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Cardiorespiratory effects produced by injecting drugs that affect GABA receptors into nuclei associated with the ventral surface of the medulla
Neuropharmacology
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1987
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CARDIORESPIRATORY EFFECTS PRODUCED BY INJECTING DRUGS THAT AFFECT GABA RECEPTORS INTO NUCLEI ASSOCIATED WITH THE VENTRAL SURFACE OF THE MEDULLA P. J. GATTI, A. M. T. DASILVA and R. A. GILLIS Departments of Pharmacology and Medicine (Pulmonary Division), Georgetown University, Schools of Medicine and Dentistry, Washington, D.C. 20007, U.S.A. (Accepled 24 February 1986)
Summary-It has recently been shown that L-glutamic acid induced stimulation of cell bodies in a circumscribed area of the rostra1 ventrolateral medulla (RVLM) in the cat, produced increases in arterial pressure (AP), decreases in heart rate (HR) and transient apnea (Gatti, Norman, DaSilva and Gillis, 1986). The purpose of the present study was to determine if this same area was sensitive to GABA receptor agonists and antagonists. Injection of the GABA agonist muscimol(200 ng), into the rostra1 ventrolateral medulla of cats anesthetized with chloralose produced a precipitous and immediate fall in arterial pressure (-95 k 4.6) and heart rate (-31 + 5.9; n = 4, P < 0.05). Maximal cardiovascular effects could only be achieved if muscimol was injected bilaterally. These effects of muscimol on arterial pressure were dose-dependent. Time-action curves for the effects of muscimol on arterial pressure and respiration were different. Hypotension occurred first and was followed later in time by a decrease in minute ventilation. Within 30 min all animals were apneic after the 200 ng dose. The cardiovascular effects of muscimol were reversed by the injection of the GABA receptor antagonist bicuculline. These data indicate that stimulation of GABA receptors in the rostra1 ventrolateral medulla produced selective cardiovascular effects and that respiratory neurons sensitive to GABA are apparently not localized with these cardiovascular neurons. Key words: muscimol, bicuculline, arterial blood pressure, heart rate, respiratory activity, cats, nucleus reticularis rostroventrolateralis, nucleus paragigantocellularis lateralis, lateral reticular nucleus.
Previous results, as well as the earlier results of Feldberg (1976), indicate that there is an important area on the ventral surface of the medulla where CNS neurotransmitters, such as y-aminobutyric acid (GABA) and glycine, produce a depressant effect on cardiorespiratory activity. This site is referred to as either Schlaetke’s area or the intermediate area (Schlaefke, 1981). Primary interest in this area stems from the findings that an important GABAergic synapse appears to exist at this site to control cardiorespiratory activity (Yamada, Norman, Hamosh and Gillis, 1982; Yamada, Moerschbaecher, Hamosh and Gillis, 1983). In an attempt to determine the specific location of GABAergic synapses at this site, the technique of injecting glutamic acid into distinct areas of the “ventral medulla” where GABAergic drugs could diffuse when placed topically on Schlaefke’s area was employed. Based on data obtained with this technique, sites have been found where injection of glutamic acid elicited cardiovascular effects. These are the “glycine sensitive area” described by Guertzenstein and Silver (1974) which seems to be synonymous with the Cl area described by ROSS, Ruggiero, Park, Joh, Sved, FemandezPardal, Saavedra and Reis (1984), the lateral reticular 423
nucleus (LRN) and the nucleus paragigantocellularis lateralis (PGL). The purpose of the present study was to determine whether a GABAergic synapse, controlling cardiorespiratory function, is present at any of these sites, as defined by the glutamic acid experiments (Gatti et al., 1986).
METHODS
Adult cats of either sex (164.6 kg) were anesthetized with alpha-chloralose, 70-80 mg/kg, (i.v.>. A femoral artery and vein were cannulated for measurement of arterial blood pressure and systemic administration of drugs, respectively. The trachea was cannulated and fitted to a Fleisch pneumotachograph. The flow signal obtained was then integrated to obtain tidal volume. Respiratory rate was obtained from fast tracings of the volume signal. Heart rate was obtained from the pulsatile blood pressure using a cardiotachometer. Blood pressure, trachea1 air flow, tidal volume, heart rate and lead II of the electrocardiogram (ECG) were monitored continuously on a Buxco Pulmonary Mechanics Analyzer (Model 6). Rectal temperature was monitored
P. J. GATTIet al.
424
and maintained between 37” and 38°C using a heat lamp. The ventral surface of the medulla was then exposed. A lon~tudinal midline incision was made in the neck. The trachea and oesophagus were retracted and the prevertebral muscles scraped from the basal plate of the skull. A portion of this plate was removed, creating a window 15 x 9 mm over the medullary surface. The dura was then cut and reflected and the cerebrospinal Ruid removed, exposing the surface of the medulla. Cotton wicks were placed at either end of the craniotomy to prevent cerebrospinal fluid from pooling on the ventral surface. The animal was then placed in a stereotaxic head holder in this position when injections were made. The GABA receptor agonist muscimol, and the antagonist bicuculline, were injected in a volume of 100-200 nl over a 15 set interval. These agents were injected through glass micropipettes pulled to an inside tip diameter of 4&50pm. Injections were made using a pressure injection system with a pump which delivered a steady stream of air which could be directed through the pipette. Only those injections which were histologically verified to be in the area of the nucleus paragigantocellularis lateralis, lateral reticular nucleus and rostra1 ventrolateral medulla were used in the analysis of the data. The injection of an equal volume of saline (0.9% NaCl) did not affect any cardiorespiratory parameter significantly. Injections were made in the nucleus paragigantocellularis lateralis and the lateral reticular nucleus with the aid of information provided in the figures of Taber’s paper (1961). Injections into the nucleus lateralis were para~gantocellularis 2.5-3.5 mm lateral to the basilar artery, 1 mm below the ventral surface of the medulla and OS-1Smm caudal to the trapezoid border. Injections into the lateral reticular nucleus were 4mm lateral to the basilar artery, 1 mm below the surface and 3.74.4mm caudal to the trapezoid border. Finally, drugs were injected into the rostal ventroIatera1 medulla. This area lies 4 mm lateral to the basilar artery and 1.5 mm caudal to the trapezoid border, and
Table 1. Cardiorespiratory Dose of muscimol per side
I ng before injection
No. of animals studied 3
After injection difference 100 ng before injection
4
After injection difference 200 ng before injection
4
After injection difference ‘P < 0.05 using the paired student’s ‘f’-test.
