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Bicuculline dialysis in the retrotrapezoid nucleus (RTN) region stimulates breathing in the awake rat
Respiration Physiology 124 (2001) 179– 193 www.elsevier.com/locate/resphysiol
Bicuculline dialysis in the retrotrapezoid nucleus (RTN) region stimulates breathing in the awake rat Eugene Nattie *, Jing Shi, Aihua Li Department of Physiology, Borwell Building, Dartmouth Medical School, Lebanon, NH 03756 -0001, USA Accepted 5 October 2000
Abstract Muscimol dialysis in the retrotrapezoid nucleus (RTN) region of awake rats reduces tidal volume during air breathing and decreases chemoreception (Nattie, Li, 2000. J. Appl. Physiol., 89, 153– 162). Is there an endogenous GABAergic inhibition of the RTN as for medullary respiratory and pressor neurons? Bicuculline microdialysis (30 min; 1 mM) into the RTN region of awake rats reversibly increased tidal volume by 11 – 16% over the period from 10 to 60 min (PB0.01; six rats). Ventilation increased but this was significant (P B 0.05) only at 5, 20, and 25 min as frequency tended to decrease during dialysis. The ventilatory response to 7% CO2 was unaffected (six rats); dialysis of vehicle alone over 4 h had no effect (five rats). It was concluded that in the awake rat there is ongoing endogenous modulation of RTN effects on tidal volume by a GABAergic process of unknown origin. The lack of effect on the response to systemic hypercapnia suggests that the RTN provides an ongoing endogenous drive to respiration by a process that is independent of its role in chemoreception. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chemosensitivity, central, GABAA receptors; Control of breathing, NTS, central chemosensitivity; Mammals, rat; Medulla, rostral ventral, chemosensitivity; Pattern of breathing, hyperventilation; Receptors, GABAA
1. Introduction While the major clusters of medullary neurons involved in the control of breathing are in the dorsal and ventral respiratory groups, the rostral ventrolateral medulla (RVLM) is a site of central chemoreception and provides an ongoing endogenous drive for respiration. In anesthetized animals, acidic stimulation of the medullary surface * Corresponding author. Tel.: +1-603-6507726; fax: +1603-6506130. E-mail address: [email protected] (E. Nattie).
at the RVLM increases respiratory output (see Bruce and Cherniack, 1987; Nattie, 1999 for references) as does focal acidification of a portion of the RVLM, the retrotrapezoid nucleus (RTN) (Coates et al., 1993; Li and Nattie, 1997). The operational definition of the RTN region includes the RTN proper, described by retrograde tracing experiments as lying ventral to the facial nucleus (Smith et al., 1989), together with adjacent neurons of the parapyramidal region and the juxtafacial portion of the nucleus paragigantocellularis lateralis (PGCL). Surface cooling at the RVLM produces apnea and virtually abolishes CO2 sensi-
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tivity in anesthetized animals (Bruce and Cherniack, 1987) with focal cooling by a small probe identifying the most sensitive site as the RTN region (Budzinska et al., 1985). Focal destruction of the RTN region by neurotoxin injection (Nattie et al., 1991; Nattie and Li, 1994; Nattie, 1999) also substantially reduces baseline respiratory output and the response to systemic hypercapnia. The importance of the endogenous drive and chemoreceptor functions of the RTN region in the absence of anesthesia is less certain. Large bilateral RVLM lesions, including the RTN, produced by electro-coagulation in the cat results in hypoventilation and a markedly diminished response to hypercapnia (Schla¨fke, 1979). In the unanesthetized goat (Ohtake et al., 1995; Forster et al., 1997), cooling of the RVLM, including the RTN region, decreases ventilation and the response to hypercapnia in the waking state but the effects are smaller than those observed with cooling of the same location under anesthesia. Awake rats, with histologically proven RTN lesions produced by prior neurotoxin injection, breathe normally at rest but have a 39% reduction in the response to systemic hypercapnia (Akilesh et al., 1997). Dialysis of the GABA-A receptor agonist, muscimol, in awake rats (Nattie and Li, 2000) decreases tidal volume and ventilation reversibly during air breathing and reduces the response to systemic hypercapnia. The size of the region affected is likely to be substantially smaller in these rat studies than in the cat or goat experiments. RVLM pressor neurons, located nearby those involved in the control of breathing, are under tonic, endogenous GABAergic inhibition that originates from the caudal ventrolateral medulla (CVLM) (Blessing and Reis, 1983; Guyenet et al., 1987). These CVLM neurons are in turn under endogenous GABAergic inhibition from unknown sites (Blessing and Reis, 1983). Some respiratory premotor neurons in the ventral respiratory group are also under tonic, endogenous GABAergic inhibition (McCrimmon et al., 1997). The purpose of this study is to evaluate whether neurons in the RTN region are also under ongoing, endogenous GABAergic inhibition. The model is an unanesthetized rat with the GABAA receptor antagonist, bicuculline, applied to the RTN region by micro-
dialysis to focally, acutely, and reversibly alter function.
2. Methods
2.1. General preparation The experiments were approved by the Institutional Animal Care and Use Committee of Dartmouth Medical School. The general methods have been recently described in detail (Nattie and Li, 2000). In brief, Sprague –Dawley rats of 300 –400 g were anesthetized with intramuscular Ketamine (100 mg/kg) and intraperitoneal Xylazine (20 mg/ kg). A guide tube (0.38 mm OD) with a dummy cannula was inserted such that the tip was just above the RTN region and secured to the skull and a sterile telemetric temperature probe was placed unattached in the abdominal cavity. The animal was allowed to recover for 3–4 days.
