Cardiorespiratory responses to chemical stimulation of the caudalmost ventrolateral medulla in the cat

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Neuroscience Letters, 129 (1991) 86-90 © 1991 ElsevierScientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100406U

NSL 07922

Cardiorespiratory responses to chemical stimulation of the caudalmost ventrolateral medulla in the cat G . A . I w a m o t o 1'2, R . D . B r t v a 1 a n d T . G . W a l d r o p 2 1Department of Veterinary Biosciences and 2Department of Physiology and Biophysics, University of Illinois, Urbana-Champaign, Urbana, IL 61801 (U.S.A.)

(Received4 February 1991; Revised version received 18 March 1991;Accepted 2 May 1991) Key words: Bloodpressure; Ventilation; Brainstem; Ventrolateral medulla; Cat

Chemical stimulation of caudal ventrolateral medulla evoked both pressor and depressor responses. The pressor sites were generally located caudal to depressor sites. Effects on heart rate were variable. Significant increases in minute ventilation were also observed, which were primarily due to changes in respiratory frequency.

The ventrolateral medulla (VLM) of the cat brain has long been identified as a pressor region. Early studies utilizing electrical stimulation (STIM) showed that an extensive ventrolateral area extending for the length of the medulla was involved in raising blood pressure [1, 19]. Recent data from m a n y species have identified cells in the rostral V L M (RVLM) which project to the spinal cord intermediolateral cell column and mediate pressor responses [3, 5, 6, 18, 20]. Recent data for the caudal ven,trolateral medulla (CVLM) are less uniform. Activation of the C V L M of the rat by electrical or chemical STIM evokes depressor responses [2, 14, 22]. However, a chemical S T I M study by G o r d o n and McCann [10] has revealed a pressor area in the rat which is caudal to the C V L M depressor area. While the caudalmost C'VLM of the cat was identified with pressor responses in older studies in using electrical S T I M [1], these data could be interpreted as stimulating fibers en passage to the R V L M . We therefore sought to determine if the cat would also show pressor responses to chemical S T I M of the caudalmost V L M as this is held to activate cell bodies while sparing axons en passage [9]. Some of these results have appeared in preliminary form [13]. Adult cats (2.3-4.0 kg, n = 22) were used in this study. Anesthesia was induced with 1-3% halothane in a mixture of 1:3 oxygen and nitrous oxide. The trachea was intubated. Arterial pressure (AP) was measured through a c o m m o n carotid artery cannula. A venous cannula was Correspondence: G.A. lwamoto, Department of Veterinary Biosciences, Collegeof Veterinary Medicine, Universityof Illinois, Urbana, IL 61801, U.S.A.

placed in the external jugular vein. The cats were then either given ~-chloralose (60 mg/kg, i.v., n = 3) or decerebrated at the midcollicular level (n = 19). Gaseous anesthetic was then discontinued. Ventilation measurements were made with a Fleisch pneumotachograph and a Gould Integrator. All cats were routinely monitored for end tidal CO2 which was maintained within a range of 3.5-4.5%. In most experiments we utilized a Radiometer ABL-3 blood analysis system to monitor CO2, 02, p H and bicarbonate. Body temperature was maintained within a range of 36.5-38°C. The caudal brainstem was then exposed for stereotaxic placement of glass micropipettes. The micropipettes ( 2 0 - 3 0 / t m tip diameter) were filled with L-glutamate ( G L U T ) in Ringer's solution (1 M, 500 or 100 mM), ?-aminobutyric acid (GABA) in Ringer's solution (500 mM), or a control solution, separated from either 1% Fast Green or 1% Pontamine Sky Blue 6BX dye by a droplet of mineral oil. Twelve animals were studied using 1 M G L U T , 3 with 500 m M and 7 with 100 mM. The 500 m M and 100 m M G L U T solutions were administered at p H 7.2-7.3. The pipette tips were placed on the surface o f the medulla at 1.5 m m rostral to 2.0 m m caudal to the level of the obex, 3.8-4.3 m m lateral to the midline. The pipettes were then advanced in steps of 0.5 or 1.0 m m from an initial depth of 3.0-4.0 m m from the dorsal surface. The chosen solution was then ejected using a pneumatic injection system (Picospritzer II, General Valve or PV 830, WPI) in total volumes of 1975 nl. Typically the injections were in 6-10 steps per STIM over a period of 5-7 s. Autoradiographic data suggesting that the extent of spread of 25 nl of tritiated

