Pressor responses to stimulation of non-NMDA receptors in the superficial laminae of the cat spinal cord

BRAIN RESEARCH ELSEVIER

Brain Research 683 (1995) 149-152

Short communication

Pressor responses to stimulation of non-NMDA receptors in the superficial laminae of the cat spinal cord Michael K.C. Wong, Janeen M. Hill, Marc P. Kaufman * Division of Cardiovascular Medicine, Departments of Internal Medicine and Human Physiology, University of California, Dat, is, CA 95616, USA Accepted 7 March 1995

Abstract Microinjection of L-glutamate (109 mM; 1 2 - 1 8 nl) or A M P A ( 1 5 0 - 3 0 0 /zM; 12 nl) into the superficial laminae of the L 7 dorsal horn of decerebrate or chloralose anesthetized cats significantly increased mean arterial pressure. In contrast, microinjection of N M D A (300 ~ M ; 12 nl) had no effect on mean arterial pressure. The pressor response to L-glutamate microinjection was blocked by prior microinjection of CNQX, an antagonist to n o n - N M D A receptors, but not by AP-5, an antagonist to N M D A receptors. We conclude that stimulation of n o n - N M D A receptors in the superficial laminae of the lumbar dorsal horn increases arterial blood pressure.

Keywords: Autonomic nervous system; Excitatory amino acid receptor; Ventilation; Dorsal horn Activation of thinly myelinated and unmyelinated afferents in the sciatic nerve is well known to increase reflexly sympathetic nerve discharge and arterial blood pressure [4,11,15]. When these thinly myelinated and unmyelinated afferents arise from the skin, they are called A-delta and C fibers, respectively. Likewise, when these afferents arise from skeletal muscle and joints, they are called group III and IV fibers. Morphological evidence suggests that thinly myelinated afferents synapse in laminae I, II and V of the dorsal horn and that unmyelinated afferents synapse in lamina II [3,5]. The neurotransmitter released by these afferents is probably glutamate [6,9,17]. In this study, we determined whether microinjection of glutamate or its analogues into the superficial laminae (i.e., I and II) of the dorsal horn increased arterial blood pressure. We also determined whether the pressor responses to the microinjection of glutamate were blocked by prior microinjection of antagonists to either non-NMDA or NMDA receptors. Cats were anesthetized initially with a mixture of 02 and N20 (3:2) and Halothane (5%). A carotid artery, external jugular vein and trachea were cannulated. Three cats were anesthetized with alpha-chloralose (50 m g / k g ; i.v.) and gaseous anesthetic was discontinued 15 min later. Additional chloralose was injected (10 m g / k g ; i.v.) hourly.

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All other cats were decerebrated according to the method described by Shik et al. [18]; afterwards the gaseous anesthetic was removed. A lumbar laminectomy was performed on all cats. The tracheal cannula of spontaneously breathing cats was connected to a heated pneumotach (Fleisch no. 00). Inspiratory airflow was integrated (Gould) to yield tidal volume, from which minute volume of ventilation (I/i) was calculated. Cats that did not breathe spontaneously or whose arterial blood gases were not within the normal range were paralyzed with vecuronium bromide (0.1 m g / k g ; i.v.) and their lungs ventilated mechanically. In these paralyzed cats, phrenic nerve activity was our index of ventilation. Phrenic nerve activity was recorded (Grass P511) and integrated (Gould) using the method described by Eldridge [8]. Glass triple barrel pipettes were filled with three of the following solutions: L-glutamate (109 mM); 6-cyano-7nitroquinoxaline-2,3-dione (CNQX; 39 /zM); D,L, 2amino-5-phosphonopentanoate (AP-5; 25 mM); c~-amino3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA; 150-300 /xM); N-methyl-D-aspartate (NMDA; 300 /~M) and fast green dye (2% in phosphate buffered saline). All chemicals were obtained from Research Biochemicals (Natick, MA). The pipette was attached to a Kopf micromanipulator and its tip positioned near the dorsal root entry zone. The pipette was advanced in 100 micron increments until microinjection of either L-glutamate (protocol 1) or AMPA (protocol 2) increased mean arterial

