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GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex
Brain Research, 297 (1984) 175-180 Elsevier
175
BRE 20107
GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex KATHRYN A. YAMADA 1, ROBIN M. McALLEN: and ARTHUR D. LOEWY 1 1Departrnent of Anatomy and Neurobiology, Washington Universi(v School of Medicine, 660 S. Euclid Avenue. St. l.oui~. MO 63110 ( U. S. A. ) and 2Department of Physiology, University Of Bristol, Bristol, BS 81 TD ( U. K. )
(Accepted November 29th, 1983) Key words." GABA - - bicuculline - - picrotoxinin - - ventral medulla oblongata - - baroreceptor reflex - - cardiovascular regulation
Application of y-aminobutyric acid (GABA) antagonist drugs (bicuculline, picrotoxinin) to the intermediate region of the ventral surface of the medulla oblongata of gallamine paralyzed, a-chloralose anesthetized cats produced a dose-dependent blockadc of the baroreceptor reflex. GABA antagonists applied to the rostral and caudal areas did not block the baroreceptor reflex. When strychnine was applied to the ventral medulla, it did not block the baroreceptor reflex. The diffusion of [3H]bicucullinewas studied by scintillation counting and autoradiography and found not to penetrate more than 2 mm from the ventral medullary surface. These rcsults suggest that a GABAergic synapse lies within the first 2 mm of the ventral medulla and is involved in baroreceptor modulation of the sympathetic outflow.
The interaction between centrally generated vaso-
tion of drugs, respectively. Rectal temperature was
motor tone and arterial baroreceptor activity is one of the major determinants involved in the neural con-
formed, and the cats were paralyzed with gallamine
trol of blood pressure. While the source of vasomotor tone is still u n k n o w n , recent experiments on cats have indicated that its transmission to the sympathetic preganglionic neurons depends critically on the activity of a group of neurons close to the ventral surface of the medulla oblongatalO,15,lg, 23. Bilateral removal of their activity, by a variety of means (drugs 7,9.23, cooling I~, lesionsm,18), reduces resting blood pressure to levels approaching that of spinal animals. Central nervous pathways of the baroreceptor reflex, however, apart from its immediate input and output nuclei, are still uncertain > . We present data here which suggest that a major synapse involved in the baroreceptor reflex pathway is located in the region of the ventral medulla, and that G A B A antagonist drugs block the reflex at this site. Cats (1.3-6.7 kg, n = 59) were anesthetized with 70-80 mg/kg i.v a-chloralose (Sigma, St. Louis). A femoral artery and vein were cannulated for measurement of blood pressure and systemic administra-
maintained at 37 _+ 1 °C. A tracheostomy was pertriethiodide (5-15 mg/kg, as needed, Davis and Geck, American Cyanamid, Pearl River, NY) and artificially ventilated with room air. End tidal CO,concentration was maintained at 5.5 + 0.5(7c (Beckmann Infrared Gas Analyzer). The ventral surface of the medulla was exposed. Raw sympathetic nerve activity was recorded from the central cut end of a right renal nerve. A unilateral carotid blind sac was preparedl~. The external carotid artery was cannulated for measurement of carotid sinus pressure and for baroreceptor stimulation. After electrophysiological identification, the contralateral carotid sinus nerve and both aortic depressor nerves were cut; the vagi were kept intact. The baroreceptor reflex was tested by inflating the blind sac with heparinized lactated Ringer's solution from an external reservoir (with a pressure of approximately 200 mm Hg) after clamping the common carotid artery. Bicuculline methiodide (BMI, Pierce Biochemicals, Rockford, IL) was dissolved in phosphate buf-
Correspondence." A. D. Loewy, Department of Anatomy and Neurobiologv, Washington University School of Medicine, 660 S. Euclid Avenue. St. Louis, MO 63110, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
176 PBS BMI 8 rain CONTROL lmM AFTERDRUG
A
300 I HEARTRATE ( beols/min )
j 200 I /
100L.