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Fig. 1. Exampleof cardiorespiratoryresponses obtained from the injectionof 20 nl of a 100 mM glutamate,solution utilizing a micropipettedriven by a pneumatic injection system. Note that blood pressure and respiratory frequencyare raised. Injection was made into the site marked by a dot just above lateral reticular nucleus (LRN) on the histology figure. 5ST, spinal tract of the trigeminal n.; 5SP, spinal nucleus of the trigeminal n.; LRN, lateral reticular nucleus; 12N, hypoglossal n.; CE, central canal; CI, nucleus centralis inferior; P, pyramidal tract; IOM, medial accessory inferior olive.

1 M G L U T to be 250 p m [7] from the site of injection (INJ) prompted us to largely limit our INJ to 20 nl. Selected STIM sites were marked after removing the pipette, ejecting the remaining chemical, replacing the pipette at the desired location and ejecting 100 nl dye. G L U T INJ were also made in two paralyzed cats (succinylcholine). The brain was finally removed and either rapidly frozen (Fast Green dye; cryostat sectioning) or placed in 4% neutral formalin (Pontamine Sky blue dye; sliding microtome with cryo stage). The locus o f the dye marks were evaluated by microscopic examination (Cresyl Violet staining). Results are expressed as mean-t-S.E.M. Statistical analysis utilized Student's t-test with P < 0.05 taken as significant. All summary data included in the text represent significant changes unless otherwise specified. Chemical STIM o f the caudal VLM led to significant changes in AP, respiratory rate and minute ventilation. Either pressor or depressor effects occurred with variable effects on heart rate (HR). Respiratory (RESP) changes occurred in a characteristic manner: Initially the tidal volume was decreased for a few RESP cycles accompanied by an increase in RESPrate, this was sometimes followed by increases in tidal volume. An example o f these data are shown in Fig. 1. The effects of STIM on mean

AP and H R are shown in summarizing diagrams. Fig. 2A shows CVLM sites investigated in 12 animals using 1 M G L U T INJ and the types of responses evoked at each site. Pressor responses averaged 17.8+3 m m H g (n = 28 sites). Depressor responses averaged -23.2 + 3.3 mmHg (n = 15 sites). Significant increases in minute ventilation occurred (839.9+42 to 1276.7+95 ml/min, n = 53 sites). This increase was caused by an increase in RESP frequency (25.2+1 to 2 8 . 7 + 6 breaths/rain). While increases in tidal volume were sometimes observed, these were statistically insignificant. The quantitative RESP results for each group are based on all sites for which measurements were made, irrespective of blood pressure response. Fig. 2B shows CVLM sites investigated using 100 m M G L U T INJ in 7 animals. Pressor responses averaged 12.3 + 1 mmHg (n = 29 sites). Depressor responses averaged - 1 6 . 7 _ 3 m m H g (n = 19 sites). Significant increases in minute ventilation were also observed with this stimulus (777.1-t-4 to 866.6+5 ml/min, n = 4 4 sites). Again, the significant increase was in the RESP frequency (25.2 + 1 to 28.7 + 2 breaths/min) but not tidal volume. Note that in all cases (Fig. 2), pressor responses were obtained at locations just dorsal to the L R N extending dorsally to an area just ventral to the trigeminal nucleus. While some pressor responses were evoked from the bor-

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Pressor Responses: • = 1" BP, J. HR: >10 mmHg • = $ BP, $ HR: <10 mrnHg

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[ ] = ~ BP, J. HR: >10 mmHg O = ~" BP. J. HR: <10 nmHg

Fig. 2. Composite of all STIM sites and responses evoked using (A) 1 M (n= 12, decerebrate animals), (B) 100 mM (n=7, decerebrate animals) and (C) 500 mM (n = 3, anesthetized animals) L-glutamate injections. IOD, dorsal accessory inferior olive; V4, 4th ventricle.

