the dorso-lateral funiculus is regulated by GABA receptors in the turtle spinal cord. J Neurophysiol 91: 40–47, 2004.[Abstract/Free Full Text]
  17. Deuchars SA, Brooke RE, Frater B, and Deuchars J. Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit KV3.1. Neuroscience 106: 433–446, 2001.[CrossRef][ISI][Medline]
  18. Deuchars SA, Milligan CJ, Stornetta RL, and Deuchars J. GABAergic neurons in the central region of the spinal cord: a novel substrate for sympathetic inhibition. J Neurosci 25: 1063–1070, 2005.[Abstract/Free Full Text]
  19. Dressnandt J, Konstanzer A, Weinzieri FX, Pfab R, and Klingelhöfer J. Intrathecal baclofen in tetanus: four cases and a review of reported cases. Intensive Care Med 23: 896–902, 1997.[CrossRef][ISI][Medline]
  20. Durgam VR, Vitela M, and Mifflin SW. Enhanced {gamma}-aminobutyric acid-B receptor agonist responses and mRNA within the nucleus of the solitary tract in hypertension. Hypertension 33: 530–536, 1999.[Abstract/Free Full Text]
  21. Filippov AK, Couve A, Pangalos MN, Walsh FS, Brown DA, and Moss SJ. Heteromeric assembly of GABABR1 and GABABR2 receptor subunits inhibits Ca2+ current in sympathetic neurons. J Neurosci 20: 2867–2874, 2000.[Abstract/Free Full Text]
  22. Fritschy JM, Meskenaite V, Weinmann O, Honer M, Benke D, and Mohler H. GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Eur J Neurosci 11: 761–768, 1999.[CrossRef][ISI][Medline]
  23. Gähwiler BH and Brown DA. GABAB-receptor-activated K+ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures. Proc Natl Acad Sci USA 82: 1558–1562, 1985.[Abstract/Free Full Text]
  24. Gaiarsa JL, Tseeb V, and Ben-Ari Y. Postnatal development of pre- and postsynaptic GABAB-mediated inhibitions in the CA3 hippocampal region of the rat. J Neurophysiol 73: 246–255, 1995.[Abstract/Free Full Text]
  25. Gootman PM, Gootman N, and Buckley BJ. Maturation of central autonomic control of circulation. Fed Proc 42: 1648–1655, 1983.[ISI][Medline]
  26. Greif GJ, Sodickson DL, Bean BP, Neer EJ, and Mende U. Altered regulation of potassium and calcium channels by GABAB and adenosine receptors in hippocampal neurons from mice lacking G{alpha}o. J Neurophysiol 83: 1010–1018, 2000.[Abstract/Free Full Text]
  27. Harasztosi C, Forsythe ID, Szucs G, Stanfield PR, and Rusznak Z. Possible modulatory role of voltage-activated Ca2+ currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus. Brain Res 839: 109–119, 1999.[CrossRef][ISI][Medline]
  28. Hasséssian H, Prat A, De-Champlain J, and Couture R. Regulation of cardiovascular sympathetic neurons by substance P and {gamma}-aminobutyric acid in the rat spinal cord. Eur J Pharmacol 202: 51–60, 1991.[CrossRef][ISI][Medline]
  29. Hayakawa K, Kimura M, and Kamata K. Mechanism underlying {gamma}-aminobutyric acid-induced antihypertensive effect in spontaneously hypertensive rats. Eur J Pharmacol 438: 107–113, 2002.[CrossRef][ISI][Medline]
  30. Hong Y and Henry JL. Cardiovascular responses to intrathecal administration of L- and D-baclofen in the rat. Eur J Pharmacol 192: 55–62, 1991.[CrossRef][ISI][Medline]
  31. Kasparov S and Paton JFR. Changes in baroreceptor vagal reflex performance in the developing rat. Pflügers Arch 434: 438–444, 1997.[CrossRef][ISI][Medline]