2.2. Protocol On each experimental day the rat would be weighed, the dummy cannula removed and the dialysis probe inserted into the guide tube. The probe tip, which protruded 1 mm beyond the guide tube tip, was 1 mm in length and 0.24 mm in diameter with membrane pores limiting movement to molecules under 6000 Da. Animals were placed into a Pappenheimer-style plethysmograph chamber (Pappenheimer, 1977; Cream et al., 1999) and allowed 30–40 min to acclimate. Ventilation (V: E), oxygen consumption (V: O ) and body temperature (TB) were measured. After acclimation, the dialysis pump was turned on with aCSF flowing at a rate of 4.0 ml/min. Dialysis continued through the entire experimental protocol in all groups. Fast green dye was added to the dialysate to facilitate visualization of the dialysate outflow and to allow a histological test for membrane leak. The membrane was examined just after each dialysis. The outflow rate was monitored semiquantitatively by visualization of the drops of dialysate coming out of the outflow tubing. Animals in this report did not have leaks in the dialysis membrane during the experiment. The 2
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rats were studied in quiet wakefulness between 09:00 and 16:00 h. They were housed on a midnight to noon circadian light/dark cycle so the data collection was during the end of the quiet/ sleep (light) circadian period and the beginning of the active/wake (dark) circadian period. This yielded reproducible data representing quiet wakefulness. Later in the active/wake period, the rats are prone to sniffing, grooming and active movement making the ventilatory data more difficult to obtain. The rats were under constant observation during the experiments, which were performed in subdued light
2.3. Experimental groups There were three experimental groups. In each, some rats received a treatment more than once. For data presentation and statistical analysis multiple trials in any rat are averaged. Group 1, a control (five rats), received three treatments. First, the dummy probe was left in place and measurements obtained every 15 min over 4 h. Second, the dummy was replaced by the dialysis probe but without any flow of dialysate and measurements obtained every 15 min over 4 h. Third, aCSF was dialyzed continuously through the 4 h period with measurements obtained every 15 min. Each rat received all treatments each on a separate day. Group 2 (six rats) tested the effects of bicuculline dialysis into the RTN region on breathing room air during wakefulness. Four sets of baseline measurements were repeated over 40 min then the dialysate was switched to 1 mM bicuculline. The time at which the new dialysate reached the tissue (wash-out of the dead space accounted for) is defined as time= 0. Measurements were obtained every 5 min during the 30 min dialysis period then every 5 min over a 60 min recovery period with the dialysate switched to aCSF. Group 3 (six rats) examined the effect of bicuculline dialysis on the response to 7% CO2. The inspired CO2 was increased by switching the inflow gas to a mixture with 7% CO2 and 93% air, which slowly increased the inspired CO2 value over 8 – 9 min, as measured in the outflow gas. Ventilatory variables from 8 to 9 and from 9 to 10 min were measured. Four sets of air breathing data were obtained over 20 min
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followed by exposure to hypercapnia then recovery. This was repeated three times in each protocol. Each rat received two treatment regimes. In one, aCSF was dialyzed throughout the experiment. In the other, after obtaining the initial CO2 response, the dialysate was switched to 1 mM bicuculline in aCSF for 30 min followed by a return to aCSF alone for the remainder of the experiment.
2.4. Data analysis and statistics Analog respiratory data were digitized (DataPac III) and a breath by breath analysis performed with the pressure deflections and the respiratory cycle time for each breath being determined over a 20–30 sec time period (DataPac III). VT (per 100 g of body weight), f, and V: E (per 100 g body weight) were calculated (SigmaPlot IV). Oxygen consumption was determined by the difference in outflow oxygen, determined by the O2 sensor, and inflow oxygen; carbon dioxide production by the difference between inflow carbon dioxide (assumed to be 0) and outflow carbon dioxide measured by infrared analyzer. The flow rate of gas through the plethysmograph was constant at 1.4 l/min. The results for V: E, VT, f, V: O , V: CO and TB in room air or 7% CO2 breathing were compared within each experimental group using a one-way repeated measures ANOVA. In some cases, the Friedman repeated measures ANOVA on ranks was required. Post-hoc tests (Dunnett’s; Student Newman –Keuls) were performed when significant differences were found. The air breathing ventilatory data (Groups 1 and 2) are evaluated in two ways. First, the absolute values of V: E, VT, and f are compared over the entire experiment. Second, these data are expressed as percent baseline, which was determined as the mean of four sets of measurements made over 20 min prior to the onset of dialysis. This allowed normalization for differences in initial values among the groups. The CO2 breathing data are evaluated in two ways. First, the absolute values of V: E, VT, and f are compared (one-way ANOVA) for the three CO2 tests before, during, and after each dialysis treatment (aCSF, 1 mM bicuculline). Second, the 2
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change in these values, calculated as the CO2 stimulated value at each CO2 challenge minus the average of the control values just before and after, are compared for the three CO2 tests before, during, and after dialysis treatment (one-way ANOVA).
2.5. Anatomy At the conclusion of the experiments, the rats were killed by an overdose of pentobarbital administered intraperitoneally after an initial sedative dose of ketamine (100 mg/kg) administered intramuscularly. The brainstems were removed and frozen immediately, sectioned (50 mm thickness), and stained with cresyl violet to identify anatomical locations of the probe tips with the assistance of an atlas (Paxinos and Watson, 1986).
3. Results
3.1. Breathing air; quiet wakefulness, control The mean (S.E.M.) absolute values of V: E, VT, and f over the entire 4 h experiment in Group 1 are shown in Fig. 1, those for V: O , V: CO , and TB in Fig. 2. The three treatments: (a) no probe in place (open triangle); (b) probe in but no dialysis (open circle); and (c) probe in with continuous dialysis of aCSF (solid circle) had no significant effect on these variables. 2
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3.2. Unilateral RTN dialysis with 1 mM bicuculline; effects on 6entilation Absolute VT, and VT expressed as per cent baseline, were significantly increased during and just after dialysis of the RTN region with 1 mM bicuculline (Figs. 3 and 4) (P B 0.001, one-way repeated measures ANOVA; P B0.05 for all times during and after dialysis compared to control except 5, and 60–90 min, Dunnett’s test). Frequency was not significantly affected (P = 0.09 one-way ANOVA). There was an apparent initial increase in frequency at 5 min of dialysis but during the bicuculline dialysis frequency tended to decrease as seen most easily on Fig. 4. Ventilation
is increased during bicuculline dialysis as shown in Figs. 3 and 4 but this does not reach significance if all data are used in the test. If the analysis is limited to 60 min after the onset of dialysis, then V: E is increased (P B0.05, one-way ANOVA; 5, 20, and 25 min points, PB 0.05 Dunnett’s test). Metabolic rate and TB were not significantly affected by bicuculline dialysis (Fig. 5).
3.3. Unilateral RTN dialysis with 1 mM bicuculline; effects on response to CO2 With aCSF treatment (data not shown) neither the absolute values of CO2 stimulated V: E, VT, and f nor the change in these values, comparing CO2 stimulated to the average of pre- and postCO2 baseline values, differed significantly among the three dialysis tests. Metabolic rate and TB were also unchanged during this experiment (data not shown). With 1 mM bicuculline treatment (Fig. 6), neither the absolute values of CO2 stimulated V: E, VT, and f nor the change in these values, comparing CO2 stimulated to the average of pre- and post-CO2 baseline values, differed significantly among the three dialysis tests. Absolute VT was increased by 1 mM bicuculline both during and after treatment as shown in Figs. 3 and 4. TB and V: CO were unaffected by this protocol but V: O did increase following bicuculline dialysis and the second CO2 test (PB 0.001; one-way repeated measures ANOVA; PB0.05 Dunnett’s test for 60–90 min values; data not shown). 2
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3.4. Anatomical location of dialysis probes Fig. 7 shows computer modified digitized 2× images of cresyl violet stained medullary cross sections at the approximate center of the tissue disruption at the site of the dialysis probe tip for group 2 animals, which received the 1 mM bicuculline without hypercapnia. The rectangles drawn on the Fig. show the location of the probe tips. In each case, the probe tip was in the region of the RTN. The results (not shown) are similar for the other two groups.