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pressure. T h e v o l u m e o f all L - g l u t a m a t e m i c r o i n j e c t i o n s w a s e i t h e r 12 ( n = 3) or 18 nl ( n = 6). T h e v o l u m e o f the C N Q X a n d A P - 5 m i c r o i n j e c t i o n s for any o n e site m a t c h e d that o f the c o r r e s p o n d i n g L-glutamate m i c r o i n j e c t i o n . T h e v o l u m e o f all N M D A a n d A M P A m i c r o i n j e c t i o n s w a s 12 nl a n d that for fast g r e e n dye w a s 100 nl. T h e s t u d y w a s c o m p r i s e d o f t w o protocols. P r o t o c o l 1: the m e a n arterial p r e s s u r e , heart rate a n d v e n t i l a t o r y res p o n s e s to m i c r o i n j e c t i o n o f L - g l u t a m a t e w e r e d e t e r m i n e d . If a r e s p o n s e w a s o b s e r v e d , 20 m i n later t - g l u t a m a t e w a s m i c r o i n j e c t e d again. If the r e s p o n s e r e p e a t e d , A P - 5 w a s m i c r o i n j e c t e d a n d 20 m i n a f t e r w a r d s , the m e a n arterial pressure, h e a r t rate a n d v e n t i l a t o r y r e s p o n s e s to L-glutam a t e m i c r o i n j e c t i o n w e r e r e - m e a s u r e d . If the p r e s s o r res p o n s e to L - g l u t a m a t e r e m a i n e d , C N Q X w a s m i c r o i n jected. T w e n t y m i n u t e s later, the c a r d i o v a s c u l a r r e s p o n s e s to L - g l u t a m a t e m i c r o i n j e c t i o n w e r e a g a i n r e - m e a s u r e d . C N Q X w a s r e m o v e d f r o m its barrel, w h i c h w a s refilled w i t h fast green, a n d the site dyed. P r o t o c o l 2: the m e a n arterial p r e s s u r e , h e a r t rate a n d v e n t i l a t o r y r e s p o n s e s to m i c r o i n j e c t i o n o f A M P A w e r e m e a s u r e d . If a p r e s s o r r e s p o n s e w a s f o u n d , the c a r d i o v a s c u l a r a n d v e n t i l a t o r y r e s p o n s e s to m i c r o i n j e c t i o n o f N M D A w e r e d e t e r m i n e d 20 m i n after the m i c r o i n j e c t i o n o f A M P A . N e x t , the site w a s d y e d w i t h fast green. A t the e n d o f e a c h e x p e r i m e n t , the l u m b a r s p i n a l c o r d w a s r e m o v e d , p l a c e d in f o r m y l s a l i n e for 4 8 - 7 2 h a n d s u b s e q u e n t l y f r o z e n a n d s e c t i o n e d ( 4 0 /~m). T h e dye spot w a s f o u n d to c o n f i r m that the i n j e c t i o n site w a s in l a m i n a e I or I1. All v a l u e s are r e p o r t e d as m e a n s _+ s t a n d a r d error. Significance was determined with a repeated measures A N O V A . W h e r e a p p r o p r i a t e , S c h e f f E ' s post h o c test w a s u s e d [12]. T h e c r i t e r i o n for statistical s i g n i f i c a n c e w a s P < 0.05. t - G l u t a m a t e , m i c r o i n j e c t e d into l a m i n a e I or II ( n i n e

-

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g

IV

V

vi

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Fig. 1. Schematic of seventh lumbar segment takcn from Rexed [14]. Superficial laminae are represented by Roman numerals 1 and II. Open triangles (n = 3) represent sites in which AMPA and NMDA were microinjected into the L7 dorsal horn of o~-ehloralose anesthetized cats. Closcd circles (n = 8) represcnt sites in which AMPA and NMDA were microinjeetcd into the L7 dorsal horn of decerebratc" cats. Open circles ( n - 9) rcpresent sites in which l--glutamate, AP-5 and CNQX were microinjected into the L7 dorsal horn of decerebrate cats. For ease of representation both contralateral and ipsilateral microinjections are indicated on one side of this schematic.

sites in e i g h t d e c e r e b r a t e cats), e v o k e d s i g n i f i c a n t and r e p e a t a b l e i n c r e a s e s in m e a n arterial p r e s s u r e (Fig. 1 (Table 1), but h a d n o effect o n h e a r t rate or p h r e n i c n e r v e activity. A P - 5 h a d n o effect o n the m a g n i t u d e o f the p r e s s o r r e s p o n s e e v o k e d b y L - g l u t a m a t e in t h e s e n i n e sites (Fig. 2). In contrast, C N Q X s i g n i f i c a n t l y ( P < 0.05) attenu a t e d the p r e s s o r r e s p o n s e e v o k e d b y L - g l u t a m a t e in e a c h o f the n i n e sites. (Fig. 2). A l p h a - c h l o r a l o s e h a s b e e n s h o w n to d e p r e s s N M D A e v o k e d r e s p o n s e s o f dorsal h o r n cells to n o x i o u s s t i m u l i