  32. Kerr DIB and Ong J. GABAB receptors. Pharmacol Ther 67: 187–246, 1995.[CrossRef][ISI][Medline]
  33. Kim B, Koh H, Kang J, Lee H, Shin I, Om S, and Kang J. Mediation of the cardiovascular response to spinal {gamma}-aminobutyric acidB receptor stimulation by adenosine A1 receptors in anesthetized rats. Neurosci Lett 296: 153–157, 2000.[CrossRef][ISI][Medline]
  34. Kohlenbach A and Schlicker E. GABAB receptor-mediated inhibition of the neurogenic vasopressor response in the pithed rat. Br J Pharmacol 100: 365–369, 1990.[ISI][Medline]
  35. Krupp J, Bordey A, and Feltz P. Electrophysiological evidence for multiple glycinergic inputs to neonatal rat sympathetic preganglionic neurons in vitro. Eur J Neurosci 9: 1711–1719, 1997.[CrossRef][ISI][Medline]
  36. Lambert NA and Wilson WA. High-threshold Ca2+ currents in rat hippocampal interneurones and their selective inhibition by activation of GABAB receptors. J Physiol 492: 115–127, 1996.[Abstract/Free Full Text]
  37. Lazorthes Y, Sallerin-Caute B, Verdie JC, Bastide R, and Carillo JP. Chronic intrathecal baclofen administration for control of severe spasticity. J Neurosurg 72: 393–402, 1990.[ISI][Medline]
  38. Li YW and Guyenet PG. Activation of GABAB receptors increases a potassium conductance in rat bulbospinal neurons of the C1 area. Am J Physiol Regul Integr Comp Physiol 271: R1304–R1310, 1996.[Abstract/Free Full Text]
  39. Li YW and Guyenet PG. Neuronal inhibition by a GABAB receptor agonist in the rostral ventrolateral medulla of the rat. Am J Physiol Regul Integr Comp Physiol 268: R428–R437, 1995.[Abstract/Free Full Text]
  40. Lipski J. Antidromic activation of neurones as an analytic tool in the study of the central nervous system. J Neurosci Methods 4: 1–32, 1981.[CrossRef][ISI][Medline]
  41. Liu Q and Wong-Riley MTT. Postnatal expression of neurotransmitters, receptors, and cytochrome oxidase in the rat pre-Bötzinger complex. J Appl Physiol 92: 923–934, 2002.[Abstract/Free Full Text]
  42. Llewellyn-Smith IJ, Cassam AK, Krenz NR, Krassioukov AV, and Weaver LC. Glutamate- and GABA-immunoreactive synapses on sympathetic preganglionic neurons caudal to spinal cord transection in rats. Neuroscience 80: 1225–1235, 1997.[CrossRef][ISI][Medline]
  43. Maltischek B, Ruegg D, Heid J, Kaupann K, Bittiger H, Frostl W, Bettler B, and Kuhn R. Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain. Mol Cell Neurosci 12: 56–64, 1998.[CrossRef][ISI][Medline]
  44. Marcotti W, Johnson SL, Holley MC, and Kros CJ. Developmental changes in the expression of potassium currents of embryonic, neonatal and mature mouse inner hair cells. J Physiol 548: 383–400, 2003.[Abstract/Free Full Text]
  45. Matsushima T, Tegner J, Hill RH, and Grillner S. GABAB receptor activation causes a depression of low- and high-voltage-activated Ca2+ currents, postinhibitory rebound, and postspike afterhyperpolarization in lamprey neurons. J Neurophysiol 70: 2606–2619, 1993.[Abstract/Free Full Text]
  46. Matzel LD, Muzzio IA, and Rogers RF. Diverse current and voltage responses to baclofen in an identified molluscan photoreceptor. J Neurophysiol 74: 506–518, 1995.[Abstract/Free Full Text]