30o[ ARTERIAL "A,&" BLOOD(mmHg)PRESSURE150[
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300[- ~ - , ~ -
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STRYCHPBS NINE 8 min CONTROL SmM AFTERDRUG
1
Fig. 1. Effect of topical application of bicuculline and strychnine to the intermediate area of the ventral medulla on the baroreceptor reflex response. A: bicuculline-induced blockade of the depressor response elicited by carotid sinus baroreceptor stimulation accompanied by hypertension and increased sympathetic nerve activity with bursting. 1 mM BMI was topically applied bilaterally to the intermediate area of ventral medullary surface to produce the response. B: bilateral topical application of 5 mM strychnine to the intermediate area did not attenuate the baroreceptor reflex response. C: diagram showing the 3 areas of the ventral medullary surface, depicted by the stippled areas: rostral, intermediate, and caudal. The black squares indicate the sites where bicuculline or picrotoxinininduced blockade of the baroreceptor reflex could be produced. Abbreviations: BMI, bicuculline methiodide; trap, trapezoid bodies; pyr, pyramidal tract; V-XII, cranial nerves. fered saline (PBS, pH 7). Strychnine, nipecotic acid, and G A B A (Sigma) were dissolved in PBS daily. Picrotoxinin (Sigma) was dissolved in 10% ethanol in PBS daily. Drugs and vehicle controls were applied bilaterally to the ventral medullary surface via square cottonoid pledgets (1.5-2.5 mm 2) containing between 10 and 50/A of the drug solution. Neither PBS nor 10% ethanol in PBS had any effects when topically applied. Statistical analyses were performed using the Student's t-test for paired data, analysis of variance and the T u k e y - K r a m e r multiple comparisons test, and linear regression by the method of A N O V A . Criterion for statistical significance was P < 0.05. BMI, a potent G A B A receptor antagonist 11, was topically applied bilaterally to the 3 areas 17 of the ventral medullary surface of 16 cats (Fig. 1C). Only at the intermediate area did BMI (10/~M to 5 mM) produce dose-dependent, reversible increase in
mean arterial blood pressure, heart rate and renal sympathetic nerve activity (Fig. 1A. 2A. B). Concomitant with these effects was an attenuation of the reflex hypotension (up to 85%) and bradycardia (up to 100%) elicited by carotid sinus baroreceptor stimulation (Fig. 1A. 2C. D) and of the baroreceptor reflex-induced silencing of sympathetic nerve activity (Fig. 1A). Since cats were paralyzed with gallamine triethiodide, which has a significant blocking action on the vagus 5. heart rate changes seen in this study reflect sympathetic rather than parasympathetic function. The pressor response to BMI started within 10-60 s of application, whereas the reduced reflex response was first measured after 1.5-7 min. with peak effects after 3.5-7 min*. Recovery was followed in 12 cats. There was no significant difference with respect to blood pressure, heart rate. reflex hypotens,on and bradycardia, or reflex timing measurements between PBS control values and recovery values after 50/~M
* In two experiments, we reduced BMI-induced sympathetic excitation by hyperventilation. This reduced arterial blood pressure and abolished the characteristic bursting pattern seen in the renal nerve recordings without affecting the reduced baroreceptor reflex response.
177
cat), or 5 mM (5 cats) BMI in the 7
perficial aspect of the ventral surface of the medulla.
cats which showed recovery (not shown). The time
In an effort to determine what possible structures
course for complete reversal was 5-17 rain after removal of the last dose of BMI and a PBS rinsing of the
may have been effected by bicuculline to cause
ventral surface.
diffusion of [SH]bicuculline methylchloride (BMC)
Picrotoxinin (PTXN), the active metabolite of picrotoxin ez. a G A B A antagonist thought to block the
graphy. Based on scintillation counts (n = 4), spread
(1 cat), 1 mM (1
blockade of the baroreceptor reflex, we followed the by two methods: scintillation counting and autoradio-
G A B A receptor-linked chloride ionophore el, was applied bilaterally to the intermediate area of 6 cats. PTXN (100 n M - 5 raM) also produced attenuation of the reflex hypotension (Fig. 2E) and bradycardia,
A~ 2OUr,
"/*i
300[ • B,cuculi,ne
B
z ~' -E,
superimposed upon a background of increasing sympathetic tone, as demonstrated by increase in arterial blood pressure and sympathetic nerve activity. Re-
C
D • 5o[
covery was observed after 1 mM PTXN in 3 of the 5 cats. BMI and PTXN are both known to be powerful convulsant agents. To test for any non-specific excit-
gg
if