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Fig. 1. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time during the 4 h duration of the control group experiment (five rats) with (a) no dialysis probe in place (), (b) dialysis probe inserted but no dialysis (), and (c) continuous dialysis of aCSF ( ).
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Fig. 2. Like Fig. 1 but showing V: O , V: CO , and TB results. 2
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Fig. 3. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time during the group 2 experiment (six rats) with initial dialysis with aCSF (dotted line) followed by 30 min period of dialysis with 1 mM bicuculline (solid line) then aCSF dialysis for the remainder (dotted line).
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Fig. 4. Mean (S.E.M.) values for V: E, VT, and f, expressed as percent of initial baseline, are shown as a function of time during the group 2 experiment with initial dialysis with aCSF (dotted line) followed by 30 min period of dialysis with 1 mM bicuculline (solid line) then aCSF dialysis for the remainder (dotted line).
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Fig. 5. V: O , V: CO , and TB results for the group 2 experiment. 2
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Fig. 6. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time for the group 3 bicuculline experiments (six rats). The lower values connected by solid line represent air breathing data. The higher values shown at three times represent the CO2 breathing data.
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Fig. 7. Cresyl violet stained cross section of the medulla of the six rats in group 2 taken at the center of the dialysis probe location. The numbers at lower left of each section refer to mm from bregma. The bar is 1 mm. VII is facial nucleus. The rectangle represents in each section the site at which tissue damage identified the location of the dialysis probe tip.
4. Discussion
4.1. Limitations of technique Microdialysis has been successfully used to deliver neuroactive substances (TRH, muscimol, CO2) to the RTN region of the medulla in the conscious rat (Cream et al., 1999; Li et al., 1999; Nattie and Li, 2000). A concern about every technique used to deliver neuroactive substances into the brain is the degree of spread within the tissue. For CO2, tissue pH has been measured at different distances from the dialysis probe, but so far only under anesthesia (Nattie and Li, 1996; Li et al., 1999). These data indicate the change in tissue pH is confined to within 500 mm of the probe tip when the CO2 in the dialysate is sufficient to cause a 15% increase in ventilation (Li et al., 1999). For TRH, muscimol, and bicuculline it is more difficult to measure the exact tissue region affected. However, drug spread has been esti-
mated using data from the literature and from the authors’ studies of the spread via dialysis of fluorescein, which has a molecular weight (332) similar to muscimol (195), bicuculline (509) and TRH (362) (see Nattie and Li, 2000). Dialysis of glutamate (Alessandri et al., 1996) at 10–1000 mM and 2.0 ml/min for 30 min showed a 35% delivery into the tissue. For the 1 mM bicuculline dialysis this would result in a maximum concentration of 0.35 mM outside the probe. The use of two probes, one for delivery, the other for collection and measurement, showed a similar finding, steady-state concentrations 1.5 mm from the delivery probe that were 10–100 times lower than in the dialysate (de Lange et al., 1995). From the literature, it appears that a considerable concentration difference of neuroactive substance exists between the inside and the outside of the dialysis probe. The use of a fluorescein marker with 1 mM concentration in the probe showed an average
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(N =5) rostral to caudal spread of 870 mm and an average radius at the cross section with the largest area of 555 mm (Cream et al., 1999). The estimated volume of distribution was 590 nl, which is 49% of the approximate volume of the RTN region (1.2 ml). From these data, it was estimated that with dialysis of 1 mM bicuculline the tissue distribution is likely to be within 500 – 900 mm of the probe tip and the concentration at the probe is likely to be 0.35 mM or less. Another concern with microdialysis is possible wash-out of neuroactive substances that might influence breathing. The control group in this experiment was designed to evaluate this concern and to show the variation in ventilatory data when obtained in the protocol over 4 h. Ventilation, VT, f, V: O , V: CO , and TB were remarkably stable over 4 h of constant dialysis with aCSF as well as over 4 h with no probe in place or with the probe in place but without any dialysis. While it seems likely that the aCSF does dialyze a variety of substances out of the RTN region this does not seem to have any detectable effect on ventilation. 2
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4.2. Effects of bicuculline dialysis in the RTN on breathing The major finding in this study is that dialysis of bicuculline at the 1 mM dose into the RTN region of the conscious rat increases VT reversibly without effect on metabolic rate or TB. The effects on V: E are complicated by the observation that f appears to increase at 5 min of dialysis then tends to decrease during the dialysis, although these changes do not reach statistical significance. The result is a significant increase in V: E at 5, 20 and 25 min. Nevertheless, the increase in VT is striking and of interest from two viewpoints. (1) It appears to be the opposite response to that described with RTN dialysis with muscimol. (2) It is consonant with other published data that suggest the presence of a tonic GABAergic inhibition of RVLM pressor neurons and respiratory neurons of the VRG and DRG but differs from results obtained with bicuculline microinjection into the preBo¨tzinger complex.
4.3. Comparison of bicuculline and muscimol effects Dialysis of muscimol (1 mM; 30 min) into the RTN region of the awake rat, using a protocol similar to that in this study, decreased VT by 16–17% (Nattie and Li, 2000). Ventilation decreased, initially, then returned to within the baseline range of values due to a subtle increase in f. With dialysis of bicuculline (1 mM; 30 min) into the RTN region, the pattern of responses is a mirror image of those obtained with muscimol. Tidal volume is increased consistently during and shortly after the period of dialysis, V: E is increased significantly at 5, 20 and 25 min the variability of this response being attributed to the tendency for f to decrease. The presence of other chemoreceptor sites both centrally (Coates et al., 1993; Nattie and Li, 1994; Li and Nattie, 1997; Nattie and Li, 1996; Li et al., 1999; Nattie, 1999) and in the periphery and the use of an animal model with a ‘closed-loop’ control system complicates the interpretation of these data. In either case, muscimol or bicuculline, the final level of V: E would reflect a balance of inhibition/disinhibition in the RTN region and hyper/hypocapnic induced excitation/ inhibition at other chemoreceptor locations. The use of a conscious rat with a ‘closed-loop’ control system to examine focal effects in the RTN may disguise the overall importance of neurons in the RTN region in the control of breathing. However, this type of experiment demonstrates the significance of having many central chemoreceptors and their importance in the intact, conscious animal.