corresponds with the Cl area of adrenergic neurons recently described in the rat (ROSS et al., 1984). The GABAergic drugs were dissolved in 1% fast green-saline solution for the purpose of marking the site of injection. To localize the site of injection, the brainstem was removed and stored in formalin until one day before sectioning, when they were transferred to 5% sucrose and refrigerated. The medulla was sectioned in 40-60 pm slices on a cryostat. When the injection site was located, those slides containing the site were stained with neutral red to distinguish cell bodies, and the injection site was photographed. The following drugs were used: muscimol (Boehringer Mannheim, GmbH, W. Germany), bicuculline methiodide and fast green (Sigma Chemical Co., St. Louis, Missouri), alpha-chloralose (Establissements Kuhlmann, Paris, France) and THIP (a gift from Dr 0. Svendsen, H. Lundbeck & Co. A/S, Copenhagen). The values stated in the text are means plus or minus the standard error of the mean @EM) and were taken at the time of the peak changes in CardiovascuIar function. Statistical analyses were performed using Student’s paired l-test with P < 0.05 being the criterion for statistical significance. Linear regression analysis was also performed to determine the line of best fit for the data and for determining whether dose-response curves had slopes significantly different (P < 0.05) from zero. RESULTS
Eflects of injection of muscimol into the rostra1ventrolateral medulla
In 11 animals, muscimol (l-200 ng) was injected bilaterally (total doses of 2-400ng) into the rostra1 ventrolateral area (i.e. glycine-sensitive area as described by Guertzenstein and Silver, 1974) and the data are summarized in Table 1. As can be seen, injection of muscimol produced significant (P < 0.05) dose-dependent decreases in arterial pressure. The other significant effects that were noted with muscimol were decreases in tidal volume (VT) with the largest dose tested (Table l), and decreases in heart
effects of muscimol after injection into the Cl area
Mean blood pressure (mmHg)
Heart rate (beats/min)
170 f 12
201 + 19
155 f 13 -15 + 3.2*
166k 17 - 35 * 5.0*
Tidal volume (ml)
Respiratory rate (breathsimin)
Respiratory minute volume (ml/min)
19 * 1.7
14 * 2.3
216 i $9
17 f 0.2 -2.0 r 1.1
12* 1.7 1.3 f 1.7
216 + 20 -60+46
156 & 18
165 i 20
19 + 0.8
18 rf: 1.1
352 + 17
97216 -59 &-12*
14oi I5 -25+11
14-i: 1.3 -5 * 1.7
16 i 3.4 -2.0 & 2.3
242 + 61 -110+55
160 f 3
135 + 19
33 + 1
12 + 4.0
400 + 40
64+6 -95 k 4.6,
103 k 15 -31 +6*
2s + 1 -8+0.0*
13k2.4 + 1.3 * 1.3
338 + 65 -61 k 21
Site of action for the depressor effect of muscimol
Fig. 1. Time-action curves of the changes in mean arterial pressure (BP) and tidal volume (VT) produced by the bilateral injection of 100 ng of muscimol(200 ng total dose) into the rostra1 ventrolateral medulla of a single animal. Note that at the time of the peak fall in mean arterial pressure, tidal volume only began to decrease.
rate with 2 of the 3 doses tested. These values for Vr and heart rate were taken at the time of peak depression in arterial pressure. The decreases in arterial pressure and heart rate occurred simultaneously and were observed within a few seconds after the bilateral injection of muscimol (Fig. 1). The maximal effect on both arterial pressure and heart rate occurred at approximately 1 min after bilateral injection of the largest dose and the effects persisted for about 30 min with the 1 ng dose, and for the duration of the experiment (i.e. for 2-3 hr) with the two larger doses. A bilateral injection of muscimol was required in order to observe a distinct effect on arterial pressure and heart rate. In some cases, a unilateral injection of the 2 larger doses of muscimol did elicit a small decrease in arterial pressure (i.e. approximately a l&15 mmHg drop in pressure). The depressant effect of muscimol on respiration followed a different time-action curve (Fig. 1). This was most clear from the data obtained using the 200 ng dose. At the time of the peak depressant effect of muscimol on cardiovascular function, there was no significant change in respiratory minute volume, despite the occurrence of a significant reduction in tidal volume (Table 1). With time, a significant decrease in respiratory minute volume occurred and by 30 min after bilateral injection of muscimol, all animals tested were apneic (Fig. 1). Respiratory arrest persisted for the duration of the experiment (i.e. for 2-3 hr). A representative experiment showing the effect of the largest dose of muscimol on cardiorespiratory activity appears as
HR (bpm) 2 3 0
t
t
Right
Left
425
muscimol 200 ng/site
Fig. 2. Representative experiment showing the effects of the injection of muscimol into the rostra1 ventrolateral medulla on tidal volume (V,), arterial blood pressure (BP) and heart rate (HR). Note that only when the muscimol was injected bilaterally did the arterial pressure fall to its lowest level. Inspiration is upward. Paper speed is 1 mmjsec.
P. J. GATTI et al.
426
Fig. 2. Respiratory arrest was not observed with the smallest dose tested. The location of the micropipette tip in the brain is shown in Fig. 3, and based on previous data, mapping the distribution of epinephrine-synthesizing neurons on the hindbrain of the rat, the tip was in an area described as the Cl area (Ross et al., 1984).
FGL
0,
LRN
RVL 1
1
I
Effect of injection of muscimol into the nucleus parag~guntocellularis later&s (PGL) In 4 animats, muscimoi (100 ng) was injected bilaterally (total dose of 200 ng) into the paragigantocellularis lateralis. At the time of the maximal decrease in arterial pressure and heart rate evoked by muscimol, injected into the Cl area, there was no significant decrease in these parameters with the injection of muscimol into the paragiganto~eIlula~s lateralis (Fig. 4). There was a delayed hypotensive and bradycardic effect with injection of drug into the nucleus paragigantocellularis lateralis. These responses were - 83 1: 4.0 mmHg (P < 0.05) and - 16 + 21 beats/min respectively, and occurred at 41 t_ 13 min after the injection of muscimoi. Respiratory depression was also noted (i.e. decrease in tidal volume of 26 + 4.6 ml) but this was delayed and only noted at 20 + 4.7 min after muscimol was administered. Efects of injection ~frn~sc~rn~l into the lateral reticular nucleus (L&V) In 3 animals, muscimol(100 ng) was injected bilaterally (total dose of 200 ng) into the lateral reticular nucleus. No significant effects on arterial pressure (Fig. 4) and heart rate (data not shown) were observed. Respiratory depression was also not noted over the first 30min after the drug was injected. Later, however, at approximately 4.5-60 min after injection, each animal exhibited a decrease in tidal volume that progressed to apnea. At this later time, there was still no decrease in arterial pressure and heart rate. Eflecfs of injection of bicuculiine met&o&& Ci area
into the
In the 3 sites tested with muscimol, the only responsive site in terms of immediate changes in arterial pressure and heart rate was the Cl area. This finding raised two questions. First, is there a tonic GABA-ergic system in the Cl area that controls arterial pressure and heart rate? And second, are the Table 2. Cardiorespiratory
Expt type Before bicuculline After 500 ng/side of bicuculline Difference
Fig. 4. Fall in mean arterial pressure (MAP) produced by the bilateral injection of IOOng of muscimol (2OOng total dose) into either the nucleus paragigantocellularis lateralis (PGL), lacteral reticular nucleus (LRN) or the nucleus reticularis
rostroventrolateralis
(RVL).