atory effects that these drugs may have been exerting another convulsant agent, strychnine, was applied bilaterally to the intermediate area in 7 cats. Strychnine (l /aM to 10 mM), a glycine receptor antagonist ~, produced small, inconsistent effects on blood pressure and heart rate (Fig. 1B, 2A, B), but had no blocking action on the baroreceptor reflex (Fig. 1B, 2C, D). These observations, coupled with the findings that the affinity of strychnine for glycine receptors is even higher than that of BMI for G A B A receptors ~,a,:~, argue that the actions of BM1 and PTXN are specific, and that a glycine synapse is not involved. Attempts to enhance the baroreflex with nipecotic acid (1-10 raM, n = 6), a competitive inhibitor of the high affinity G A B A uptake system 12, were unsuccessful. This may be due to any one of several factors, e.g. the domination of a glial reuptake system rather than a neuronal reuptake mechanism at this CNS site, inadequate penetration of the drug, or simply, the normal action of G A B A at this synapse is already maximal. Although drugs topically applied to the ventral medullary surface elicited effects in as little as a few seconds, it is not known what anatomical structure(s) may mediate these evoked responses. Electron micrographic studies of the ventral surface of the medulla oblongata~ have revealed an extensive pore and vascular system by which drugs could easily diffuse dorsally to a site of action deeper than the most su-
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Fig. 2. Effects of various drugs on baseline blood pressure and heart rate, and on reflex-induced changes in blood pressure and heart rate. Dose-response curves for bicuculline (@) and strychnine ([3) applied to the intermediate area of the ventral surface on mean arterial pressure (A) and heart rate (B). Note the marked pressor response produced by BMI (B). • Significant slope and no significant deviation from linearity using linear regression by the method of analysis of variance. PBS, phosphate buffered saline control. Dosc-rcsponse curves for bicuculline (@) and strychnine (~) applied to the intermediate area on the vasomotor (C) and cardiac components (D) of the baroreceptor reflex. Bicuculline produced a dosedependent attenuation of the baroreceptor reflex response. E shows the effect of various drugs on the vasomotor component of the baroreceptor reflex response as a percentage change from the control value. * 5 mM dose of bicuculline and picrotoxinin produced a significant difference from control, determined by analysis of variance and the Tukey-Kramer multiple comparisons test run on the set of doses for each drug condition, P < 0.05. This analysis was used for the dose ranges tested for each drug at the intermediate area. For all other drug conditions only the highest dose found effective at the intermediate area (5 mM) was used. In these cases, the effects of bicucufline on the reflex response were not found to be significantlydifferent from control using the Student's t-test for paired data.
1"78 of [3H]BMC (198-940 nCi) topically applied to the intermediate area f o r m e d a gradient (Fig. 3). The highest concentration (2.15-2.33 nCi/mg tissue) was found at the ventral surface, 50% of that concentration was found 0.5-0.9 m m below the surface, 10% at
ceptor blockade, A t the lower end of the dose range used, tissue values of bicuculline within the first 2 mm of the ventral m e d u l l a would a p p r o a c h those found to displace G A B A (IC50 = 3 fzM 24) from its receptors in in vitro binding studies. Second, structures such as the nucleus ambiguus and the nucleus of tractus solitarius can be excluded as the site where B M I blocks the reflex. Earlier studiesT,9,15,23 have implicity as-
1,8-2.5 mm below the surface, and no detectable [3H]BMC 3.5-3.7 m m below the surface. A u t o r a d i o graphic visualization of the [3H]BMC diffusion using tritium-sensitive film (Ultrofilm; L K B B r o m m a , Sweden) revealed grain densities only in the first 1.5 m m below the ventral surface of the m e d u l l a (n = 5, exposure = 4-8 weeks).
sumed that when drugs are applied topically to the ventral medullary surface, the main site of action is on the superficial neurons of the ventral medulla. O u r data indicates that [3H}bicuculline can reach d e e p e r structures including the A1 or C [ cell groups and the inferior olivary nucleus as well as the ventral surface itself. Clearly, this means that topical appli-
F r o m these results we can form two conclusions. First, the concentrations of B M I seen in the superficial 2 m m of brainstem are a p p r o p r i a t e for G A B A re-
Nucleus
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0.4 0.8 1.2
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mm from Ventral Surface of Medulla Oblongota Fig. 3. Diffusion of [3H]bicuculline methylchloride (BMC, New England Nuclear, Boston, MA; spec. act. = 82.0 Ci/mmol) was studied after application to the intermediate area of the ventral medullary surface. [3H]BMC was topically applied via cottonoid pledgets. After 6.5-11 min of drug exposure, the pledgers were removed and the brainstem was blotted, dissected out and frozen in Freon chilled on dry ice. 20 am horizontal sections were cut at 20 °C in a cryostat, weighed, solubilized (1 N NaOH, 60 °C), and counted (Packard Instruments). Fig. 3A shows the degree of penetration of [3H]BMC as determined by scintillation counting. Ten to twenty 20/~m horizontal sections were pooled in order to determine nCi/mg tissue. The/~M concentrations of bicucutline were calculated based on the assumption that one gram of tissue is equivalent to one ml of solvent. Fig. 3B illustrates the anatomical landmarks of the medulla corresponding to the levels at which the scintillation counts were made.