4.4. Endogenous GABAergic inhibition Pre-sympathetic vasomotor neurons in the RVLM are under GABAergic inhibition, in part related to the baroreceptor reflex, via the caudal ventrolateral medulla (CVLM; Yamada et al., 1984; Guyenet et al., 1987) and in part due to non-baroreceptor related inhibition from the CVLM (Sun and Guyenet, 1985). Bicuculline application to the RVLM produces a pressor response (Sun and Guyenet, 1985; Amano and Kubo, 1993). The CVLM neurons themselves are
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also under tonic GABAergic inhibition from unknown sources (Blessing and Reis, 1983). Effects of bicuculline application on breathing can be classified into types of responses; (1) changes in rhythm; and (2) increases in respiratory drive. Focal application of bicuculline into the preBo¨tzinger region bilaterally in anesthetized cats slows frequency and results in an apneustic pattern (Pierrefiche et al., 1998). Similar effects have been reported following bicuculline injections into the ventrolateral medulla of anesthetized 4 – 8-weekold opossums (Farber, 1995). In contrast, systemic administration of bicuculline, or the equivalent, increased respiratory output in anesthetized kittens (Sica et al., 1993), anesthetized rabbits (Delpierre et al., 1996), and in an arterially perfused in situ brainstem-spinal cord preparation of the adult rat (Hayashi and Lipski, 1992). Further, microinjection of bicuculline on some inspiratory and expiratory premotor neurons of the ventral respiratory group that project to the spinal cord of anesthetized dogs amplified their discharge pattern in a multiplicative manner (McCrimmon et al., 1997). This effect was termed ‘gain modulation’ and while the source of the endogenous GABAergic input is as yet undefined the concept is that many respiratory neurons, and the respiratory system output, is under some type of ongoing endogenous GABAergic modulation in resting conditions. The results support this concept of an ongoing endogenous GABA mediated modulation of the respiratory system here via effects on neurons within the RTN region. It has been previously proposed that the RTN neurons provide a source of endogenous drive to the respiratory system. These current data suggest, along the lines proposed by McCrimmon and colleagues, that the RTN neurons are also under endogenous GABAergic inhibition. It was emphasized that the data are obtained in conscious rats adding to the potential importance of this GABAergic modulation. That this effect does not involve the response to systemic hypercapnia is surprising. Prior studies that lesion or inhibit RTN neurons under anesthesia (Nattie et al., 1991; Nattie and Li, 1994; Nattie, 1999) or in the awake rat (Akilesh et al., 1997) show that the response to hypercapnia is decreased.
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Focal acidification of the RTN region by acetazolamide microinjection increases respiratory output (Coates et al., 1993) and focal application of CO2 to the RTN region increases respiratory output in the anesthetized (Li and Nattie, 1997) and in the awake rat (Li et al., 1999). Thus, the RTN is involved in chemoreception and provides an endogenous ongoing drive to the respiratory system. Muscimol dialysis into the RTN inhibits the response to systemic hypercapnia by 19–22% (Nattie and Li, 2000). It was suggested that many or all RTN neurons have GABA receptors and are susceptible to inhibition with exogenously applied muscimol. However, only some of these neurons receive endogenous GABAergic input. These would be the neurons involved in providing an ongoing drive to the respiratory system; not those involved in chemoreception. Bicuculline seems only to affect the ongoing drive component of RTN function.
4.5. Effects of dialysis of TRH in the RTN, comparison to muscimol TRH has also been dialyzed into the RTN of conscious rats (Cream et al., 1999). Both 1 and 10 mM doses increased V: E, TB, V: O , and arousal; the effects being greater and longer lasting with the larger dose. It was suggested that the RTN is involved with the integration of signals, which match ventilation to metabolic rate. The observed stimulation of breathing was appropriate for the observed increase in metabolism. With muscimol, the decrease in ventilation occurs without a change in metabolic rate suggesting the presence of hypoventilation, a disruption of the link between ventilation and metabolism. Similarly, with bicuculline, the increase in ventilation occurs without an increase in metabolism, again a disruption in the link of ventilation and metabolic rate. 2
4.6. Conclusions and physiological significance It was concluded that the excitatory input of the RTN to neurons that determine the tidal volume and the overall level of ventilation in the awake resting state is modulated by an ongoing, endogenous GABAergic influence. The anatomi-
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cal source is unknown. Preliminary retrograde tracing data suggest that afferents converge into the RTN region from numerous sites within the brainstem, many known to be involved with autonomic regulation (Cream, unpublished observations).
Acknowledgements This research was supported by HL 28066.
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Forster, H.V., Ohtake, P.J., Pan, L.G., Lowry, T.F., 1997. Effect on breathing of surface ventrolateral medullary cooling in awake, anesthetized and asleep goats. Respir. Physiol. 110, 187– 197. Guyenet, P.G., Filtz, T.M., Donaldson, S.R., 1987. Role of excitatory amino acids in rat vagal and sympathetic baroreflexes. Brain Res. 407, 272– 284. Hayashi, F., Lipski, J., 1992. The role of inhibitory amino acids in control of respiratory motor output in an arterially perfused rat. Respir. Physiol. 89, 47 – 63. Li, A., Nattie, E.E., 1997. Focal central chemoreceptor sensitivity in the RTN studied with a CO2 diffusion pipette in vivo. J. Appl. Physiol. 83, 420– 428. Li, A., Randall, M., Nattie, E.E., 1999. CO2 microdialysis in the retrotrapezoid nucleus of the rat increases breathing in wakefulness but not in sleep. J. Appl. Physiol. 87, 910– 919. McCrimmon, D.R., Zuperku, E.J., Hayashi, F., Dogas, Z., Hinrichsen, C.F.L., Stuth, E.A., Tonkovic-Capin, M., Krolo, M., Hopp, F.A., 1997. Modulation of the synaptic drive to respiratory premotor and motor neurons. Respir. Physiol. 110, 161– 176. Nattie, E.E., 1999. CO2, brainstem chemoreceptors, and breathing. Prog. Neurobiol. 59, 299– 331. Nattie, E.E., Li, A., 1994. Retrotrapezoid nucleus lesions decrease phrenic activity and CO2 sensitivity in rats. Respir. Physiol. 97, 63 – 77. Nattie, E.E., Li, A., 1996. Central chemoreception in the region of the ventral respiratory group of the rat. J. Appl. Physiol. 81, 1987– 1995. Nattie, E., Li, A., 2000. Muscimol dialysis in the retrotrapezoid nucleus (RTN) region inhibits breathing in the awake rat. J. Appl. Physiol. 89, 153– 162. Nattie, E.E., Li, A., St. John, W.M., 1991. Lesions in retrotrapezoid nucleus decrease ventilatory output in anesthetized or decerebrate cats. J. Appl. Physiol. 71, 1364– 1375. Ohtake, P.J., Forster, H.V., Pan, L.G., Lowry, T.F., Korducki, M.J., Aaron, E.A., Weiss, E.M., 1995. Ventilatory responses to cooling the ventrolateral medullary surface of awake and anesthetized goats. J. Appl. Physiol. 78, 247– 257. Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J. Physiol. London 266, 191– 207. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Pierrefiche, O., Schwarzacher, S.W., Bischoff, A.M., Richter, D.W., 1998. Blockade of synaptic inhibition within the pre-Bo¨tzinger complex in the cat suppresses respiratory rhythm generation in vivo. J. Physiol. Lond. 509, 245– 254. Schla¨fke, M.E., 1979. Elimination of central chemosensitivity by coagulation of a bilateral area on the ventral medullary surface in awake cats. Pflu¨gers Arch. 379, 231– 241. Sica, A.L., Siddiqi, Z.A., Hundley, B.W., Gootman, P.M.,
E. Nattie et al. / Respiration Physiology 124 (2001) 179–193 Steele, A.M., 1993. Effects of GABAA receptor antagonism on inspiratory activities in kittens. Neurosci. Lett. 160, 149– 152. Smith, J.C., Morrison, D.E., Ellenberger, H.H., Otto, M.R., Feldman, J., 1989. Brainstem projections to the major respiratory neuron populations in the medulla of the cat. J. Comp. Neurol. 281, 69–96.