Table 1 Effects of microinjcction of AMPA and NMDA as well as repeated microinjections of L-glutamatc on cardiovascular and ventilatory function. Values are means ± SE; n, number of sites; baseline MAP and HR, baseline prior to microinjection of L-glutamate or analogue; baseline l)l, minute ventilation prior to microinjection; baseline phrenic, averaged phrenic discharge 30 s prior to microinjection; response MAP and HR, peak MAP and HR occurring after the microinjection of L-glutamate or analogue; responsc ~)~, minute ventilation after the microinjection; response phrenic, averaged phrenic discharge 30 s following microinjection. Onset and peak latencies were calculated from the end of microinjection, which took 1-3 s GLUI (n = 9 )

GLU2 (n = 9 )

AMPA (n = 11)

NMDA (n = 11)

baseline response latency to onset (s) latency to peak (s)

96 ± 7 104 ± 7 * 6± 2 I1 ± 2

98 ± 8 106 ± 8 * 7+2 13 ± 3

116 ± 7 127 ± 7 * 8± 2 20 ± 6

114 ± 8 116 + 9 -

baseline response

183 + 16 185 + 17

185 + 17 187 ± 17

163 ± 13 169 ± 12 (n ~ 10) 612 ± 68 638 + 68

(n = 7) 20 _+3 22 ± 2

(n - 7) 23 _+ 3 24 ± 3

168 + 12 173 ± 10 (n = 10) 643 ± 74 649 + 94

-

-

Mean arterial pressure (mmHg)

Heart rate (beats/min)

Minute ventilation (ml/min) baseline response Phrenic nerve activity (% of maximum discharge) baseline response Significant difference ( P < 0.05) between baseline and response.

M.K.C. Wong et al. /Brain Research 683 (1995) 149-152 120

A

,-r

g

ta_

too

80

L-Glu

L-Olu + AP-5

L-Olu 4CNQX

L-GIu

L-Olu + AP-5

L-GIu + CNQX

L-Glu

L-GIu + AP-5

L-GIu + CNQX

210

B

170 C

4o

~3 30 "< =E 'z, ~E

20

g 10

Fig. 2. Summary data (means ± S.E.) of the effects of L-glutamate (L-GIu) microinjected into the superficial laminae of the L7 dorsal horn on mean arterial pressure (MAP) (A), heart rate (HR) (B) and phrenic nerve activity (PNA) (C). L-GIu was microinjected before the NMDA receptor antagonist, AP-5, 20 min after AP-5 and 20 min after the non-NMDA receptor antagonist, CNQX. CNQX significantly ( P < 0.05) attenuated the pressor response to L-GIu. Open bars, baseline means; solid bars, response means; brackets, standard errors. * Significant differences ( P < 0.05) between baseline and response means. Horizontal brackets, significant differences ( P < 0.05) between response to L-GIu before AP-5 and after CNQX and to L-GIu after AP-5 and after CNQX. None of the baseline values for MAP, HR or PNA were significantly different from cach other.

[13]. We, therefore, compared the effects of microinjection of NMDA on mean arterial pressure, heart rate and ventilation in eight sites in five decerebrate unanesthetized cats with the effects of microinjection of NMDA on these variables in three sites in three chloralose anesthetized cats (Fig. 1). Prior microinjection of AMPA evoked a pressor response in each of the 11 sites, which were obtained from eight cats. Subsequent microinjection of NMDA did not increase mean arterial pressure, heart rate or ventilation in either decerebrate unanesthetized or chloralose anes-

