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Microinjections of glutamate within trigeminal subnucleus interpolaris alters adrenal and autonomic function in the cat
Brain Research, 622 (1993) 155-162
155
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19192
Microinjections of glutamate within trigeminal subnucleus interpolaris alters adrenal and autonomic function in the cat David A. Bereiter Departments of Neuroscience and Surgery Brown University/Rhode Island Hospital, Providence, RI 02903 (USA) (Accepted 20 April 1993)
Key words." Adrenal catecholamine; Autonomic control; Excitatory amino acid; Spinal trigeminal nucleus
The influence of rostral portions of the trigeminal sensory complex on adrenal and autonomic function was assessed by microinjections of L-glutamate (500 or 5 mM, 100 nl) directed at subnucleus interpolaris (Vi) or at the nucleus principalis/subnucleus oralis level (Vp/Vo) in chloralose-anesthetized cats. Microinjections of glutamate (500 mM) within Vi evoked prompt (by + 1 min) dose-related increases in the adrenal secretion of epinephrine (+11.4+2.5 ng/min, P<0.001), adrenal blood flow (+0.19+0.06 ml/min, P<0.05), mean arterial pressure (+6.6_+3.0 mmHg, P < 0.025) and heart rate (+8.0+2.7 beats/min, P < 0.01, n = 16). Microinjections of lower doses of L-glutamate (5 mM, n = 7) within Vi had no effect. Microinjections of 500 mM glutamate within V p / V o (n = 15) or within the spinal trigeminal tract (n = 13) had no consistent effect on adrenal or autonomic function. Plasma concentrations of ACTH were not altered significantly by glutamate regardless of dose or of the site of injection. The results suggest that local release of glutamate within Vi, but not within Vp/Vo, influences adrenal and autonomic function. Together with previous results obtained after injections of glutamate within subnucleus caudalis, these data indicate that glutaminergic input to both Vi and to more caudal portions of the spinal trigeminal nucleus contribute to the control of autonomic function such as that which often accompanies trigeminal nociception.
INTRODUCTION The contribution of rostral portions of the trigeminal sensory complex in mediating the discriminative and reflex aspects of orofacial pain has remained controversial. Early clinical reports that surgical interruption of the descending spinal trigeminal tract caused a selective loss of facial pain and thermal sensation 38 suggested that trigeminal subnucleus caudalis (Vc) was necessary for trigeminal pain. More recent results, however, indicate that trigeminal tractotomy at the level of the obex does not abolish pain reactions evoked by perioral or intraoral stimuli in humans 45 or in animals 16'31'41'46. Also, considerable anatomical 2'24'29'36'37'39 and electrophysiological evidence 4,12,17,19,20,27,34 indicates that input from tooth pulp and corneal nerves are processed within rostral portions of the trigeminal brain stem complex. Although the mechanisms that underlie the sensory-discriminative aspects of trigeminal nociception have received much attention, stimulation of
trigeminal nociceptors also evoke autonomic reflex or affective responses 6'33. However, the anatomical pathways and the neurochemical mechanisms that underlie the changes in autonomic function evoked by trigeminal nociceptors are not well defined. Early tractotomy studies 18 suggested that afferent projections to the most rostral portions of the trigeminal brain stem complex alone were not sufficient to support the autonomic responses to corneal stimulation. Excitatory amino acid transmitters are well associated with the integration of nociceptive input within the spinal cord (see ref. 43). Similarly, anatomical 3'44'~5'22'35'4° and physiological32 evidence suggests that glutamate may function as a neurotransmitter within the spinal trigeminal system. In previous studies 7'8, microinjections of glutamate within Vc evoked adrenal and autonomic responses similar qualitatively to those seen after noxious thermal stimulation of the corneal surface in the anesthetized cat 6. The purpose of the present study was to determine if local release of L-glutamate, by microinjection, within rostral portions of the trigeminal sensory complex af-
Correspondence." D.A. Bereiter, Brown University/Rhode Island Hospital, Division of Surgical Research, Neuroendocrine Laboratory, Providence, RI 02903, USA. Fax: (1) (401) 444-8052.