effects of mu~imol due to the activation of GABA receptors in the Cl area? In an attempt to answer both questions, the GABA receptor antagonist drug bicuculline methiodide was injected into the Cl area of naive cats and cats that were exhibiting hypotension and bradycardia evoked by muscimol. In 5 naive animals, bicuculline methiodide (BMI) was injected bilaterally (500 ng/side) and the data are summarized in Table 2. As can be seen, injection of bicuculline methiodide produced a significant (P < 0.05) increase in arterial blood pressure. In addition, bicuculline methiodide also produced significant (P < 0.05) decreases in heart rate and respiratory minute volume. In order for these changes to occur, bicuculline methiodide had to be injected bilaterally; unilateral injections were ineffective in eliciting significant changes in cardiorespiratory function. The increase in arterial pressure and decreases in heart rate and respiratory minute volume occurred simultaneously and were observed within 5 min after the bilateral injection of bicuculline methiodide. The
effects of bicuculline after injection into the CI area
No. of z&mats studied
Mean blood preSSWe (mm&z)
Heart rate t~ats/min)
Tidal volume WI
5
152+_20
166 + 14
22 + 4.3
14 i 1.8
303 zk38
180 + 16
126 + 12
19 + 3.6
12+_ 1.7
197 + 36
+ 28 + 7.0’
-40 4 13’
-2.7 rt 3.3
-2.0 * 1.1
-106+_30*
*P =z0.05 using the paired Student’s t-test.
Respiratory rate (breatha/min)
Respiratory minute volume (ml/min)
Fig. 3. Photomicrograph showing the micropipette track in the brainstem of an animal in which muscimol was injected into the rostra1 ventrolateral medulla. ION = inferior olivary nucleus; RF = retrofacial nucleus x 42; bar = 0.5 mm.
427
Site of action for the depressor effect of muscimol
429
MUSCIMOL 2M) ng/ side R L
Fig. 5. Representative experiment showing the antagonism between muscimol and bicuculline in the rostra1 ventrolateral medulla on arterial pressure (BP) and heart rate (HR). The traces on the left illustrate the immediate hypotensive effect of 400 ng of muscimol (injected on the left (L) and right (R) sides). Five minutes after the peak fall in arterial pressure (right traces) bicuculline (3 pg) was injected unilaterally on the right side in the rostra1 ventrolateral medulla. Note the immediate antagonism produced by this unilateral injection of bicuculline. Paper speed was 1mm/set. This animal was artificially respired.
maximal effects on cardiorespiratory function occurred approximately 10 min after bilateral injection of the drug and the effects persisted for about 20-30min. In a few experiments, a smaller dose of bicuculline was tested (i.e. 100 ng) but was observed to produce no effects on cardiorespiratory activity. Bicuculline methiodide (l-3.4 pg/side) was injected into the Cl area of 3 cats that had received an injection of muscimol(200 ng/side) into the same site. These experiments were performed in artificiallyrespired animals in order to prevent drug-induced respiratory effects from influencing the arterial pressure and heart rate. Bilateral injections of muscimol decreased arterial pressure and heart rate by 100 +_13.3 mmHg and -30 f 0.0 beats/min. At the time of the maximal decreases in arterial pressure and heart rate, bicuculline methiodide was administered and it restored arterial pressure and heart rate to normal in each animal tested. Only a unilateral injection of bicuculline methiodide was required to produce a complete reversal of the decreases in arterial pressure and heart rate induced by muscimol. THIP, I.“. 200
r
A representative experiment showing the reversal of the effects of muscimol by bicuculline methiodide appears as Fig. 5. Eflects of injection of bicuculline methiodide into the Cl area of cats receiving an intravenous injection of THZP It is known that THIP (4,5,6,7,tetrahydroisoxazolo [5,4-C] pyridine-3-ol), a GABA receptor agonist, crosses the blood brain barrier after intravenous injection (Waszczak, Hruska and Walters, 1980). To determine whether THIP was acting at the rostal ventrolateral medulla to produce hypotension and bradycardia, this agent was first administered intravenously to artificially respired animals and maximal decreases in arterial pressure and heart rate were obtained. These decreases were 65 f 14 mmHg (P < 0.05) and 66 + 38 beats/min, respectively and occurred with doses of 2-4mg/kg (i.v.). Next, bicuculline methiodide was injected into the rostra1 ventrolateral medulla to see if reversal of the intravenous effects of THIP would occur. A unilateral bieuculline RV LM
4
BP
Fig. 6. Representative experiment showing the antagonism between intravenously-administered THIP and intracerebrally-administered bicuculline on arterial blood pressure (BP) and heart rate (HR). The left portion of the tracing illustrates the cardiovascular depression produced by the intravenous injection of THIP (4 mg/kg). Note the near production of cardiovascular collapse by this agent. Bicuculline (1.7 ng) was then injected unilaterally into the rostra1 ventrolateral medulla (RVLM) at the second arrow. Note the dramatic and immediate antagonism of the effects of THIP by bicuculline. This animal was artificially respired. Paper speed was 1 mm/set.