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Sun, M.-K., Guyenet, P.G., 1985. GABA-mediated baroreceptor inhibition of reticulospinal neurons. Am. J. Physiol. 249, R672– R680. Yamada, K., McAllen, R.M., Loewy, A.D., 1984. GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex. Brain Res. 297, 175– 180.
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Bicuculline dialysis in the retrotrapezoid nucleus (RTN) region stimulates breathing in the awake rat Eugene Nattie *, Jing Shi, Aihua Li Department of Physiology, Borwell Building, Dartmouth Medical School, Lebanon, NH 03756 -0001, USA Accepted 5 October 2000
Abstract Muscimol dialysis in the retrotrapezoid nucleus (RTN) region of awake rats reduces tidal volume during air breathing and decreases chemoreception (Nattie, Li, 2000. J. Appl. Physiol., 89, 153– 162). Is there an endogenous GABAergic inhibition of the RTN as for medullary respiratory and pressor neurons? Bicuculline microdialysis (30 min; 1 mM) into the RTN region of awake rats reversibly increased tidal volume by 11 – 16% over the period from 10 to 60 min (PB0.01; six rats). Ventilation increased but this was significant (P B 0.05) only at 5, 20, and 25 min as frequency tended to decrease during dialysis. The ventilatory response to 7% CO2 was unaffected (six rats); dialysis of vehicle alone over 4 h had no effect (five rats). It was concluded that in the awake rat there is ongoing endogenous modulation of RTN effects on tidal volume by a GABAergic process of unknown origin. The lack of effect on the response to systemic hypercapnia suggests that the RTN provides an ongoing endogenous drive to respiration by a process that is independent of its role in chemoreception. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chemosensitivity, central, GABAA receptors; Control of breathing, NTS, central chemosensitivity; Mammals, rat; Medulla, rostral ventral, chemosensitivity; Pattern of breathing, hyperventilation; Receptors, GABAA
1. Introduction While the major clusters of medullary neurons involved in the control of breathing are in the dorsal and ventral respiratory groups, the rostral ventrolateral medulla (RVLM) is a site of central chemoreception and provides an ongoing endogenous drive for respiration. In anesthetized animals, acidic stimulation of the medullary surface * Corresponding author. Tel.: +1-603-6507726; fax: +1603-6506130. E-mail address: [email protected] (E. Nattie).
at the RVLM increases respiratory output (see Bruce and Cherniack, 1987; Nattie, 1999 for references) as does focal acidification of a portion of the RVLM, the retrotrapezoid nucleus (RTN) (Coates et al., 1993; Li and Nattie, 1997). The operational definition of the RTN region includes the RTN proper, described by retrograde tracing experiments as lying ventral to the facial nucleus (Smith et al., 1989), together with adjacent neurons of the parapyramidal region and the juxtafacial portion of the nucleus paragigantocellularis lateralis (PGCL). Surface cooling at the RVLM produces apnea and virtually abolishes CO2 sensi-
0034-5687/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 4 - 5 6 8 7 ( 0 0 ) 0 0 2 1 2 - 7
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tivity in anesthetized animals (Bruce and Cherniack, 1987) with focal cooling by a small probe identifying the most sensitive site as the RTN region (Budzinska et al., 1985). Focal destruction of the RTN region by neurotoxin injection (Nattie et al., 1991; Nattie and Li, 1994; Nattie, 1999) also substantially reduces baseline respiratory output and the response to systemic hypercapnia. The importance of the endogenous drive and chemoreceptor functions of the RTN region in the absence of anesthesia is less certain. Large bilateral RVLM lesions, including the RTN, produced by electro-coagulation in the cat results in hypoventilation and a markedly diminished response to hypercapnia (Schla¨fke, 1979). In the unanesthetized goat (Ohtake et al., 1995; Forster et al., 1997), cooling of the RVLM, including the RTN region, decreases ventilation and the response to hypercapnia in the waking state but the effects are smaller than those observed with cooling of the same location under anesthesia. Awake rats, with histologically proven RTN lesions produced by prior neurotoxin injection, breathe normally at rest but have a 39% reduction in the response to systemic hypercapnia (Akilesh et al., 1997). Dialysis of the GABA-A receptor agonist, muscimol, in awake rats (Nattie and Li, 2000) decreases tidal volume and ventilation reversibly during air breathing and reduces the response to systemic hypercapnia. The size of the region affected is likely to be substantially smaller in these rat studies than in the cat or goat experiments. RVLM pressor neurons, located nearby those involved in the control of breathing, are under tonic, endogenous GABAergic inhibition that originates from the caudal ventrolateral medulla (CVLM) (Blessing and Reis, 1983; Guyenet et al., 1987). These CVLM neurons are in turn under endogenous GABAergic inhibition from unknown sites (Blessing and Reis, 1983). Some respiratory premotor neurons in the ventral respiratory group are also under tonic, endogenous GABAergic inhibition (McCrimmon et al., 1997). The purpose of this study is to evaluate whether neurons in the RTN region are also under ongoing, endogenous GABAergic inhibition. The model is an unanesthetized rat with the GABAA receptor antagonist, bicuculline, applied to the RTN region by micro-
dialysis to focally, acutely, and reversibly alter function.