151

thetized cats. Specifically, microinjection of NMDA in the latter had no effect on mean arterial pressure (123 + 11 mmHg to 127 + 14 mmHg), heart rate (160 + 22 bpm to 163 + 21 bpm) or minute ventilation (409 __+37 m l / m i n to 418 _ 45 ml/min). However, microinjection of AMPA in these anesthetized cats increased mean arterial pressure (122 ___12 mmHg to 131 + 14 mmHg), but had no effect on heart rate (158 + 22 bpm to 163 _ 23 bpm) or ventilation (405 + 37 m l / m i n to 397 + 35 ml/min). Microinjection of NMDA in decerebrate unanesthetized cats also had no effect on mean arterial pressure (111 ± 11 mmHg to 111 + 11 mmHg), heart rate (165 + 18 bpm to 171 + 16 bpm) or ventilation ( 6 9 8 _ 80 ml/min to 733 + 75 ml/min). Again, however, microinjection of AMPA in these decerebrate cats increased mean arterial pressure (115 _ 10 mmHg to 126 ___10 mmHg), but had no effect on either heart rate (172 ___15 bpm to 177 + 13 bpm) or ventilation (745 + 83 m l / m i n to 757 ___120 ml/min). Because our results were not affected by the presence or absence of chloralose, we pooled the data from the anesthetized and decerebrate cats (Table 1). Statistical analysis revealed that for the 11 pooled sites microinjection of AMPA, but not NMDA, significantly ( P < 0.05) increased mean arterial pressure (Table 1). This analysis also revealed that neither microinjection of AMPA nor NMDA increased heart rate or ventilation (Table 1). Our findings in cats have shown that microinjection of L-glutamate and AMPA into the superficial laminae of the dorsal horn of the lower lumbar spinal cord evoked a pressor response that was caused by the stimulation of non-NMDA receptors. Nevertheless, the magnitude of this pressor response was modest and may be attributable to the small volumes microinjected into the L7 dorsal horn. We used these relatively small volumes in an attempt to limit spread of the injectate within the superficial laminae of the dorsal horn. We reasoned that spread was, in fact, minimal because repositioning the tip of the pipette 100 microns in the medial-lateral or dorsal-ventral planes abolished the pressor response to microinjection of L-glutamate or AMPA. Our attempts to limit spread of the injectate may also explain why our microinjections of either glutamate or AMPA did not increase heart rate or ventilation. We speculate that the threshold for evoking a pressor response in our preparations was considerably lower than that for evoking a ventilatory or cardioaccelerator response. This speculation has a parallel in the human literature in which the pressor component of the muscle chemoreflex evoked from the leg has a lower threshold than does the cardioaccelerator component [1,2]. Our microinjections of excitatory amino acids into the superficial dorsal horn probably activated reflex arcs arising from the hindlimb. Nevertheless, interpretation of our data must be limited by the fact that our microinjections did not selectively activate pathways involving afferents transducing a particular sensory modality or arising from a

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M.K.C. Wong et al. / Brain Research 683 (1995) 149-152

p a r t i c u l a r tissue. D e s p i t e this l i m i t a t i o n , o u r f i n d i n g s indicate t h a t n o n - N M D A r e c e p t o r s in the s u p e r f i c i a l l a m i n a e o f the dorsal h o r n play an i m p o r t a n t role in c a u s i n g L - g l u t a m a t e i n d u c e d i n c r e a s e s in m e a n arterial pressure. N o n - N M D A r e c e p t o r s h a v e b e e n s h o w n to b e f u n c t i o n ally i m p o r t a n t in s e v e r a l s t u d i e s i n v o l v i n g the l u m b a r s p i n a l cord. F o r e x a m p l e , the r e s p o n s e s o f dorsal h o r n n e u r o n s to n o c i c e p t i v e i n p u t in i n t a c t a n e s t h e t i z e d rats is m e d i a t e d b y n o n - N M D A r e c e p t o r s [7]. L i k e w i s e , s y n a p t i c t r a n s m i s s i o n f r o m dorsal root a f f e r e n t s o n t o dorsal h o r n cells in r o d e n t slice p r e p a r a t i o n s is a t t e n u a t e d b y C N Q X , w h i c h b l o c k s n o n - N M D A r e c e p t o r s [16,20]. In a d d i t i o n , the reflex p r e s s o r r e s p o n s e to m u s c u l a r c o n t r a c t i o n in intact a n e s t h e t i z e d cats is a t t e n u a t e d m a r k e d l y b y C N Q X [10]. F i n a l l y , w e f o u n d n o e v i d e n c e that m i c r o i n j e c t i o n o f N M D A into the s u p e r f i c i a l l a m i n a e i n c r e a s e d arterial pressure. W e are u n w i l l i n g , h o w e v e r , to e x c l u d e a c a p a c i t y for N M D A r e c e p t o r s to e v o k e a p r e s s o r effect f r o m the dorsal horn. E x p r e s s i o n o f this c a p a c i t y m a y b e c o m p l e x a n d m a y d e p e n d o n the c o - a c t i v a t i o n o f n o n - N M D A or t a c h y k i n i n r e c e p t o r s [19]. T h i s r e s e a r c h w a s s u p o r t e d b y N a t i o n a l Institutes o f H e a l t h G r a n t H L 3 0 7 1 0 . W e t h a n k Ms. P e n n y J o n e s for t y p i n g the m a n u s c r i p t .

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