P. 1.GATTIet al.
430
injection of bicuculline methiodide (l-3 pg) fully counteracted the hypotensive and bradycardic effects of THIP. Increases of 87 + 25 mmHg (P < 0.05) and 73 _I 58 ~ats/min, respectively, were noted. A dramatic representation of this interaction is illustrated in Fig. 6. DISCUSSION
The present data with the GABA receptor agonist drug, muscimol, injected into the lateral reticular nucleus and nucleus paragigantocellularis lateralis clearly indicate that the GABA-sensitive site, associated with Schlaefke’s area (also known as the intermediate area), is the Cl area. (Also referred to as nucleus reticularis rostroventrolateralis Ross et al., 1984). Injection of muscimol at this site produced an immediate (within 1 min) fall in arterial pressure and heart rate. In contrast, injection of the largest dose of muscimol into either the lateral reticular nucleus or nucleus paragigantocellularis lateralis had no significant effect on cardiorespiratory activity at the 1 min time period. The fact that muscimol was in fact acting at Cl by activating GABA receptors was indicated by the finding that the GABA receptor antagonist drug, bicuculline, injected at the same site, reversed the hypotensive effect of muscimol. In addition, it appeared that there was a tonic GABAergic inhibitor tone present at Cl, as bicuculline, injected at this site in naive animals, resulted in a hypertensive response. It has been proposed that the important nucleus responsible for cardiorespiratory effects, occurring upon application of chemicals at the ventral surface of the medulla is the paragigantocellularis lateralis (Schlaefke and See, 1980; Keeler, Shutts, Chase and Helke, 1984; Brown and Guyenet, 1984; Brown and Guyenet, 1985). This is not the case in the cat as injection of muscimol in a dose that exerts a profound hypotensive and bradycardic effect at Cl had no effect on these indices of cardiovascular function at the time when cardiova~ular changes occurred from injection into Cl. The present data fit with corresponding data obtained by Ross and colleagues (1984) in the rat. These investigators reported that injection of GABA into the Cl area caused transient dose-dependent hypotension and bradycar~a and injection of bicuculline into the Cl area caused elevation of arterial pressure. Similarly, Willette, Krieger, Barcas and Sapru (1983) reported that muscimol, injected in this general region of the vehtrolateral medulla, produced a pronounced fall in arterial pressure. Most importantly, Ruggiero, Meely, Anwar and Reis (1985) have demonstrated with immun~ytochemical techniques that GABAergic neurons are present in the Cl area of the rat. In the present study, it was possible to demonstrate that a GABA receptor agonist, administered systemically, also acted in the Cl area to produce hypo-
tension and bradycardia. This was shown by administering THIP by the intravenous route and reversing its hypotensive and bradycardic effect by injecting bicuculline into the Cl area. It should be noted that bicuculline could be acting as a physiological antagonist to THIP (and muscimol). If so, then a neuroexcitatory drug such as strychnine should also counteract the effects of THIP (and muscimol). However, this does not seem to be the case because injection of bicuculline alone had very little effect on arterial pressure and heart rate (see Table 2). The respiratory depressant effect of muscimol probably does not occur at any of these 3 sites. When injected into the Cl area, muscimol did decrease respiratory activity but the response was delayed relative to the time-action curve for the cardiovascular depressant effect of the drug. This implies that the respiratory effect is due to diffusion of muscimol to a site some distance from the Cl area. It is tentatively concluded that the site responsible for eliciting the respiratory depressant effect of muscimol is either on the ventral surface or close to the surface. Schlaefke and See (1980) reported cells located superficially between 0 and 100 pm below the surface with a maximum density at 3.5pm. These cells may be responsible for the respiratory depression that occurs after muscimol is placed on Schlaefke’s area. Several investigators have proposed that a major direct sympathoexcitatory outflow pathway from the ventrolateral medulla to the intermediolateral nucleus originates in the nucleus paragigantocellularis lateralis (PGL). This may be true for the rat (Brown and Guyenet, 1985) but, it is probably not true for the cat. Reasons for discounting this nucleus as a major source of sympathoexcitatory activity in the cat are as follows: (1) injection of glutamic acid into the nucleus paragigantocellularis lateralis in a previous study did not elicit a hypertensive response; instead, hypotension was observed; and (2) injection of muscimol into the nucleus paragigantocellularis lateralis in the present study had no immediate effect on cardiorespiratory activity. Hence, the nucleus paragigantocellularis lateralis of the cat differs from that of the rat in that activation of GABA receptors did not appear to inhibit sympathoexcitatory neurons (Sun and Guyenet, 1985), and totally different, changes in arterial pressure occurred when glutamic acid was injected into this nucleus (Gatti er al., 1986). It is conceivable that a tonic GABAergic system is present at the nucleus paragigantocellularis lateralis and/or at the lateral reticular nucleus. However, this system would have to be maximally active, and hence analogous to the system that has previously been described in the forebrain (Williford et ai., 1981). In an earlier study of GABAergic mechanisms in the forebrain controlling central sympathetic outflow, no effect of muscimol on arterial pressure was observed when it was restricted to the forebrain ventricles. However, injections of bicuculline into, and restricted to the forebrain ventricles evoked hypertension and
Site of action for the depressor effect of muscimol
tachycardia (Williford, Hamilton, DiMicco, Nor-, man, Yamada, Quest, Zavadil and Gillis, 1981). It was concluded that GABA is an important neurotransmitter in the forebrain region controlling central sympathetic outflow. The same may be true of the nucleus paragigantocellularis lateralis and the lateral reticular nucleus but this needs to be examined by injecting bicuculline, not muscimol, into these nuclei. To summarize, the present data indicate that changes in cardiorespiratory activity produced by applying drugs that affect GABAergic mechanisms to the intermediate area of the ventrai surface of the medulla occur from two different sites. Cardiovascular effects appear to be due to the action of drugs on neurons located in the Cl area, while respiratory effects appear to be due to the action of drugs on neurons located on the surface or close to the surface. A separation of cardiovascular effects from respiratory effects fits with earlier data obtained by Schlaefke and See (1980). They used either superficial coagulation or cooling of the intermediate area and found that there were separate neuronal systems responsibie for eliciting changes in cardiovascular and respiratory function. In fact, their data suggested that respiratory effects were elicited from superficial neurons, while arterial pressure effects were elicited from deeper structures. Previous results with the injection of r..-glutamic acid also indicated a separation of ventral medullary sites controlling cardiorespiratory activity. Injection of L-glutamic acid into the Cl area, the nucleus paragigantocellularis lateralis and the lateral reticular nucleus elicited changes in cardiovascular function and did not evoke respiratory changes that resembled those seen with surface application to the inte~ediate area (Gatti et al., 1986). Acknowledgements-This
work was supported by a grant from Marion Laboratories, Inc., Kansas City, Missouri.
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Keeler 3. R., Shults C. W., Chase T. N. and Helke C. J. (1984) The ventral surface of the medulla in the rap _-_. ~ha~acoiogic and autoradiographic lo~li~tion of ~~2~~~ cardiovascular e&cts. Brain Res. 297: Ross C. A:, Ruggiero D. A., Park D. H., Joh T. H., Sved A. F.. Femandez-Pardal J., Saavedra J. N. and Ppic .-_._ D. J.. (1984) Tonic vasomotor control by the rostra1 ventrolateral medulla: effect of electrical or chemical stimilation of the area containing Cl adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin. J. Neurosci. 4: 474494. Ruggiero D. A., Meely M. P., Anwar M. and Reis D. J. (1985) Newlv identified GABAeraic neurons in regions_”of __ the vdntrola~eral medulla which regulate blood piessure. Brain Res. 339: 171-177.