2. Methods
2.1. General preparation The experiments were approved by the Institutional Animal Care and Use Committee of Dartmouth Medical School. The general methods have been recently described in detail (Nattie and Li, 2000). In brief, Sprague –Dawley rats of 300 –400 g were anesthetized with intramuscular Ketamine (100 mg/kg) and intraperitoneal Xylazine (20 mg/ kg). A guide tube (0.38 mm OD) with a dummy cannula was inserted such that the tip was just above the RTN region and secured to the skull and a sterile telemetric temperature probe was placed unattached in the abdominal cavity. The animal was allowed to recover for 3–4 days.
2.2. Protocol On each experimental day the rat would be weighed, the dummy cannula removed and the dialysis probe inserted into the guide tube. The probe tip, which protruded 1 mm beyond the guide tube tip, was 1 mm in length and 0.24 mm in diameter with membrane pores limiting movement to molecules under 6000 Da. Animals were placed into a Pappenheimer-style plethysmograph chamber (Pappenheimer, 1977; Cream et al., 1999) and allowed 30–40 min to acclimate. Ventilation (V: E), oxygen consumption (V: O ) and body temperature (TB) were measured. After acclimation, the dialysis pump was turned on with aCSF flowing at a rate of 4.0 ml/min. Dialysis continued through the entire experimental protocol in all groups. Fast green dye was added to the dialysate to facilitate visualization of the dialysate outflow and to allow a histological test for membrane leak. The membrane was examined just after each dialysis. The outflow rate was monitored semiquantitatively by visualization of the drops of dialysate coming out of the outflow tubing. Animals in this report did not have leaks in the dialysis membrane during the experiment. The 2
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rats were studied in quiet wakefulness between 09:00 and 16:00 h. They were housed on a midnight to noon circadian light/dark cycle so the data collection was during the end of the quiet/ sleep (light) circadian period and the beginning of the active/wake (dark) circadian period. This yielded reproducible data representing quiet wakefulness. Later in the active/wake period, the rats are prone to sniffing, grooming and active movement making the ventilatory data more difficult to obtain. The rats were under constant observation during the experiments, which were performed in subdued light
2.3. Experimental groups There were three experimental groups. In each, some rats received a treatment more than once. For data presentation and statistical analysis multiple trials in any rat are averaged. Group 1, a control (five rats), received three treatments. First, the dummy probe was left in place and measurements obtained every 15 min over 4 h. Second, the dummy was replaced by the dialysis probe but without any flow of dialysate and measurements obtained every 15 min over 4 h. Third, aCSF was dialyzed continuously through the 4 h period with measurements obtained every 15 min. Each rat received all treatments each on a separate day. Group 2 (six rats) tested the effects of bicuculline dialysis into the RTN region on breathing room air during wakefulness. Four sets of baseline measurements were repeated over 40 min then the dialysate was switched to 1 mM bicuculline. The time at which the new dialysate reached the tissue (wash-out of the dead space accounted for) is defined as time= 0. Measurements were obtained every 5 min during the 30 min dialysis period then every 5 min over a 60 min recovery period with the dialysate switched to aCSF. Group 3 (six rats) examined the effect of bicuculline dialysis on the response to 7% CO2. The inspired CO2 was increased by switching the inflow gas to a mixture with 7% CO2 and 93% air, which slowly increased the inspired CO2 value over 8 – 9 min, as measured in the outflow gas. Ventilatory variables from 8 to 9 and from 9 to 10 min were measured. Four sets of air breathing data were obtained over 20 min
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followed by exposure to hypercapnia then recovery. This was repeated three times in each protocol. Each rat received two treatment regimes. In one, aCSF was dialyzed throughout the experiment. In the other, after obtaining the initial CO2 response, the dialysate was switched to 1 mM bicuculline in aCSF for 30 min followed by a return to aCSF alone for the remainder of the experiment.
2.4. Data analysis and statistics Analog respiratory data were digitized (DataPac III) and a breath by breath analysis performed with the pressure deflections and the respiratory cycle time for each breath being determined over a 20–30 sec time period (DataPac III). VT (per 100 g of body weight), f, and V: E (per 100 g body weight) were calculated (SigmaPlot IV). Oxygen consumption was determined by the difference in outflow oxygen, determined by the O2 sensor, and inflow oxygen; carbon dioxide production by the difference between inflow carbon dioxide (assumed to be 0) and outflow carbon dioxide measured by infrared analyzer. The flow rate of gas through the plethysmograph was constant at 1.4 l/min. The results for V: E, VT, f, V: O , V: CO and TB in room air or 7% CO2 breathing were compared within each experimental group using a one-way repeated measures ANOVA. In some cases, the Friedman repeated measures ANOVA on ranks was required. Post-hoc tests (Dunnett’s; Student Newman –Keuls) were performed when significant differences were found. The air breathing ventilatory data (Groups 1 and 2) are evaluated in two ways. First, the absolute values of V: E, VT, and f are compared over the entire experiment. Second, these data are expressed as percent baseline, which was determined as the mean of four sets of measurements made over 20 min prior to the onset of dialysis. This allowed normalization for differences in initial values among the groups. The CO2 breathing data are evaluated in two ways. First, the absolute values of V: E, VT, and f are compared (one-way ANOVA) for the three CO2 tests before, during, and after each dialysis treatment (aCSF, 1 mM bicuculline). Second, the 2
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change in these values, calculated as the CO2 stimulated value at each CO2 challenge minus the average of the control values just before and after, are compared for the three CO2 tests before, during, and after dialysis treatment (one-way ANOVA).
2.5. Anatomy At the conclusion of the experiments, the rats were killed by an overdose of pentobarbital administered intraperitoneally after an initial sedative dose of ketamine (100 mg/kg) administered intramuscularly. The brainstems were removed and frozen immediately, sectioned (50 mm thickness), and stained with cresyl violet to identify anatomical locations of the probe tips with the assistance of an atlas (Paxinos and Watson, 1986).