156 fects adrenal secretory or autonomic function in the cat. MATERIALS AND METHODS
General methods Adult cats of either sex (2.9-5.9 kg) were deprived of food overnight but were given free access to water. After an initial tranquilizing dose of ketamine-HCl (30 mg/kg, i.m.) to permit insertion of a cephalic vein catheter, anesthesia was achieved by a-chloralose (75 mg/kg, i.v.) given as a full dose prior to surgery. Supplemental doses of chloralose (7.5 mg/kg, i.v.) were given every 60-90 min. Catheters were placed in the descending aorta (via the femoral artery) to monitor arterial pressure, in the cephalic vein for the infusion of normal saline during blood sampling and for the administration of all drugs, and in the femoral artery for the collection of peripheral blood samples. Adrenal venous blood was sampled directly from a catheter placed in the left lumboadrenal vein. After tracheostomy, animals were respired artificially (15 strokes per min) with O2-supplemented room air and expiratory CO 2 was monitored continuously and maintained within normal range (3.0-4.5%). Heart rate was monitored from a standard 3-lead electrocardiogram. Animals were paralyzed with gallamine triethiodide (5 mg/kg, i.v.) after completion of all surgical procedures and supplemental doses (3 mg/kg, i.v.) were given hourly. Depth of anesthesia was determined before surgery and prior to gallamine administration by pupillary constriction and loss of withdrawal reflexes. Body temperature was kept at 38°C with a heating blanket. All surgical incisions were infiltrated with 2% lidocaine jelly.
Microinjection technique After placement of all catheters, the animal was secured in a stereotaxic frame (Kopf, Tujunga, CA), the muscles overlying the occipital bone were reflected and a portion of the bone was removed with a dental drill. Microinjections were delivered via a 28 gauge stainless steel cannula or from a glass micropipette positioned stereotaxically above the targeted brain stem site at a 40 ° angle off vertical. The injection cannula was filled with either a high (500 mM) or low (5 mM) concentration of L-glutamate (pH 8.0 in artificial cerebrospinal fluid plus 2% fast green dye) and delivered in a 100 nl volume over approximately 1 min. The patency of the injection cannula was confirmed upon removal from the brain at the end of the 10 min sampling period. A bolus injection of chloralose (25 mg/kg) was given at the end of the experiment and the animal was perfused through the heart with normal saline followed by 10% buffered formalin. The sites of microinjection were identified in transverse sections (40/zm) by the deposit of dye and mapped onto a series of brain stem outlines adapted from Berman t°.
Experimental design A total of 38 cats each received one to three microinjections of glutamate directed at the nucleus principalis (Vp), subnucleus oralis (Vo) or at subnucleus interpolaris (Vi). Each microinjection was delivered in a total volume of 100 nl ipsilateral to the adrenal vein catheter on the left side of the brain stem. Peripheral arterial (0.5-1.0 ml) and adrenal venous (0.4-1.0 ml) blood samples were collected over 20-60 s at - 5 and 0 min (prestimulus controls) and at 1, 3, 6 and 10 min after the onset of each microinjection. The volume of adrenal venous blood collected per unit time permitted an estimate of total adrenal blood flow. The timing for the first postinjection adrenal venous sample began 10-30 s prior to + 1 min depending on the flow rate for each animal. At least 30 min elapsed between subsequent microinjections.
electrochemical detection as described previously 6. The intra-assay and interassay coefficients of variation for a cat plasma pool containing 2.29 ng/extraction volume of norepinephrine were 2.25 and 5.82%, respectively; and for a plasma pool containing 2.45 ng/extraction volume of epinephrine were 2.78 and 4.49%, respectively; and for a plasma pool containing 2.24 ng/extraction volume of dopamine were 4.38 and 4.72%, respectively. The adrenal secretory rate for each catecholamine species was calculated by the formula: [adrenal plasma concentration ( n g / m l ) - p e r i p h e r a l plasma concentration (ng/ml)]×adrenal plasma flow ( m l / m i n ) = ng/min for epinephrine, norepinephrine and dopamine. Plasma adrenocorticotropin (ACTH) was determined by direct radioimmunoassay 6 using an antibody generated against a conjugate of ACTH 1-24 that reacts completely with ACTH 1-39 and ACTH 1-24, but not with either the N-terminal end (ACTH 1-13) or with the C-terminal end (ACTH 18-39) fragments of the AC~H molecule. Charcoal-stripped cat plasma was added to the ACTH 1-39 standards (Bachem, Torrance, CA) to control for matrix effects. Intra-assay and interassay coefficients of variation for a cat plasma pool with an ACTH concentration of 101 pg/ml were 4.9% and 14.6%, respectively.