ders of LRN, many of the non-responsive sites were in the LRN region. Some of the depressor sites were admixed within the general population of pressor sites. However, the most consistent pattern is shown in Fig. 2B in which the depressor region is located dorsal to the L R N but generally rostral to the pressor sites. Since anesthesia could impose somewhat different conditions, we included chloralose-anesthetized cats (n = 3), while closely following the methods of others [15] in utilizing 500 mM G L U T INJ in these cases. The effects of these INJ are summarized in Fig. 2C. While pressor responses (mean +29.7-t-6.7 mmHg, n = l l ) could be evoked from the CVLM, these responses were often accompanied by a fall in heart rate (mean -45 -t- 12 beats/ min). These results are significantly greater than those of the other preparations. INJ of Ringer's solution vehicle and pH corrected Ringer's solution controls (at the same pH as the 1 M G L U T solution) were ineffective in producing these changes. Pressor responses were obtained in paralyzed

cats demonstrating that this result was not secondary to movement. In agreement with Gordon and McCann [10], INJ of GABA (n= 15 sites) into putative CVLM pressor sites had no effect. The results clearly show that putative axon sparing chemical STIM in the CVLM may give rise to pressor responses and increases in ventilation in addition to more widely known depressor responses. The pressor area is generally caudal to the depressor area elucidated by others [2, 14, 16, 20]. The results confirm and extend the observations of Gordon and McCann [10] in rats but with some apparent differences. The pressor and depressors sites are not as clearly separated in the cat as apparently equivalent regions between individual animals could give rise to either type of response. This is particularly true at the level of the obex. A lack of a clear separation in the cat may be due to not having stimulated in precisely equivalent areas to those of the rat or may simply be a species variation. Furthermore, having pressor and depressor sites so close to one another may ac-

89 c o u n t for the pressor responses n o t being as great in magnitude as those f o u n d in the rat. However, the m a j o r point o f the data was simply that depressor responses are not the rule as one uses chemical S T I M in the area at and below the level o f the obex in the cat. The pressor responses are n o t due t o differences in anesthetic state since they m a y be obtained u n d e r chloralose anesthesia as well as midcollicular decerebrate conditions. O u r anesthetized animals gave us o u r m o s t consistent pressor responses. As might have been predicted f r o m the result o f Stornetta et al. [20], our S T I M sites in the L R N included m a n y which were non-responsive. A l t h o u g h it is possible pressor activity f r o m this region m a y be the result o f inhibiting neurons o f depressor areas, we consider this unlikely. It was possible to obtain pressor or depressor responses simply by changing stimulus locations. Furthermore, as G o r d o n and M c C a n n [10] noted, there were very few responses indicative o f a depressor response followed by a pressor response, m u c h as one would expect if initial excitation o f a depressor area was followed by inhibition. The sites producing consistent depressor responses were located generally rostral to those p r o d u c i n g pressor responses. The responses could be obtained at a variety o f G L U T concentrations and volumes including < 20 nl o f what entered the pipette as 100 m M . It is also unlikely [7] that the pressor responses were the result o f spread to another area, such as the rostral V L M . The effects o f chemical S T I M a p p e a r to correlate with results o f single unit recordings which have indicated that the area is a putative site o f integration for pressor responses to somatic nerve electrical S T I M [4] and muscular contraction [11]. The present results contrast s o m e w h a t with those o f other investigators using similar S T I M in the rostral L R N and 'glycine-sensitive area' o f the cat [8]. In this study [8], S T I M o f these areas gave rise o f pressor responses with decreases in heart rate and ventilation. The pattern o f consistent pressor responses a c c o m p a n y e d by bradycardia was only observed in our anesthetized animals while decreases in ventilation were rarely observed. Differences in anesthesia or I N J volumes between studies m a y a c c o u n t for some o f the differences observed. However, it is m o s t likely that differences between these results are because our S T I M sites are caudal to those previously investigated. While the S T I M sites are in the general vicinity o f the caudal area o f Loeschcke (area L2) [15, 19], these neurons are p r o b a b l y not involved. The area we have stimulated is quite lateral to area L2 and is in the order o f 1 m m f r o m the surface. In any case, the p r i m a r y effect we have observed is on R E S P frequency and not tidal volume as the case with area L2.

It is possible that c o m p a r e d to m o r e rostral V L M the c a u d a l m o s t V L M in the cat m a y represent a site m o r e involved with integrating responses involving increases in m o t o r activity and metabolic d e m a n d such as exercise. The region is perhaps primarily k n o w n as a possible integrative site for s o m a t o m o t o r activity o f which R E S P m o v e m e n t m a y be a part. Additional experimentation will determine if this area m a y be further differentiated functionally. Supported by N I H G r a n t s HL06296, H L 3 8 7 2 6 and the A m e r i c a n Heart Assn.

HL37400,

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