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F’rinted in Great
Vol. 26, Britain.
All
No. rights
5, pp.
423431,
1987
002%3908/87
reserved
Copyright
0
1987
$3.00
+ 0.00
Pergamon Journals Ltd
CARDIORESPIRATORY EFFECTS PRODUCED BY INJECTING DRUGS THAT AFFECT GABA RECEPTORS INTO NUCLEI ASSOCIATED WITH THE VENTRAL SURFACE OF THE MEDULLA P. J. GATTI, A. M. T. DASILVA and R. A. GILLIS Departments of Pharmacology and Medicine (Pulmonary Division), Georgetown University, Schools of Medicine and Dentistry, Washington, D.C. 20007, U.S.A. (Accepled 24 February 1986)
Summary-It has recently been shown that L-glutamic acid induced stimulation of cell bodies in a circumscribed area of the rostra1 ventrolateral medulla (RVLM) in the cat, produced increases in arterial pressure (AP), decreases in heart rate (HR) and transient apnea (Gatti, Norman, DaSilva and Gillis, 1986). The purpose of the present study was to determine if this same area was sensitive to GABA receptor agonists and antagonists. Injection of the GABA agonist muscimol(200 ng), into the rostra1 ventrolateral medulla of cats anesthetized with chloralose produced a precipitous and immediate fall in arterial pressure (-95 k 4.6) and heart rate (-31 + 5.9; n = 4, P < 0.05). Maximal cardiovascular effects could only be achieved if muscimol was injected bilaterally. These effects of muscimol on arterial pressure were dose-dependent. Time-action curves for the effects of muscimol on arterial pressure and respiration were different. Hypotension occurred first and was followed later in time by a decrease in minute ventilation. Within 30 min all animals were apneic after the 200 ng dose. The cardiovascular effects of muscimol were reversed by the injection of the GABA receptor antagonist bicuculline. These data indicate that stimulation of GABA receptors in the rostra1 ventrolateral medulla produced selective cardiovascular effects and that respiratory neurons sensitive to GABA are apparently not localized with these cardiovascular neurons. Key words: muscimol, bicuculline, arterial blood pressure, heart rate, respiratory activity, cats, nucleus reticularis rostroventrolateralis, nucleus paragigantocellularis lateralis, lateral reticular nucleus.
Previous results, as well as the earlier results of Feldberg (1976), indicate that there is an important area on the ventral surface of the medulla where CNS neurotransmitters, such as y-aminobutyric acid (GABA) and glycine, produce a depressant effect on cardiorespiratory activity. This site is referred to as either Schlaetke’s area or the intermediate area (Schlaefke, 1981). Primary interest in this area stems from the findings that an important GABAergic synapse appears to exist at this site to control cardiorespiratory activity (Yamada, Norman, Hamosh and Gillis, 1982; Yamada, Moerschbaecher, Hamosh and Gillis, 1983). In an attempt to determine the specific location of GABAergic synapses at this site, the technique of injecting glutamic acid into distinct areas of the “ventral medulla” where GABAergic drugs could diffuse when placed topically on Schlaefke’s area was employed. Based on data obtained with this technique, sites have been found where injection of glutamic acid elicited cardiovascular effects. These are the “glycine sensitive area” described by Guertzenstein and Silver (1974) which seems to be synonymous with the Cl area described by ROSS, Ruggiero, Park, Joh, Sved, FemandezPardal, Saavedra and Reis (1984), the lateral reticular 423
nucleus (LRN) and the nucleus paragigantocellularis lateralis (PGL). The purpose of the present study was to determine whether a GABAergic synapse, controlling cardiorespiratory function, is present at any of these sites, as defined by the glutamic acid experiments (Gatti et al., 1986).
METHODS
Adult cats of either sex (164.6 kg) were anesthetized with alpha-chloralose, 70-80 mg/kg, (i.v.>. A femoral artery and vein were cannulated for measurement of arterial blood pressure and systemic administration of drugs, respectively. The trachea was cannulated and fitted to a Fleisch pneumotachograph. The flow signal obtained was then integrated to obtain tidal volume. Respiratory rate was obtained from fast tracings of the volume signal. Heart rate was obtained from the pulsatile blood pressure using a cardiotachometer. Blood pressure, trachea1 air flow, tidal volume, heart rate and lead II of the electrocardiogram (ECG) were monitored continuously on a Buxco Pulmonary Mechanics Analyzer (Model 6). Rectal temperature was monitored
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and maintained between 37” and 38°C using a heat lamp. The ventral surface of the medulla was then exposed. A lon~tudinal midline incision was made in the neck. The trachea and oesophagus were retracted and the prevertebral muscles scraped from the basal plate of the skull. A portion of this plate was removed, creating a window 15 x 9 mm over the medullary surface. The dura was then cut and reflected and the cerebrospinal Ruid removed, exposing the surface of the medulla. Cotton wicks were placed at either end of the craniotomy to prevent cerebrospinal fluid from pooling on the ventral surface. The animal was then placed in a stereotaxic head holder in this position when injections were made. The GABA receptor agonist muscimol, and the antagonist bicuculline, were injected in a volume of 100-200 nl over a 15 set interval. These agents were injected through glass micropipettes pulled to an inside tip diameter of 4&50pm. Injections were made using a pressure injection system with a pump which delivered a steady stream of air which could be directed through the pipette. Only those injections which were histologically verified to be in the area of the nucleus paragigantocellularis lateralis, lateral reticular nucleus and rostra1 ventrolateral medulla were used in the analysis of the data. The injection of an equal volume of saline (0.9% NaCl) did not affect any cardiorespiratory parameter significantly. Injections were made in the nucleus paragigantocellularis lateralis and the lateral reticular nucleus with the aid of information provided in the figures of Taber’s paper (1961). Injections into the nucleus lateralis were para~gantocellularis 2.5-3.5 mm lateral to the basilar artery, 1 mm below the ventral surface of the medulla and OS-1Smm caudal to the trapezoid border. Injections into the lateral reticular nucleus were 4mm lateral to the basilar artery, 1 mm below the surface and 3.74.4mm caudal to the trapezoid border. Finally, drugs were injected into the rostal ventroIatera1 medulla. This area lies 4 mm lateral to the basilar artery and 1.5 mm caudal to the trapezoid border, and
Table 1. Cardiorespiratory Dose of muscimol per side
I ng before injection
No. of animals studied 3
After injection difference 100 ng before injection
4
After injection difference 200 ng before injection
4
After injection difference ‘P < 0.05 using the paired student’s ‘f’-test.