Statistical analyses The responses were analyzed after first grouping the anatomical location of each site of microinjection according to the description of Berman 1°. The statistical analyses included five treatment groups: (a) microinjections of 500 mM glutamate at sites within Vp or Vo, transverse planes P 4.0-P 7.1 (n = 15); (b) microinjections of 500 mM glutamate within Vi, planes P 10.8-P 13.5 (n = 16); (c) microinjections of 500 mM glutamate within the spinal trigeminal tract (Vtr), predominantly at planes P 10.8-P 13.5 (n = 13); (d) microinjections of 5 mM glutamate within the boundaries of Vi (n = 7); and (e) microinjections of 500 mM glutamate at sites located outside of these trigeminal brain stem regions (n = 14). As a point of reference in the transverse plane, the obex level of the cat brain stem would correspond approximately to a plane of P 14.5 of the atlas of Berman 1°. Thus, all sites of microinjection were at least 1 mm rostral to the obex. All of the data included in these analyses were collected from animals where the mean arterial pressure remained above 70 mmHg and the patency of the injection cannula was verified at the end of the experiment. The adrenal secretion of catecholamines, peripheral plasma concentrations of catecholamines and ACTH, adrenal blood flow, adrenal vascular conductance, mean arterial pressure, and heart rate responses to injections of L-glutamate were assessed by two-way analysis of variance (location and time) corrected for repeated measures 44. The analyses compared the absolute values to the prestimulus value (mean of - 5 and 0 min values) for each variable and individual comparisons used the Newman-Keuls test after analysis of variance. Adrenal catecholamine secretory rates and plasma concentrations of ACTH were assessed after logarithmic transformation to reduce the error variance. In addition to these group comparisons, each microinjection site was encoded on the brain stem outlines (see Fig. 2) for the evoked change in adrenal secretion of epinephrine. The 95% confidence limit for prestimulus ( - 5 and 0 min) variability was calculated across all sites of injection of glutamate (n = 65) as an absolute difference (za = 2.33 ng/min) and as a percentage difference (A% = 52.4%). The symbols on the brain stem outlines reflect the peak (maximum) change in secretion of epinephrine that was seen during the 10 min postinjection sampling period when compared against these 95% confidence limits. All data in the text and in the figures represent the mean + S.E.M. Spearman rank correlation (r s) analysis was used to assess possible relationships between response variables.
Biochemical determinations
RESULTS
Peripheral arterial and adrenal venous blood samples were collected on ice in tubes containing EDTA (30 /zl/ml whole blood). After centrifugation the plasma was stored at - 60°C for subsequent analyses. The plasma concentrations of catecholamines were determined from 100/xl of plasma after alumina extraction by HPLC with
Adrenal secretion of catecholamines Microinjections of the high dose (500 mM) of glutamate within Vi evoked a significant (P < 0.001) in-
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Fig. 1. Dose-related effects of microinjections of L-glutamate within subnucleus interpolaris on the adrenal secretion of epinephrine (top panel) and on the adrenal epinephrine/norepinephrine secretory ratio. Symbols: closed circles = high dose (500 mM, 100 nl, n = 16); open circles = low dose (5 mM, 100 nl, n = 7). * P < 0.05, ** P < 0.01 versus prestimulus value; a p < 0.05, b p < 0.01 versus low dose group; shaded bar on abscissa indicates period of microinjection.