179 cation of drugs is a useful way to screen drugs but that
are f o u n d c e n t e r e d in the i n t e r m e d i a t e area, and
m o r e refined t e c h n i q u e s will be n e e d e d to localize
mostly within 1.5 m m of the ventral m e d u l l a r y sur-
exactly w h e r e the G A B A e r g i c synapse lies that m o d -
face l, the exact a r e a affected in the p r e s e n t study.
ulates b a r o r e c e p t o r activity. In s u m m a r y , our d a t a suggest that within 2 m m of
T h e p r e s e n t study does not tell us a b o u t the vagal
the ventral m e d u l l a r y surface at the i n t e r m e d i a t e
have m a s k e d its e x p r e s s i o n , nor does it tell us h o w
area there is a G A B A e r g i c synapse i n v o l v e d in b a r e -
m u c h b a r o r e c e p t o r inhibition of s y m p a t h e t i c activity
r e c e p t o r m o d u l a t i o n of v a s o m o t o r t o n e , T h e sim-
o c c u r r e d at m o r e rostral or spinal levels2, ~. I n d e e d ,
plest i n t e r p r e t a t i o n is that this synapse is an integral
such alternate p a t h w a y s may account for the r e m a i n -
part of the b a r o r e c e p t o r reflex p a t h w a y , but we can-
der of the reflex we w e r e u n a b l e to block.
limb of the b a r o r e c e p t o r reflex as g a l l a m i n e w o u l d
not exclude the possibility that this b l o c k a d e of the G A B A inhibition is of a d i f f e r e n t origin which may
The study was s u p p o r t e d by U S P H S G r a n t s H L -
have affected b a r o r e c e p t o r reflex gain. P r e s u m a b l y
25449, HL-07275 and a G r a n t - i n - A i d (83-656) f r o m
this synapse acts on those cells of the i n t e r m e d i a t e
the A m e r i c a n H e a r t A s s o c i a t i o n , with funds contrib-
area
uted in part by the Missouri
previously
shown
to
mediate
vasomotor
Heart
Association.
t o n e Is,2). and which may have a direct p r o j e c t i o n to
A . D . L . is an E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i -
the s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n s in the spinal
can H e a r t A s s o c i a t i o n .
c o r d 1,14. Interestingly, such spinally p r o j e c t i n g cells
1 Amendt, K., Czachurski, J., Dembowsky, K. and Seller, H., Ncurones within the 'chemosensitive area' on the ventral surface of the brainstem which project to the intermediotateral column, t~fhiger's Arch. ges. Physiol.. 375 119781 289--292. 2 Cootc. J. H. and Macleod, V. H., Evidence for the involvement in the baroreceptor reflex of a descending inhibitory pathway, J. Physiol. (Lend,), 241 (1974) 477-496. 3 Curtis, D. R., Duggan, A. W., Felix, D. and Johnston, G. A. R., Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 ( 1971 ) 69-96. 4 Curtis, D. R., Duggan, A, W,, Felix, D., Johnston, G. A. R. and McLennan, H., Antogonism between bicuculline and GABA in the cat brain, Brain Research, 33 119711 57-73. 5 Della Bella, D., Rognoni, F. and Gopal, K., Curare-like drugs and cardiovagal synapses: comparative study in vitro on isolated guinea-pig vagus-heart preparation, J. Pharm. Pharmacol.. 13 119611 93-97. 6 Dermietzel, R., Central chemosensitivity, morphological studies. In H. |l. Loeschcke (Ed.), Acid-Base Homeostasis of the Brain Extracellular Fluid and the Respirato O, Control System. Thiemc. Stuttgart. 1976, pp. 52-66. 7 Feldberg. W. and Guertzenstcin, P. G., Vasodepressor effects obtained by drugs acting on the ventral surface of the brain stem. J. Physiol. (Lond.I. 258 (1976) 337-355. 8 Gebber, G. L., Taylor, D. G. and Weaver, L. C., Electro* physiological studies on organization of central vasopressor pathaays. Amer. J. Phys'iol,, 224 11973) 470-481. 9 Guerlzenstein. P. G.. Blood pressure effects obtained by drugs applied to the ventral surface of the brain stem, J. Physiol. (Lend. ~. 229 (1973) 395-408. 111 Guertzcnstein. P. G. and Silver, A., Fall in blood pressure produced from discrete regions of the ventral surface of the medulla by glycine and lesions, J. Physiol. (Lend.), 242 (1974) 489-503.