corresponds with the Cl area of adrenergic neurons recently described in the rat (ROSS et al., 1984). The GABAergic drugs were dissolved in 1% fast green-saline solution for the purpose of marking the site of injection. To localize the site of injection, the brainstem was removed and stored in formalin until one day before sectioning, when they were transferred to 5% sucrose and refrigerated. The medulla was sectioned in 40-60 pm slices on a cryostat. When the injection site was located, those slides containing the site were stained with neutral red to distinguish cell bodies, and the injection site was photographed. The following drugs were used: muscimol (Boehringer Mannheim, GmbH, W. Germany), bicuculline methiodide and fast green (Sigma Chemical Co., St. Louis, Missouri), alpha-chloralose (Establissements Kuhlmann, Paris, France) and THIP (a gift from Dr 0. Svendsen, H. Lundbeck & Co. A/S, Copenhagen). The values stated in the text are means plus or minus the standard error of the mean @EM) and were taken at the time of the peak changes in CardiovascuIar function. Statistical analyses were performed using Student’s paired l-test with P < 0.05 being the criterion for statistical significance. Linear regression analysis was also performed to determine the line of best fit for the data and for determining whether dose-response curves had slopes significantly different (P < 0.05) from zero. RESULTS
Eflects of injection of muscimol into the rostra1ventrolateral medulla
In 11 animals, muscimol (l-200 ng) was injected bilaterally (total doses of 2-400ng) into the rostra1 ventrolateral area (i.e. glycine-sensitive area as described by Guertzenstein and Silver, 1974) and the data are summarized in Table 1. As can be seen, injection of muscimol produced significant (P < 0.05) dose-dependent decreases in arterial pressure. The other significant effects that were noted with muscimol were decreases in tidal volume (VT) with the largest dose tested (Table l), and decreases in heart
effects of muscimol after injection into the Cl area
Mean blood pressure (mmHg)
Heart rate (beats/min)
170 f 12
201 + 19
155 f 13 -15 + 3.2*
166k 17 - 35 * 5.0*
Tidal volume (ml)
Respiratory rate (breathsimin)
Respiratory minute volume (ml/min)
19 * 1.7
14 * 2.3
216 i $9
17 f 0.2 -2.0 r 1.1
12* 1.7 1.3 f 1.7
216 + 20 -60+46
156 & 18
165 i 20
19 + 0.8
18 rf: 1.1
352 + 17
97216 -59 &-12*
14oi I5 -25+11
14-i: 1.3 -5 * 1.7
16 i 3.4 -2.0 & 2.3
242 + 61 -110+55
160 f 3
135 + 19
33 + 1
12 + 4.0
400 + 40
64+6 -95 k 4.6,
103 k 15 -31 +6*
2s + 1 -8+0.0*
13k2.4 + 1.3 * 1.3
338 + 65 -61 k 21
Site of action for the depressor effect of muscimol
Fig. 1. Time-action curves of the changes in mean arterial pressure (BP) and tidal volume (VT) produced by the bilateral injection of 100 ng of muscimol(200 ng total dose) into the rostra1 ventrolateral medulla of a single animal. Note that at the time of the peak fall in mean arterial pressure, tidal volume only began to decrease.
rate with 2 of the 3 doses tested. These values for Vr and heart rate were taken at the time of peak depression in arterial pressure. The decreases in arterial pressure and heart rate occurred simultaneously and were observed within a few seconds after the bilateral injection of muscimol (Fig. 1). The maximal effect on both arterial pressure and heart rate occurred at approximately 1 min after bilateral injection of the largest dose and the effects persisted for about 30 min with the 1 ng dose, and for the duration of the experiment (i.e. for 2-3 hr) with the two larger doses. A bilateral injection of muscimol was required in order to observe a distinct effect on arterial pressure and heart rate. In some cases, a unilateral injection of the 2 larger doses of muscimol did elicit a small decrease in arterial pressure (i.e. approximately a l&15 mmHg drop in pressure). The depressant effect of muscimol on respiration followed a different time-action curve (Fig. 1). This was most clear from the data obtained using the 200 ng dose. At the time of the peak depressant effect of muscimol on cardiovascular function, there was no significant change in respiratory minute volume, despite the occurrence of a significant reduction in tidal volume (Table 1). With time, a significant decrease in respiratory minute volume occurred and by 30 min after bilateral injection of muscimol, all animals tested were apneic (Fig. 1). Respiratory arrest persisted for the duration of the experiment (i.e. for 2-3 hr). A representative experiment showing the effect of the largest dose of muscimol on cardiorespiratory activity appears as
HR (bpm) 2 3 0
t
t
Right
Left
425
muscimol 200 ng/site
Fig. 2. Representative experiment showing the effects of the injection of muscimol into the rostra1 ventrolateral medulla on tidal volume (V,), arterial blood pressure (BP) and heart rate (HR). Note that only when the muscimol was injected bilaterally did the arterial pressure fall to its lowest level. Inspiration is upward. Paper speed is 1 mmjsec.
P. J. GATTI et al.
426
Fig. 2. Respiratory arrest was not observed with the smallest dose tested. The location of the micropipette tip in the brain is shown in Fig. 3, and based on previous data, mapping the distribution of epinephrine-synthesizing neurons on the hindbrain of the rat, the tip was in an area described as the Cl area (Ross et al., 1984).