c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t was m a x i m a l by + 1 min, r e m a i n e d e l e v a t e d at + 6 min a n d r e t u r n e d t o w a r d s p r e s t i m u l u s v a l u e s by + 10 min. Mic r o i n j e c t i o n s o f t h e lower d o s e o f g l u t a m a t e (5 m M ) within Vi h a d no effect (Fig. 1, t o p panel). A l t h o u g h the mean prestimulus adrenal secretory rate of e p i n e p h r i n e was n u m e r i c a l l y l o w e r in t h e g r o u p o f cats t h a t r e c e i v e d t h e 5 m M d o s e o f g l u t a m a t e c o m p a r e d to t h e g r o u p t h a t r e c e i v e d 500 m M injections, this differe n c e was n o t significant. A n i n c r e a s e in t h e a d r e n a l s e c r e t i o n of e p i n e p h r i n e e v o k e d by Vi injections o f 500
m M g l u t a m a t e was a c o n s i s t e n t f i n d i n g as 15 o f t h e 16 sites d e m o n s t r a t e d a m a x i m u m i n c r e a s e t h a t e x c e e d e d t h e 95% c o n f i d e n c e limit for t h e v a r i a b i l i t y b e t w e e n t h e - 5 a n d 0 m i n p r e s t i m u l u s v a l u e s (Fig. 2, p l a n e s P 11.6 a n d P 13.5, + 1 . 0 - + 3 . 0 to obex). O f t h e seven i n j e c t i o n sites within Vi t h a t r e c e i v e d 5 m M g l u t a m a t e n o n e c a u s e d an i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t e x c e e d e d t h e 9 5 % c o n f i d e n c e limit for p r e s t i m u l u s v a r i a b i l i t y ( N o t e : t h e s e 7 sites a r e n o t e n c o d e d o n Fig. 2). A f t e r injections o f 500 m M glutam a t e within t h e V t r only 1 o f 13 sites c a u s e d an i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t e x c e e d e d t h e 9 5 % c o n f i d e n c e limit for p r e s t i m u l u s variability. A l t h o u g h t h e m e a n g r o u p r e s p o n s e for all sites l o c a t e d w i t h i n V p / V o d i d n o t i n d i c a t e a signific a n t i n c r e a s e in t h e s e c r e t i o n o f e p i n e p h r i n e (see T a b l e I), several i n d i v i d u a l sites (5 o f 15) l o c a t e d m a i n l y within t h e d o r s o m e d i a l p o r t i o n o f V p / V o d i d r e s p o n d to 500 m M g l u t a m a t e with an i n c r e a s e in s e c r e t i o n (Fig. 2, p l a n e s P 4.6 a n d P 6.0). T h r e e sites w e r e l o c a t e d v e n t r a l to t h e spinal t r i g e m i n a l n u c l e u s at t h e level o f Vi (Fig. 2, p l a n e s P l l . 6 - P 13.5) in [he r e g i o n o f t h e ~audal v e n t r o l a t e r a l m e d u l l a r y A 1 g r o u p a n d collectively r e s p o n d e d to 500 m M g l u t a m a t e injections with a d e c r e a s e ( - 2 9 . 7 + 9 . 6 % ) in t h e a d r e n a l secretion o f e p i n e p h r i n e ( d a t a n o t shown). M i c r o i n j e c t i o n s o f 500 m M g l u t a m a t e within Vi c a u s e d a small b u t significant ( + 1.4 + 0.5 n g / m i n , P < 0.05) i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f n o r e p i n e p h r i n e , w h e r e a s o t h e r t r e a t m e n t g r o u p s h a d no r e s p o n s e ( T a b l e I). C a l c u l a t i o n o f t h e a d r e n a l epinephrine/norepinephrine secretory ratio revealed t h a t 500 m M g l u t a m a t e i n j e c t i o n s w i t h i n Vi i n c r e a s e d t h e r a t i o f r o m 3.2 + 0.5 to a m a x i m u m o f 4.4 + 0.6 ( P < 0.001, n = 16) by + 6 min, i n d i c a t i n g a p r e f e r e n tial i n c r e a s e in t h e s e c r e t i o n o f e p i n e p h r i n e a b o v e t h a t o f n o r e p i n e p h r i n e (Fig. 1, l o w e r panel). T h e m e a n
TABLE I Summary of the adrenal and autonomic responses to microinjections of L-glutamate (500 raM, 100 nl) within the principal sensory nucleus / subnucleus oralis (Vp / Vo), the subnucleus interpolaris (Vi) or the spinal trigeminal tract (Vtr)
Peak = maximum value compared to prestimulus observed during the 10 min sampling period following microiniection. Adrenal Epi, adrenal secretion of epinephrine; adrenal NE, adrenal secretion of norepinephrine; ACTH, adrenocorticotropic hormone; ABF, adrenal blood flow; MAP, mean arterial pressure. Vp / Iio (n = 15) Prestimulus
AdrenalEpi(ng/min) AdrenalNE(ng/min) ACTH (pg/ml) ABF(ml/min) MAP(mmHg) Heart rate (beats/min)
5.3 _+ 1.1 4.1 + 0.8 97 +_21 0.70± 0.06 107 + 4 186 ± 5
* P < 0.05, * * P < 0.01 versus prestimulus value.