11 Johnston, G. A. R., Neuropharmacology of amino acid inhibitory transmitters, Ann. Rev. Pharmacol. Toxicol., 18 ( 19781 26%289. 12 Krogsgaard-Larsen, P. and Johnston, G. A. R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J, Neurochem.. 25 (19751 797-802. 13 Krnjevic, K,, Chemical nature of synaptic transmission in vertebrates, Physiol. Rev.. 54 (1974) 41 ~-541). 14 Loewy, A. D. and McKellar, S,, Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat, Brain Research, 211 (1981) 146--152. 15 McAIlen, R. M., Nell, J. J. and Loewy, A. D., Effects of kainic acid applied to the ventral surface of the medulla obIongata on vasomotor tone, the baroreceptor reflex and hypothalamic autonomic responses, Brain Research, 238 (i982) 65-76. 16 Moissejeff, E., Zur Kenntnis des Carotissinus-reflexes, Z. ges. exp. Med., 53 11927) 696--7(14. 17 Schlaefke, M. E., Central chemoscnsitivity: a respiratory drive, Rev. Physiol. Biochern. Pharmacol., 911 11981) 171-244. 18 Schl~fke, M. E. and Loeschcke, l:l. H., Lokalisation eines an der Regulation yon Atmung und Kreislauf beleiligten gebietes an der ventralen Oberflfiche der Medulla oblongata durch Kalteblockade. pfh~ger's Arch. ,ees, Phvfiol., 297 (1967) 201-22tl. 19 Schl~tke, M. E. and See, W. R.~ Ventral medullary surface stimulus response in relation to ventilator~ and cardiovascular effects. In H. P. Koepchen, S. M. [lilton and A. Trzebski (Eds.), Central biteraction Betu'ee~z Respiratory and Cardiovascular ('ontrol 5),stems. Springer, Berlin, 1981/. pp. 56-63. 211 Spyer, K. M., Neural organisation and contn~l of the barereceptor reflex, Rev, Physiol. Biochem. Pharmacol.. 88 (1981) 23-124. 21 "Ficku, M. K,, Van Ness, P. C., Haycock, J. W., Levv. W.
180 B. and Olson, R. W., Dihydropicrotoxinin binding sites in rat brain: comparison to GABA receptors, Brain Research, 150 (1978) 642-647. 22 Wang, S. C. and Ward, J. W., Analeptics, Pharmac. Ther. B, 3 (1977) 123-165. 23 Yamada, K. A., Norman, W. P., Hamosh, P. and Gillis, R. A., Medullary ventral surface GABA receptors affect r e s -
piratory and cardiovascular function, Brain Research, 248 (1982) 71-78. 24 Zukin, S. R.. Young, A. B. and Snyder, S. H., Gammaaminobutyric acid binding to receptor sites in the rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 7l (1974) 4802--4807.
175
BRE 20107
GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex KATHRYN A. YAMADA 1, ROBIN M. McALLEN: and ARTHUR D. LOEWY 1 1Departrnent of Anatomy and Neurobiology, Washington Universi(v School of Medicine, 660 S. Euclid Avenue. St. l.oui~. MO 63110 ( U. S. A. ) and 2Department of Physiology, University Of Bristol, Bristol, BS 81 TD ( U. K. )
(Accepted November 29th, 1983) Key words." GABA - - bicuculline - - picrotoxinin - - ventral medulla oblongata - - baroreceptor reflex - - cardiovascular regulation
Application of y-aminobutyric acid (GABA) antagonist drugs (bicuculline, picrotoxinin) to the intermediate region of the ventral surface of the medulla oblongata of gallamine paralyzed, a-chloralose anesthetized cats produced a dose-dependent blockadc of the baroreceptor reflex. GABA antagonists applied to the rostral and caudal areas did not block the baroreceptor reflex. When strychnine was applied to the ventral medulla, it did not block the baroreceptor reflex. The diffusion of [3H]bicucullinewas studied by scintillation counting and autoradiography and found not to penetrate more than 2 mm from the ventral medullary surface. These rcsults suggest that a GABAergic synapse lies within the first 2 mm of the ventral medulla and is involved in baroreceptor modulation of the sympathetic outflow.
The interaction between centrally generated vaso-
tion of drugs, respectively. Rectal temperature was
motor tone and arterial baroreceptor activity is one of the major determinants involved in the neural con-
formed, and the cats were paralyzed with gallamine
trol of blood pressure. While the source of vasomotor tone is still u n k n o w n , recent experiments on cats have indicated that its transmission to the sympathetic preganglionic neurons depends critically on the activity of a group of neurons close to the ventral surface of the medulla oblongatalO,15,lg, 23. Bilateral removal of their activity, by a variety of means (drugs 7,9.23, cooling I~, lesionsm,18), reduces resting blood pressure to levels approaching that of spinal animals. Central nervous pathways of the baroreceptor reflex, however, apart from its immediate input and output nuclei, are still uncertain > . We present data here which suggest that a major synapse involved in the baroreceptor reflex pathway is located in the region of the ventral medulla, and that G A B A antagonist drugs block the reflex at this site. Cats (1.3-6.7 kg, n = 59) were anesthetized with 70-80 mg/kg i.v a-chloralose (Sigma, St. Louis). A femoral artery and vein were cannulated for measurement of blood pressure and systemic administra-
maintained at 37 _+ 1 °C. A tracheostomy was pertriethiodide (5-15 mg/kg, as needed, Davis and Geck, American Cyanamid, Pearl River, NY) and artificially ventilated with room air. End tidal CO,concentration was maintained at 5.5 + 0.5(7c (Beckmann Infrared Gas Analyzer). The ventral surface of the medulla was exposed. Raw sympathetic nerve activity was recorded from the central cut end of a right renal nerve. A unilateral carotid blind sac was preparedl~. The external carotid artery was cannulated for measurement of carotid sinus pressure and for baroreceptor stimulation. After electrophysiological identification, the contralateral carotid sinus nerve and both aortic depressor nerves were cut; the vagi were kept intact. The baroreceptor reflex was tested by inflating the blind sac with heparinized lactated Ringer's solution from an external reservoir (with a pressure of approximately 200 mm Hg) after clamping the common carotid artery. Bicuculline methiodide (BMI, Pierce Biochemicals, Rockford, IL) was dissolved in phosphate buf-
Correspondence." A. D. Loewy, Department of Anatomy and Neurobiologv, Washington University School of Medicine, 660 S. Euclid Avenue. St. Louis, MO 63110, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
176 PBS BMI 8 rain CONTROL lmM AFTERDRUG
A
300 I HEARTRATE ( beols/min )
j 200 I /
100L.