3. Results
3.1. Breathing air; quiet wakefulness, control The mean (S.E.M.) absolute values of V: E, VT, and f over the entire 4 h experiment in Group 1 are shown in Fig. 1, those for V: O , V: CO , and TB in Fig. 2. The three treatments: (a) no probe in place (open triangle); (b) probe in but no dialysis (open circle); and (c) probe in with continuous dialysis of aCSF (solid circle) had no significant effect on these variables. 2
2
3.2. Unilateral RTN dialysis with 1 mM bicuculline; effects on 6entilation Absolute VT, and VT expressed as per cent baseline, were significantly increased during and just after dialysis of the RTN region with 1 mM bicuculline (Figs. 3 and 4) (P B 0.001, one-way repeated measures ANOVA; P B0.05 for all times during and after dialysis compared to control except 5, and 60–90 min, Dunnett’s test). Frequency was not significantly affected (P = 0.09 one-way ANOVA). There was an apparent initial increase in frequency at 5 min of dialysis but during the bicuculline dialysis frequency tended to decrease as seen most easily on Fig. 4. Ventilation
is increased during bicuculline dialysis as shown in Figs. 3 and 4 but this does not reach significance if all data are used in the test. If the analysis is limited to 60 min after the onset of dialysis, then V: E is increased (P B0.05, one-way ANOVA; 5, 20, and 25 min points, PB 0.05 Dunnett’s test). Metabolic rate and TB were not significantly affected by bicuculline dialysis (Fig. 5).
3.3. Unilateral RTN dialysis with 1 mM bicuculline; effects on response to CO2 With aCSF treatment (data not shown) neither the absolute values of CO2 stimulated V: E, VT, and f nor the change in these values, comparing CO2 stimulated to the average of pre- and postCO2 baseline values, differed significantly among the three dialysis tests. Metabolic rate and TB were also unchanged during this experiment (data not shown). With 1 mM bicuculline treatment (Fig. 6), neither the absolute values of CO2 stimulated V: E, VT, and f nor the change in these values, comparing CO2 stimulated to the average of pre- and post-CO2 baseline values, differed significantly among the three dialysis tests. Absolute VT was increased by 1 mM bicuculline both during and after treatment as shown in Figs. 3 and 4. TB and V: CO were unaffected by this protocol but V: O did increase following bicuculline dialysis and the second CO2 test (PB 0.001; one-way repeated measures ANOVA; PB0.05 Dunnett’s test for 60–90 min values; data not shown). 2
2
3.4. Anatomical location of dialysis probes Fig. 7 shows computer modified digitized 2× images of cresyl violet stained medullary cross sections at the approximate center of the tissue disruption at the site of the dialysis probe tip for group 2 animals, which received the 1 mM bicuculline without hypercapnia. The rectangles drawn on the Fig. show the location of the probe tips. In each case, the probe tip was in the region of the RTN. The results (not shown) are similar for the other two groups.
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Fig. 1. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time during the 4 h duration of the control group experiment (five rats) with (a) no dialysis probe in place (), (b) dialysis probe inserted but no dialysis (), and (c) continuous dialysis of aCSF ( ).
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Fig. 2. Like Fig. 1 but showing V: O , V: CO , and TB results. 2
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Fig. 3. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time during the group 2 experiment (six rats) with initial dialysis with aCSF (dotted line) followed by 30 min period of dialysis with 1 mM bicuculline (solid line) then aCSF dialysis for the remainder (dotted line).
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Fig. 4. Mean (S.E.M.) values for V: E, VT, and f, expressed as percent of initial baseline, are shown as a function of time during the group 2 experiment with initial dialysis with aCSF (dotted line) followed by 30 min period of dialysis with 1 mM bicuculline (solid line) then aCSF dialysis for the remainder (dotted line).
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Fig. 5. V: O , V: CO , and TB results for the group 2 experiment. 2
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Fig. 6. Mean 9 S.E.M. absolute values for ventilation (V: E), tidal volume (VT), and respiratory frequency (f) are shown as a function of time for the group 3 bicuculline experiments (six rats). The lower values connected by solid line represent air breathing data. The higher values shown at three times represent the CO2 breathing data.
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Fig. 7. Cresyl violet stained cross section of the medulla of the six rats in group 2 taken at the center of the dialysis probe location. The numbers at lower left of each section refer to mm from bregma. The bar is 1 mm. VII is facial nucleus. The rectangle represents in each section the site at which tissue damage identified the location of the dialysis probe tip.
4. Discussion
4.1. Limitations of technique Microdialysis has been successfully used to deliver neuroactive substances (TRH, muscimol, CO2) to the RTN region of the medulla in the conscious rat (Cream et al., 1999; Li et al., 1999; Nattie and Li, 2000). A concern about every technique used to deliver neuroactive substances into the brain is the degree of spread within the tissue. For CO2, tissue pH has been measured at different distances from the dialysis probe, but so far only under anesthesia (Nattie and Li, 1996; Li et al., 1999). These data indicate the change in tissue pH is confined to within 500 mm of the probe tip when the CO2 in the dialysate is sufficient to cause a 15% increase in ventilation (Li et al., 1999). For TRH, muscimol, and bicuculline it is more difficult to measure the exact tissue region affected. However, drug spread has been esti-
mated using data from the literature and from the authors’ studies of the spread via dialysis of fluorescein, which has a molecular weight (332) similar to muscimol (195), bicuculline (509) and TRH (362) (see Nattie and Li, 2000). Dialysis of glutamate (Alessandri et al., 1996) at 10–1000 mM and 2.0 ml/min for 30 min showed a 35% delivery into the tissue. For the 1 mM bicuculline dialysis this would result in a maximum concentration of 0.35 mM outside the probe. The use of two probes, one for delivery, the other for collection and measurement, showed a similar finding, steady-state concentrations 1.5 mm from the delivery probe that were 10–100 times lower than in the dialysate (de Lange et al., 1995). From the literature, it appears that a considerable concentration difference of neuroactive substance exists between the inside and the outside of the dialysis probe. The use of a fluorescein marker with 1 mM concentration in the probe showed an average
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(N =5) rostral to caudal spread of 870 mm and an average radius at the cross section with the largest area of 555 mm (Cream et al., 1999). The estimated volume of distribution was 590 nl, which is 49% of the approximate volume of the RTN region (1.2 ml). From these data, it was estimated that with dialysis of 1 mM bicuculline the tissue distribution is likely to be within 500 – 900 mm of the probe tip and the concentration at the probe is likely to be 0.35 mM or less. Another concern with microdialysis is possible wash-out of neuroactive substances that might influence breathing. The control group in this experiment was designed to evaluate this concern and to show the variation in ventilatory data when obtained in the protocol over 4 h. Ventilation, VT, f, V: O , V: CO , and TB were remarkably stable over 4 h of constant dialysis with aCSF as well as over 4 h with no probe in place or with the probe in place but without any dialysis. While it seems likely that the aCSF does dialyze a variety of substances out of the RTN region this does not seem to have any detectable effect on ventilation. 2
2
4.2. Effects of bicuculline dialysis in the RTN on breathing The major finding in this study is that dialysis of bicuculline at the 1 mM dose into the RTN region of the conscious rat increases VT reversibly without effect on metabolic rate or TB. The effects on V: E are complicated by the observation that f appears to increase at 5 min of dialysis then tends to decrease during the dialysis, although these changes do not reach statistical significance. The result is a significant increase in V: E at 5, 20 and 25 min. Nevertheless, the increase in VT is striking and of interest from two viewpoints. (1) It appears to be the opposite response to that described with RTN dialysis with muscimol. (2) It is consonant with other published data that suggest the presence of a tonic GABAergic inhibition of RVLM pressor neurons and respiratory neurons of the VRG and DRG but differs from results obtained with bicuculline microinjection into the preBo¨tzinger complex.