FGL
0,
LRN
RVL 1
1
I
Effect of injection of muscimol into the nucleus parag~guntocellularis later&s (PGL) In 4 animats, muscimoi (100 ng) was injected bilaterally (total dose of 200 ng) into the paragigantocellularis lateralis. At the time of the maximal decrease in arterial pressure and heart rate evoked by muscimol, injected into the Cl area, there was no significant decrease in these parameters with the injection of muscimol into the paragiganto~eIlula~s lateralis (Fig. 4). There was a delayed hypotensive and bradycardic effect with injection of drug into the nucleus paragigantocellularis lateralis. These responses were - 83 1: 4.0 mmHg (P < 0.05) and - 16 + 21 beats/min respectively, and occurred at 41 t_ 13 min after the injection of muscimoi. Respiratory depression was also noted (i.e. decrease in tidal volume of 26 + 4.6 ml) but this was delayed and only noted at 20 + 4.7 min after muscimol was administered. Efects of injection ~frn~sc~rn~l into the lateral reticular nucleus (L&V) In 3 animals, muscimol(100 ng) was injected bilaterally (total dose of 200 ng) into the lateral reticular nucleus. No significant effects on arterial pressure (Fig. 4) and heart rate (data not shown) were observed. Respiratory depression was also not noted over the first 30min after the drug was injected. Later, however, at approximately 4.5-60 min after injection, each animal exhibited a decrease in tidal volume that progressed to apnea. At this later time, there was still no decrease in arterial pressure and heart rate. Eflecfs of injection of bicuculiine met&o&& Ci area
into the
In the 3 sites tested with muscimol, the only responsive site in terms of immediate changes in arterial pressure and heart rate was the Cl area. This finding raised two questions. First, is there a tonic GABA-ergic system in the Cl area that controls arterial pressure and heart rate? And second, are the Table 2. Cardiorespiratory
Expt type Before bicuculline After 500 ng/side of bicuculline Difference
Fig. 4. Fall in mean arterial pressure (MAP) produced by the bilateral injection of IOOng of muscimol (2OOng total dose) into either the nucleus paragigantocellularis lateralis (PGL), lacteral reticular nucleus (LRN) or the nucleus reticularis
rostroventrolateralis
(RVL).
effects of mu~imol due to the activation of GABA receptors in the Cl area? In an attempt to answer both questions, the GABA receptor antagonist drug bicuculline methiodide was injected into the Cl area of naive cats and cats that were exhibiting hypotension and bradycardia evoked by muscimol. In 5 naive animals, bicuculline methiodide (BMI) was injected bilaterally (500 ng/side) and the data are summarized in Table 2. As can be seen, injection of bicuculline methiodide produced a significant (P < 0.05) increase in arterial blood pressure. In addition, bicuculline methiodide also produced significant (P < 0.05) decreases in heart rate and respiratory minute volume. In order for these changes to occur, bicuculline methiodide had to be injected bilaterally; unilateral injections were ineffective in eliciting significant changes in cardiorespiratory function. The increase in arterial pressure and decreases in heart rate and respiratory minute volume occurred simultaneously and were observed within 5 min after the bilateral injection of bicuculline methiodide. The
effects of bicuculline after injection into the CI area
No. of z&mats studied
Mean blood preSSWe (mm&z)
Heart rate t~ats/min)
Tidal volume WI
5
152+_20
166 + 14
22 + 4.3
14 i 1.8
303 zk38
180 + 16
126 + 12
19 + 3.6
12+_ 1.7
197 + 36
+ 28 + 7.0’
-40 4 13’
-2.7 rt 3.3
-2.0 * 1.1
-106+_30*
*P =z0.05 using the paired Student’s t-test.
Respiratory rate (breatha/min)
Respiratory minute volume (ml/min)
Fig. 3. Photomicrograph showing the micropipette track in the brainstem of an animal in which muscimol was injected into the rostra1 ventrolateral medulla. ION = inferior olivary nucleus; RF = retrofacial nucleus x 42; bar = 0.5 mm.
427
Site of action for the depressor effect of muscimol
429
MUSCIMOL 2M) ng/ side R L
Fig. 5. Representative experiment showing the antagonism between muscimol and bicuculline in the rostra1 ventrolateral medulla on arterial pressure (BP) and heart rate (HR). The traces on the left illustrate the immediate hypotensive effect of 400 ng of muscimol (injected on the left (L) and right (R) sides). Five minutes after the peak fall in arterial pressure (right traces) bicuculline (3 pg) was injected unilaterally on the right side in the rostra1 ventrolateral medulla. Note the immediate antagonism produced by this unilateral injection of bicuculline. Paper speed was 1mm/set. This animal was artificially respired.
maximal effects on cardiorespiratory function occurred approximately 10 min after bilateral injection of the drug and the effects persisted for about 20-30min. In a few experiments, a smaller dose of bicuculline was tested (i.e. 100 ng) but was observed to produce no effects on cardiorespiratory activity. Bicuculline methiodide (l-3.4 pg/side) was injected into the Cl area of 3 cats that had received an injection of muscimol(200 ng/side) into the same site. These experiments were performed in artificiallyrespired animals in order to prevent drug-induced respiratory effects from influencing the arterial pressure and heart rate. Bilateral injections of muscimol decreased arterial pressure and heart rate by 100 +_13.3 mmHg and -30 f 0.0 beats/min. At the time of the maximal decreases in arterial pressure and heart rate, bicuculline methiodide was administered and it restored arterial pressure and heart rate to normal in each animal tested. Only a unilateral injection of bicuculline methiodide was required to produce a complete reversal of the decreases in arterial pressure and heart rate induced by muscimol. THIP, I.“. 200
r
A representative experiment showing the reversal of the effects of muscimol by bicuculline methiodide appears as Fig. 5. Eflects of injection of bicuculline methiodide into the Cl area of cats receiving an intravenous injection of THZP It is known that THIP (4,5,6,7,tetrahydroisoxazolo [5,4-C] pyridine-3-ol), a GABA receptor agonist, crosses the blood brain barrier after intravenous injection (Waszczak, Hruska and Walters, 1980). To determine whether THIP was acting at the rostal ventrolateral medulla to produce hypotension and bradycardia, this agent was first administered intravenously to artificially respired animals and maximal decreases in arterial pressure and heart rate were obtained. These decreases were 65 f 14 mmHg (P < 0.05) and 66 + 38 beats/min, respectively and occurred with doses of 2-4mg/kg (i.v.). Next, bicuculline methiodide was injected into the rostra1 ventrolateral medulla to see if reversal of the intravenous effects of THIP would occur. A unilateral bieuculline RV LM
4
BP
Fig. 6. Representative experiment showing the antagonism between intravenously-administered THIP and intracerebrally-administered bicuculline on arterial blood pressure (BP) and heart rate (HR). The left portion of the tracing illustrates the cardiovascular depression produced by the intravenous injection of THIP (4 mg/kg). Note the near production of cardiovascular collapse by this agent. Bicuculline (1.7 ng) was then injected unilaterally into the rostra1 ventrolateral medulla (RVLM) at the second arrow. Note the dramatic and immediate antagonism of the effects of THIP by bicuculline. This animal was artificially respired. Paper speed was 1 mm/set.