30o[ ARTERIAL "A,&" BLOOD(mmHg)PRESSURE150[
~' __---.---------
B
300[- ~ - , ~ -
| 200[
C
~
tOOL
~// PONS~-~j~
iii[
iNTERMEDIATE ~ I L"~ IIZ
0
CAROTID 300 f SINUSPRESSURE 150 (mmHg) 0 RENAL l~)I NERVE ACTIVITY (pV) 0L
STRYCHPBS NINE 8 min CONTROL SmM AFTERDRUG
1
Fig. 1. Effect of topical application of bicuculline and strychnine to the intermediate area of the ventral medulla on the baroreceptor reflex response. A: bicuculline-induced blockade of the depressor response elicited by carotid sinus baroreceptor stimulation accompanied by hypertension and increased sympathetic nerve activity with bursting. 1 mM BMI was topically applied bilaterally to the intermediate area of ventral medullary surface to produce the response. B: bilateral topical application of 5 mM strychnine to the intermediate area did not attenuate the baroreceptor reflex response. C: diagram showing the 3 areas of the ventral medullary surface, depicted by the stippled areas: rostral, intermediate, and caudal. The black squares indicate the sites where bicuculline or picrotoxinininduced blockade of the baroreceptor reflex could be produced. Abbreviations: BMI, bicuculline methiodide; trap, trapezoid bodies; pyr, pyramidal tract; V-XII, cranial nerves. fered saline (PBS, pH 7). Strychnine, nipecotic acid, and G A B A (Sigma) were dissolved in PBS daily. Picrotoxinin (Sigma) was dissolved in 10% ethanol in PBS daily. Drugs and vehicle controls were applied bilaterally to the ventral medullary surface via square cottonoid pledgets (1.5-2.5 mm 2) containing between 10 and 50/A of the drug solution. Neither PBS nor 10% ethanol in PBS had any effects when topically applied. Statistical analyses were performed using the Student's t-test for paired data, analysis of variance and the T u k e y - K r a m e r multiple comparisons test, and linear regression by the method of A N O V A . Criterion for statistical significance was P < 0.05. BMI, a potent G A B A receptor antagonist 11, was topically applied bilaterally to the 3 areas 17 of the ventral medullary surface of 16 cats (Fig. 1C). Only at the intermediate area did BMI (10/~M to 5 mM) produce dose-dependent, reversible increase in
mean arterial blood pressure, heart rate and renal sympathetic nerve activity (Fig. 1A. 2A. B). Concomitant with these effects was an attenuation of the reflex hypotension (up to 85%) and bradycardia (up to 100%) elicited by carotid sinus baroreceptor stimulation (Fig. 1A. 2C. D) and of the baroreceptor reflex-induced silencing of sympathetic nerve activity (Fig. 1A). Since cats were paralyzed with gallamine triethiodide, which has a significant blocking action on the vagus 5. heart rate changes seen in this study reflect sympathetic rather than parasympathetic function. The pressor response to BMI started within 10-60 s of application, whereas the reduced reflex response was first measured after 1.5-7 min. with peak effects after 3.5-7 min*. Recovery was followed in 12 cats. There was no significant difference with respect to blood pressure, heart rate. reflex hypotens,on and bradycardia, or reflex timing measurements between PBS control values and recovery values after 50/~M
* In two experiments, we reduced BMI-induced sympathetic excitation by hyperventilation. This reduced arterial blood pressure and abolished the characteristic bursting pattern seen in the renal nerve recordings without affecting the reduced baroreceptor reflex response.