1,7 (n = 16) Peak
6.5 ± 1.5 5.5 + 1.1
157 + 61 0.75± 0.09 107 + 3 189 ± 6
Vtr (n = 13)
Prestimulus
Peak
Prestimulus
Peak
7.7 + 2.3 2.5 + 0.5 115 _+23 0.74± 0.07 108 ± 6 181 + 4
19.1 + 4.4** 3.9 + 0.6 * 99 + 12 0.93+ 0.1"* 115 ± 6 * 189 + 5 **
5.9 + 1.3 7.3 _+ 3.2 129 + 20 0.66± 0.09 103 ± 3 173 ± 6
6.7 + 2.0 8.7 ± 3.9 127 + 22 0.62± 0.1 103 ± 4 173 ± 7
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Fig. 2. Sites of microinjection of L-glutamate (500 mM, 100 nl) encoded for the evoked change in the adrenal secretion of epinephrine. A, increase; II, decrease; D, no change. 5M, trigeminal motor n.; 7N, facial nerve; CX, external cuneate n.; IO, inferior olivary n.; LRN, lateral reticular nucleus; NTS, n. tractus solitarius; SO, superior olivary n.; Vi, trigeminal subnucleus interpolaris; Vo, subnucleus oralis; Vp, principal trigeminal n.; Vtr, spinal trigeminal tract. Bar = 1.0 ram; numbers refer to anterior-posterior plane of section adapted from Berman z°.
prestimulus adrenal secretion of dopamine for all sites was 0.14_+ 0.03 ng/min (n = 65) and no significant responses to microinjections of glutamate were seen among the five treatment groups. The prestimulus peripheral arterial plasma concentrations of epinephrine, norepinephrine and dopamine averaged 0.05_+ 0.06 ng/ml, 0.39 _+0.03 n g / m l and 0.06 _+0.1 ng/ml (n = 65), respectively, and were not affected significantly by microinjections of glutamate (data not shown).
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Microinjections of 500 mM glutamate within Vi evoked a sustained increase in total adrenal blood flow (P < 0.005), whereas 5 mM glutamate caused a smaller non-significant increase (Fig. 3, top panel). Analysis of variance revealed a significant (P < 0.025) interaction effect indicating a dose-related influence on total adrenal blood flow by injections within Vi. However, it was noted that even the low dose of glutamate evoked small increases in adrenal blood flow and in adrenal vascular conductance. In contrast to the sustained increase in total adrenal blood flow, mean arterial pressure (MAP) increased transiently after microinjections of 500 mM glutamate into Vi (+ 7.2 _+ 2.5 mmHg, P < 0.001) and returned towards prestimulus values by + 3 min (Fig. 3, middle panel). By 10 min after Vi injections of 500 mM glutamate, MAP fell below prestimulus values, whereas MAP remained below prestimulus values throughout the sampling period after 5 mM glutamate, a result that was consistent with the trigeminal depressor response 23. Correspondingly, the calculated adrenal vascular conductance (adrenal blood flow/mean arterial pressure, Fig. 3, bottom panel) showed a sustained increase (P < 0.001) after injec-
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Fig. 3. Dose-related effects of microinjections of L-glutamate within subnucleus interpolaris on total adrenal blood flow (top panel), mean arterial pressure (MAP, middle panel), and on adrenal vascular conductance (lower panel), e, high dose (500 mM, 100 nl, n = 16); ©, low dose (5 mM, 100 nl, n = 7 ) . * P < 0 . 0 5 , * * P < 0 . 0 1 versus prestimulus value; shaded bar on abscissa indicates period of microinjection.