  47. McKenna KE and Schramm LP. Baclofen inhibits sympathetic preganglionic neurons in an isolated spinal cord preparation. Neurosci Lett 47: 85–88, 1984.[CrossRef][ISI][Medline]
  48. Miranda FJ, Torregrosa G, Salom JB, Campos V, Alabadi JA, and Alborch E. Inhibitory effect of GABA on cerebrovascular sympathetic neurotransmission. Brain Res 492: 45–52, 1989.[CrossRef][ISI][Medline]
  49. Nonogaki K, Mizuno K, Sakamoto N, and Iguchi A. Effects of central GABA receptors activation on catecholamine secretion in rats. Life Sci 55: PL239–PL243, 1994.[CrossRef][ISI][Medline]
  50. Ochs GA. Intrathecal baclofen. Baillieres Clin Neurol 2: 73–86, 1993.[ISI][Medline]
  51. Parkman HP, Stapelfeldt WH, Williams CL, Lennon VA, and Szurszewski JH. Enteric GABA-containing nerves projecting to the guinea-pig inferior mesenteric ganglion modulate acetylcholine release. J Physiol 471: 191–207, 1993.[Abstract/Free Full Text]
  52. Peng SC, Ho CM, Ho ST, Tsai SK, and Su CK. The role of intraspinal adenosine receptors in sympathetic regulation. Eur J Pharmacol 492: 49–55, 2004.[CrossRef][ISI][Medline]
  53. Persson B and Henning M. Central cardiovascular and biochemical effects of baclofen in the conscious rat. J Pharm Pharmacol 32: 417–422, 1980.[ISI][Medline]
  54. Shen E, Wu SY, and Dun NJ. Spontaneous and transmitter-induced rhythmic activity in neonatal rat sympathetic preganglionic neurons in vitro. J Neurophysiol 71: 1197–1205, 1994.[Abstract/Free Full Text]
  55. Shen W and Slaughter MM. Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons. J Physiol 516: 711–718, 1999.[Abstract/Free Full Text]
  56. Slonimski M, Abram SE, and Zuniga RE. Intrathecal baclofen in pain management. Reg Anesth 29: 269–276, 2004.
  57. Slugg RM, Zheng SX, Fang Y, Kelly MJ, and Rønnekleiv OK. Baclofen inhibits guinea pig magnocellular neurones via activation of an inwardly rectifying K+ conductance. J Physiol 551.1: 295–308, 2003.
  58. Spanswick D and Logan SD. Spontaneous rhythmic activity in the intermediolateral cell nucleus of the neonate rat thoracolumbar spinal cord in vitro. Neuroscience 39: 395–403, 1990.[CrossRef][ISI][Medline]
  59. Su CK. Rhythmic sympathetic nerve discharges in an in vitro neonatal rat brain stem-spinal cord preparation. J Appl Physiol 87: 1066–1075, 1999.[Abstract/Free Full Text]
  60. Su CK. Intraspinal amino acid neurotransmitter activities are involved in the generation of rhythmic sympathetic nerve discharge in newborn rat spinal cord. Brain Res 904: 112–125, 2001.[CrossRef][ISI][Medline]
  61. Su CK, Mellen NM, and Feldman JL. Intrinsic and extrinsic factors affecting phrenic motoneuronal excitability in neonatal rats. Brain Res 774: 62–68, 1997.[CrossRef][ISI][Medline]
  62. Su CK, Phoon SL, and Yen CT. Identification of active thoracic spinal segments responsible for tonic and bursting sympathetic discharge in neonatal rats. Brain Res 966: 288–299, 2003.[CrossRef][ISI][Medline]
  63. Takeda M, Tanimoto T, Ikeda M, Kadoi J, and Matsumoto S. Activation of GABAB receptors inhibits the excitability of rat small diameter trigeminal root ganglion neurons. Neuroscience 123: 491–505, 2004.[CrossRef]