177
cat), or 5 mM (5 cats) BMI in the 7
perficial aspect of the ventral surface of the medulla.
cats which showed recovery (not shown). The time
In an effort to determine what possible structures
course for complete reversal was 5-17 rain after removal of the last dose of BMI and a PBS rinsing of the
may have been effected by bicuculline to cause
ventral surface.
diffusion of [SH]bicuculline methylchloride (BMC)
Picrotoxinin (PTXN), the active metabolite of picrotoxin ez. a G A B A antagonist thought to block the
graphy. Based on scintillation counts (n = 4), spread
(1 cat), 1 mM (1
blockade of the baroreceptor reflex, we followed the by two methods: scintillation counting and autoradio-
G A B A receptor-linked chloride ionophore el, was applied bilaterally to the intermediate area of 6 cats. PTXN (100 n M - 5 raM) also produced attenuation of the reflex hypotension (Fig. 2E) and bradycardia,
A~ 2OUr,
"/*i
300[ • B,cuculi,ne
B
z ~' -E,
superimposed upon a background of increasing sympathetic tone, as demonstrated by increase in arterial blood pressure and sympathetic nerve activity. Re-
C
D • 5o[
covery was observed after 1 mM PTXN in 3 of the 5 cats. BMI and PTXN are both known to be powerful convulsant agents. To test for any non-specific excit-
gg
if
atory effects that these drugs may have been exerting another convulsant agent, strychnine, was applied bilaterally to the intermediate area in 7 cats. Strychnine (l /aM to 10 mM), a glycine receptor antagonist ~, produced small, inconsistent effects on blood pressure and heart rate (Fig. 1B, 2A, B), but had no blocking action on the baroreceptor reflex (Fig. 1B, 2C, D). These observations, coupled with the findings that the affinity of strychnine for glycine receptors is even higher than that of BMI for G A B A receptors ~,a,:~, argue that the actions of BM1 and PTXN are specific, and that a glycine synapse is not involved. Attempts to enhance the baroreflex with nipecotic acid (1-10 raM, n = 6), a competitive inhibitor of the high affinity G A B A uptake system 12, were unsuccessful. This may be due to any one of several factors, e.g. the domination of a glial reuptake system rather than a neuronal reuptake mechanism at this CNS site, inadequate penetration of the drug, or simply, the normal action of G A B A at this synapse is already maximal. Although drugs topically applied to the ventral medullary surface elicited effects in as little as a few seconds, it is not known what anatomical structure(s) may mediate these evoked responses. Electron micrographic studies of the ventral surface of the medulla oblongata~ have revealed an extensive pore and vascular system by which drugs could easily diffuse dorsally to a site of action deeper than the most su-
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Fig. 2. Effects of various drugs on baseline blood pressure and heart rate, and on reflex-induced changes in blood pressure and heart rate. Dose-response curves for bicuculline (@) and strychnine ([3) applied to the intermediate area of the ventral surface on mean arterial pressure (A) and heart rate (B). Note the marked pressor response produced by BMI (B). • Significant slope and no significant deviation from linearity using linear regression by the method of analysis of variance. PBS, phosphate buffered saline control. Dosc-rcsponse curves for bicuculline (@) and strychnine (~) applied to the intermediate area on the vasomotor (C) and cardiac components (D) of the baroreceptor reflex. Bicuculline produced a dosedependent attenuation of the baroreceptor reflex response. E shows the effect of various drugs on the vasomotor component of the baroreceptor reflex response as a percentage change from the control value. * 5 mM dose of bicuculline and picrotoxinin produced a significant difference from control, determined by analysis of variance and the Tukey-Kramer multiple comparisons test run on the set of doses for each drug condition, P < 0.05. This analysis was used for the dose ranges tested for each drug at the intermediate area. For all other drug conditions only the highest dose found effective at the intermediate area (5 mM) was used. In these cases, the effects of bicucufline on the reflex response were not found to be significantlydifferent from control using the Student's t-test for paired data.
1"78 of [3H]BMC (198-940 nCi) topically applied to the intermediate area f o r m e d a gradient (Fig. 3). The highest concentration (2.15-2.33 nCi/mg tissue) was found at the ventral surface, 50% of that concentration was found 0.5-0.9 m m below the surface, 10% at
ceptor blockade, A t the lower end of the dose range used, tissue values of bicuculline within the first 2 mm of the ventral m e d u l l a would a p p r o a c h those found to displace G A B A (IC50 = 3 fzM 24) from its receptors in in vitro binding studies. Second, structures such as the nucleus ambiguus and the nucleus of tractus solitarius can be excluded as the site where B M I blocks the reflex. Earlier studiesT,9,15,23 have implicity as-
1,8-2.5 mm below the surface, and no detectable [3H]BMC 3.5-3.7 m m below the surface. A u t o r a d i o graphic visualization of the [3H]BMC diffusion using tritium-sensitive film (Ultrofilm; L K B B r o m m a , Sweden) revealed grain densities only in the first 1.5 m m below the ventral surface of the m e d u l l a (n = 5, exposure = 4-8 weeks).