159 tions of 500 mM glutamate into Vi, whereas injections of 5 mM glutamate evoked smaller non-significant responses. Thus, the increase in total adrenal blood flow seen after injections of glutamate within Vi is likely due in part to active vasodilatation of the adrenal vascular bed and cannot be explained by the evoked increase in MAP. Heart rate increased ( + 8 . 0 + 2.7 beats/min, P < 0.001) by + 1 min after injection of 500 mM glutamate within Vi and returned towards prestimulus values by + 3 min. Neither heart rate nor the other cardiovascular variables were affected significantly by injections of 500 mM glutamate into V p / V o or into Vtr (Table I).
Arterial plasma concentrations of ACTH Mean arterial plasma concentrations of A C T H were not affected consistently by microinjections of 500 mM glutamate regardless of the site of injection (Table I), however, several individual sites did cause changes in plasma A C T H that exceeded the 95% confidence limits (A = 20.4 p g / m l , A% = 18.3%) for prestimulus variability. For example, 4 of 15 sites located within V p / V o , mainly in the dorsomedial portion, caused an increase in plasma A C T H and two sites caused a decrease in ACTH. After injection of 500 mM glutamate within Vi, 3 of 16 sites evoked an increase in plasma ACTH, but 7 of 16 sites caused a decrease in plasma ACTH. Among the 13 sites located within Vtr, three sites caused an increase in plasma A C T H and five sites caused a decrease in plasma ACTH.
Correlation analyses Spearman rank-order correlation analyses of the responses from the 16 sites of microinjection of 500 mM glutamate within Vi indicated that the magnitude of the evoked change in adrenal secretion of epinephrine was not correlated with that of total adrenal blood flow (r s = 0.185, P > 0.05), adrenal vascular conductance (r s = 0.175, P > 0.05), or with that of MAP (rs = 0.206, P > 0.05). Also, the magnitude of the evoked change in total adrenal blood flow was not correlated with that of MAP (r s = -0.156, P > 0.05) and the change in MAP was not correlated with the change in heart rate (r s = 0.196, P > 0.05). DISCUSSION The results indicated that microinjections of glutamate within Vi evoked significant dose-related increases in the adrenal secretion of catecholamines and in cardiovascular function, whereas injections of glutamate within the most rostral portions of the trigeminal sensory complex ( V p / V o ) had no consistent effects.
Although the volume of each injection was small (100 nl), uncertainty about the extent of the diffusion of the injected substance is always a concern for microinjection studies. It is not likely that the autonomic responses evoked by injections within Vi were due to the spread of glutamate to nearby structures since numerous injection sites were located laterally within the adjacent spinal trigeminal tract and were generally without effect. Glutamate injections within three sites located immediately ventral to Vi caused small decreases in the adrenal secretion of catecholamines in contrast to the significant increase in secretion evoked by sites within the adjacent Vi. In previous studies somewhat larger injection volumes (160 nl) were used, however, an apparent laminar organization was seen within Vc for glutamate-evoked adrenal and autonomic responses 7'8. Thus, the dose-related nature of the responses and the restricted anatomical localization of responsive sites suggested that the adrenal and autonomic responses evoked by microinjections of glutamate within Vi were the result of local action and were not due to widespread diffusion. Although the changes in adrenal and autonomic function evoked by microinjections of glutamate within Vi or Vc were similar qualitatively to those seen after noxious stimulation of trigeminal receptors 1'5'6, a role for glutamate as a neurotransmitter in trigeminal nociception has not been well established. Anatomical studies revealed that 2 0 - 3 0 % of trigeminal ganglion ceils in the rat were positive for glutamate-like immunoreactivity 22 and axon terminals containing glutamate-like immunoreactivity were found throughout the trigeminal brain stem complex ~4. At caudal levels of the Vsp glutamate-positive fibers and terminals were associated with tooth pulp nerves 3'~5. In receptor binding studies in the rat, the density of both N M D A (N-methyl-o-aspartate) and n o n - N M D A glutamate receptor subtypes within the Vsp had a rank-order of Vc > Vi > Vo 4°, a finding consistent with the order of magnitude of the autonomic responses evoked by microinjections of glutamate. It is not yet known if the adrenal and autonomic responses evoked by microinjections of glutamate within Vi or within Vc 7,s demonstrate a preference for one glutamate receptor subtype over another. Salt and Hill 32 have shown that iontophoretic application of a non-selective glutamate receptor antagonist within Vc blocked the neural responses to noxious mechanical but not to noxious thermal stimuli. The first systematic study of the Vsp and nociceptor-evoked autonomic reflex activity was reported by Gerard TM who noted increases in arterial pressure and pupillary dilatation that accompanied corneal stimula-