P. 1.GATTIet al.
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injection of bicuculline methiodide (l-3 pg) fully counteracted the hypotensive and bradycardic effects of THIP. Increases of 87 + 25 mmHg (P < 0.05) and 73 _I 58 ~ats/min, respectively, were noted. A dramatic representation of this interaction is illustrated in Fig. 6. DISCUSSION
The present data with the GABA receptor agonist drug, muscimol, injected into the lateral reticular nucleus and nucleus paragigantocellularis lateralis clearly indicate that the GABA-sensitive site, associated with Schlaefke’s area (also known as the intermediate area), is the Cl area. (Also referred to as nucleus reticularis rostroventrolateralis Ross et al., 1984). Injection of muscimol at this site produced an immediate (within 1 min) fall in arterial pressure and heart rate. In contrast, injection of the largest dose of muscimol into either the lateral reticular nucleus or nucleus paragigantocellularis lateralis had no significant effect on cardiorespiratory activity at the 1 min time period. The fact that muscimol was in fact acting at Cl by activating GABA receptors was indicated by the finding that the GABA receptor antagonist drug, bicuculline, injected at the same site, reversed the hypotensive effect of muscimol. In addition, it appeared that there was a tonic GABAergic inhibitor tone present at Cl, as bicuculline, injected at this site in naive animals, resulted in a hypertensive response. It has been proposed that the important nucleus responsible for cardiorespiratory effects, occurring upon application of chemicals at the ventral surface of the medulla is the paragigantocellularis lateralis (Schlaefke and See, 1980; Keeler, Shutts, Chase and Helke, 1984; Brown and Guyenet, 1984; Brown and Guyenet, 1985). This is not the case in the cat as injection of muscimol in a dose that exerts a profound hypotensive and bradycardic effect at Cl had no effect on these indices of cardiovascular function at the time when cardiova~ular changes occurred from injection into Cl. The present data fit with corresponding data obtained by Ross and colleagues (1984) in the rat. These investigators reported that injection of GABA into the Cl area caused transient dose-dependent hypotension and bradycar~a and injection of bicuculline into the Cl area caused elevation of arterial pressure. Similarly, Willette, Krieger, Barcas and Sapru (1983) reported that muscimol, injected in this general region of the vehtrolateral medulla, produced a pronounced fall in arterial pressure. Most importantly, Ruggiero, Meely, Anwar and Reis (1985) have demonstrated with immun~ytochemical techniques that GABAergic neurons are present in the Cl area of the rat. In the present study, it was possible to demonstrate that a GABA receptor agonist, administered systemically, also acted in the Cl area to produce hypo-
tension and bradycardia. This was shown by administering THIP by the intravenous route and reversing its hypotensive and bradycardic effect by injecting bicuculline into the Cl area. It should be noted that bicuculline could be acting as a physiological antagonist to THIP (and muscimol). If so, then a neuroexcitatory drug such as strychnine should also counteract the effects of THIP (and muscimol). However, this does not seem to be the case because injection of bicuculline alone had very little effect on arterial pressure and heart rate (see Table 2). The respiratory depressant effect of muscimol probably does not occur at any of these 3 sites. When injected into the Cl area, muscimol did decrease respiratory activity but the response was delayed relative to the time-action curve for the cardiovascular depressant effect of the drug. This implies that the respiratory effect is due to diffusion of muscimol to a site some distance from the Cl area. It is tentatively concluded that the site responsible for eliciting the respiratory depressant effect of muscimol is either on the ventral surface or close to the surface. Schlaefke and See (1980) reported cells located superficially between 0 and 100 pm below the surface with a maximum density at 3.5pm. These cells may be responsible for the respiratory depression that occurs after muscimol is placed on Schlaefke’s area. Several investigators have proposed that a major direct sympathoexcitatory outflow pathway from the ventrolateral medulla to the intermediolateral nucleus originates in the nucleus paragigantocellularis lateralis (PGL). This may be true for the rat (Brown and Guyenet, 1985) but, it is probably not true for the cat. Reasons for discounting this nucleus as a major source of sympathoexcitatory activity in the cat are as follows: (1) injection of glutamic acid into the nucleus paragigantocellularis lateralis in a previous study did not elicit a hypertensive response; instead, hypotension was observed; and (2) injection of muscimol into the nucleus paragigantocellularis lateralis in the present study had no immediate effect on cardiorespiratory activity. Hence, the nucleus paragigantocellularis lateralis of the cat differs from that of the rat in that activation of GABA receptors did not appear to inhibit sympathoexcitatory neurons (Sun and Guyenet, 1985), and totally different, changes in arterial pressure occurred when glutamic acid was injected into this nucleus (Gatti er al., 1986). It is conceivable that a tonic GABAergic system is present at the nucleus paragigantocellularis lateralis and/or at the lateral reticular nucleus. However, this system would have to be maximally active, and hence analogous to the system that has previously been described in the forebrain (Williford et ai., 1981). In an earlier study of GABAergic mechanisms in the forebrain controlling central sympathetic outflow, no effect of muscimol on arterial pressure was observed when it was restricted to the forebrain ventricles. However, injections of bicuculline into, and restricted to the forebrain ventricles evoked hypertension and
Site of action for the depressor effect of muscimol
tachycardia (Williford, Hamilton, DiMicco, Nor-, man, Yamada, Quest, Zavadil and Gillis, 1981). It was concluded that GABA is an important neurotransmitter in the forebrain region controlling central sympathetic outflow. The same may be true of the nucleus paragigantocellularis lateralis and the lateral reticular nucleus but this needs to be examined by injecting bicuculline, not muscimol, into these nuclei. To summarize, the present data indicate that changes in cardiorespiratory activity produced by applying drugs that affect GABAergic mechanisms to the intermediate area of the ventrai surface of the medulla occur from two different sites. Cardiovascular effects appear to be due to the action of drugs on neurons located in the Cl area, while respiratory effects appear to be due to the action of drugs on neurons located on the surface or close to the surface. A separation of cardiovascular effects from respiratory effects fits with earlier data obtained by Schlaefke and See (1980). They used either superficial coagulation or cooling of the intermediate area and found that there were separate neuronal systems responsibie for eliciting changes in cardiovascular and respiratory function. In fact, their data suggested that respiratory effects were elicited from superficial neurons, while arterial pressure effects were elicited from deeper structures. Previous results with the injection of r..-glutamic acid also indicated a separation of ventral medullary sites controlling cardiorespiratory activity. Injection of L-glutamic acid into the Cl area, the nucleus paragigantocellularis lateralis and the lateral reticular nucleus elicited changes in cardiovascular function and did not evoke respiratory changes that resembled those seen with surface application to the inte~ediate area (Gatti et al., 1986). Acknowledgements-This
work was supported by a grant from Marion Laboratories, Inc., Kansas City, Missouri.
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