sumed that when drugs are applied topically to the ventral medullary surface, the main site of action is on the superficial neurons of the ventral medulla. O u r data indicates that [3H}bicuculline can reach d e e p e r structures including the A1 or C [ cell groups and the inferior olivary nucleus as well as the ventral surface itself. Clearly, this means that topical appli-
F r o m these results we can form two conclusions. First, the concentrations of B M I seen in the superficial 2 m m of brainstem are a p p r o p r i a t e for G A B A re-
Nucleus
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tractus solitarius
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0.4 0.8 1.2
17
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2.5
31
4.0
5.0
6.0
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mm from Ventral Surface of Medulla Oblongota Fig. 3. Diffusion of [3H]bicuculline methylchloride (BMC, New England Nuclear, Boston, MA; spec. act. = 82.0 Ci/mmol) was studied after application to the intermediate area of the ventral medullary surface. [3H]BMC was topically applied via cottonoid pledgets. After 6.5-11 min of drug exposure, the pledgers were removed and the brainstem was blotted, dissected out and frozen in Freon chilled on dry ice. 20 am horizontal sections were cut at 20 °C in a cryostat, weighed, solubilized (1 N NaOH, 60 °C), and counted (Packard Instruments). Fig. 3A shows the degree of penetration of [3H]BMC as determined by scintillation counting. Ten to twenty 20/~m horizontal sections were pooled in order to determine nCi/mg tissue. The/~M concentrations of bicucutline were calculated based on the assumption that one gram of tissue is equivalent to one ml of solvent. Fig. 3B illustrates the anatomical landmarks of the medulla corresponding to the levels at which the scintillation counts were made.
179 cation of drugs is a useful way to screen drugs but that
are f o u n d c e n t e r e d in the i n t e r m e d i a t e area, and
m o r e refined t e c h n i q u e s will be n e e d e d to localize
mostly within 1.5 m m of the ventral m e d u l l a r y sur-
exactly w h e r e the G A B A e r g i c synapse lies that m o d -
face l, the exact a r e a affected in the p r e s e n t study.
ulates b a r o r e c e p t o r activity. In s u m m a r y , our d a t a suggest that within 2 m m of
T h e p r e s e n t study does not tell us a b o u t the vagal
the ventral m e d u l l a r y surface at the i n t e r m e d i a t e
have m a s k e d its e x p r e s s i o n , nor does it tell us h o w
area there is a G A B A e r g i c synapse i n v o l v e d in b a r e -
m u c h b a r o r e c e p t o r inhibition of s y m p a t h e t i c activity
r e c e p t o r m o d u l a t i o n of v a s o m o t o r t o n e , T h e sim-
o c c u r r e d at m o r e rostral or spinal levels2, ~. I n d e e d ,
plest i n t e r p r e t a t i o n is that this synapse is an integral
such alternate p a t h w a y s may account for the r e m a i n -
part of the b a r o r e c e p t o r reflex p a t h w a y , but we can-
der of the reflex we w e r e u n a b l e to block.
limb of the b a r o r e c e p t o r reflex as g a l l a m i n e w o u l d
not exclude the possibility that this b l o c k a d e of the G A B A inhibition is of a d i f f e r e n t origin which may
The study was s u p p o r t e d by U S P H S G r a n t s H L -
have affected b a r o r e c e p t o r reflex gain. P r e s u m a b l y
25449, HL-07275 and a G r a n t - i n - A i d (83-656) f r o m
this synapse acts on those cells of the i n t e r m e d i a t e
the A m e r i c a n H e a r t A s s o c i a t i o n , with funds contrib-
area
uted in part by the Missouri
previously
shown
to
mediate
vasomotor
Heart
Association.
t o n e Is,2). and which may have a direct p r o j e c t i o n to
A . D . L . is an E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i -
the s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n s in the spinal
can H e a r t A s s o c i a t i o n .
c o r d 1,14. Interestingly, such spinally p r o j e c t i n g cells
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180 B. and Olson, R. W., Dihydropicrotoxinin binding sites in rat brain: comparison to GABA receptors, Brain Research, 150 (1978) 642-647. 22 Wang, S. C. and Ward, J. W., Analeptics, Pharmac. Ther. B, 3 (1977) 123-165. 23 Yamada, K. A., Norman, W. P., Hamosh, P. and Gillis, R. A., Medullary ventral surface GABA receptors affect r e s -
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