4.3. Comparison of bicuculline and muscimol effects Dialysis of muscimol (1 mM; 30 min) into the RTN region of the awake rat, using a protocol similar to that in this study, decreased VT by 16–17% (Nattie and Li, 2000). Ventilation decreased, initially, then returned to within the baseline range of values due to a subtle increase in f. With dialysis of bicuculline (1 mM; 30 min) into the RTN region, the pattern of responses is a mirror image of those obtained with muscimol. Tidal volume is increased consistently during and shortly after the period of dialysis, V: E is increased significantly at 5, 20 and 25 min the variability of this response being attributed to the tendency for f to decrease. The presence of other chemoreceptor sites both centrally (Coates et al., 1993; Nattie and Li, 1994; Li and Nattie, 1997; Nattie and Li, 1996; Li et al., 1999; Nattie, 1999) and in the periphery and the use of an animal model with a ‘closed-loop’ control system complicates the interpretation of these data. In either case, muscimol or bicuculline, the final level of V: E would reflect a balance of inhibition/disinhibition in the RTN region and hyper/hypocapnic induced excitation/ inhibition at other chemoreceptor locations. The use of a conscious rat with a ‘closed-loop’ control system to examine focal effects in the RTN may disguise the overall importance of neurons in the RTN region in the control of breathing. However, this type of experiment demonstrates the significance of having many central chemoreceptors and their importance in the intact, conscious animal.
4.4. Endogenous GABAergic inhibition Pre-sympathetic vasomotor neurons in the RVLM are under GABAergic inhibition, in part related to the baroreceptor reflex, via the caudal ventrolateral medulla (CVLM; Yamada et al., 1984; Guyenet et al., 1987) and in part due to non-baroreceptor related inhibition from the CVLM (Sun and Guyenet, 1985). Bicuculline application to the RVLM produces a pressor response (Sun and Guyenet, 1985; Amano and Kubo, 1993). The CVLM neurons themselves are
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also under tonic GABAergic inhibition from unknown sources (Blessing and Reis, 1983). Effects of bicuculline application on breathing can be classified into types of responses; (1) changes in rhythm; and (2) increases in respiratory drive. Focal application of bicuculline into the preBo¨tzinger region bilaterally in anesthetized cats slows frequency and results in an apneustic pattern (Pierrefiche et al., 1998). Similar effects have been reported following bicuculline injections into the ventrolateral medulla of anesthetized 4 – 8-weekold opossums (Farber, 1995). In contrast, systemic administration of bicuculline, or the equivalent, increased respiratory output in anesthetized kittens (Sica et al., 1993), anesthetized rabbits (Delpierre et al., 1996), and in an arterially perfused in situ brainstem-spinal cord preparation of the adult rat (Hayashi and Lipski, 1992). Further, microinjection of bicuculline on some inspiratory and expiratory premotor neurons of the ventral respiratory group that project to the spinal cord of anesthetized dogs amplified their discharge pattern in a multiplicative manner (McCrimmon et al., 1997). This effect was termed ‘gain modulation’ and while the source of the endogenous GABAergic input is as yet undefined the concept is that many respiratory neurons, and the respiratory system output, is under some type of ongoing endogenous GABAergic modulation in resting conditions. The results support this concept of an ongoing endogenous GABA mediated modulation of the respiratory system here via effects on neurons within the RTN region. It has been previously proposed that the RTN neurons provide a source of endogenous drive to the respiratory system. These current data suggest, along the lines proposed by McCrimmon and colleagues, that the RTN neurons are also under endogenous GABAergic inhibition. It was emphasized that the data are obtained in conscious rats adding to the potential importance of this GABAergic modulation. That this effect does not involve the response to systemic hypercapnia is surprising. Prior studies that lesion or inhibit RTN neurons under anesthesia (Nattie et al., 1991; Nattie and Li, 1994; Nattie, 1999) or in the awake rat (Akilesh et al., 1997) show that the response to hypercapnia is decreased.
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Focal acidification of the RTN region by acetazolamide microinjection increases respiratory output (Coates et al., 1993) and focal application of CO2 to the RTN region increases respiratory output in the anesthetized (Li and Nattie, 1997) and in the awake rat (Li et al., 1999). Thus, the RTN is involved in chemoreception and provides an endogenous ongoing drive to the respiratory system. Muscimol dialysis into the RTN inhibits the response to systemic hypercapnia by 19–22% (Nattie and Li, 2000). It was suggested that many or all RTN neurons have GABA receptors and are susceptible to inhibition with exogenously applied muscimol. However, only some of these neurons receive endogenous GABAergic input. These would be the neurons involved in providing an ongoing drive to the respiratory system; not those involved in chemoreception. Bicuculline seems only to affect the ongoing drive component of RTN function.
4.5. Effects of dialysis of TRH in the RTN, comparison to muscimol TRH has also been dialyzed into the RTN of conscious rats (Cream et al., 1999). Both 1 and 10 mM doses increased V: E, TB, V: O , and arousal; the effects being greater and longer lasting with the larger dose. It was suggested that the RTN is involved with the integration of signals, which match ventilation to metabolic rate. The observed stimulation of breathing was appropriate for the observed increase in metabolism. With muscimol, the decrease in ventilation occurs without a change in metabolic rate suggesting the presence of hypoventilation, a disruption of the link between ventilation and metabolism. Similarly, with bicuculline, the increase in ventilation occurs without an increase in metabolism, again a disruption in the link of ventilation and metabolic rate. 2
4.6. Conclusions and physiological significance It was concluded that the excitatory input of the RTN to neurons that determine the tidal volume and the overall level of ventilation in the awake resting state is modulated by an ongoing, endogenous GABAergic influence. The anatomi-
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cal source is unknown. Preliminary retrograde tracing data suggest that afferents converge into the RTN region from numerous sites within the brainstem, many known to be involved with autonomic regulation (Cream, unpublished observations).
Acknowledgements This research was supported by HL 28066.
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