160 tion in the cat. Beginning at the level of the obex successively more rostral cuts of the descending spinal trigeminal tract were made until cornea-evoked autonomic reflex activity was abolished. Gerard noted that cornea-evoked autonomic activity persisted until the tract was lesioned at mid-Vi levels. Matsuo et al. 25 assessed the increase in volume of saliva secreted from the submandibular gland of the decerebrate rat after noxious thermal or mechanical stimulation of the perioral region. Lesions of Vi or of Vc greatly attenuated salivary flow, whereas lesions of the nucleus tractus solitarius (NTS) or of the parabrachial nuclei had no effect on the volume of saliva. Panneton 2s reported that injections of lidocaine within the Vsp at the level of the obex blocked the cardiorespiratory responses to nasal stimulation in the muskrat. In recent studies 9, we have found that the adrenal and autonomic responses evoked by chemical stimulation of the cornea were attenuated greatly by ipsilateral injections of lidocaine within the middle to caudal portions of Vi in the cat. Further, in this same study c-fos protein was localized immunocytochemically within ventrolateral portions of Vi and more caudally within Vc but was not detected in V p / V o regions. Electrophysiological studies 19'2° have revealed numerous nociceptive neurons within the middle and caudal regions of the Vi in the cat. Collectively, these results suggest that neurons within the Vi as well as within the Vc portions of Vsp contribute to the integration of sensory input relevant for trigeminal-evoked autonomic output. The present study also suggests that glutaminergic input to V p / V o regions has little influence on autonomic function. Although nociceptive input from perioral or intraoral structures clearly activates V p / V o neurons 12'17'34, this input may be mediated through non-glutaminergic pathways. Whereas the function of the nociceptive neurons within V p / V o is not known, it is also possible that these ceils mediate sensory-discriminative aspects of trigeminal nociception and that reflex autonomic adjustments to trigeminal nociceptive stimulation does not include a significant relay through the most rostral portions of the Vsp as originally suggested by Gerard TM. It is not yet known to what extent the central neural pathways that mediate the sensory-discriminative aspects of trigeminal input overlap with those that mediate the autonomic responses to noxious trigeminal stimuli. Although the adrenal and autonomic responses to Vi injections of glutamate were similar qualitatively to those seen after microinjections into Vc 7'8, significant differences also were noted. Glutamate injections within Vi did not affect plasma A C T H , whereas similar injections within laminae I - I I of Vc caused a reliable
increase 8. The adrenal secretion of catecholamines was accompanied by significant increases in total adrenal blood flow and in adrenal vascular conductance after injections of glutamate into Vi, whereas injections into laminae I - I I or V - V I of Vc had no effect total adrenal blood flow or on adrenal vascular conductance 7. The differences in adrenal and autonomic responses observed after injections of glutamate at different rostrocaudal sites within the trigeminal sensory complex suggests an underlying organization for control of autonomic function that cannot be explained simply by the density of glutamate receptor subtypes 4°. Also, the qualitative differences in endocrine and autonomic responses to injections of glutamate within Vi versus Vc argue against the notion that these results are due simply to widespread diffusion of glutamate between these two subnuclei. Efferent projections from Vi and from Vc to the hypothalamus, periaqueductal gray, parabrachial nuclei and to the NTS have been well described 11'13'2°'21'26'3°'42, whereas significant projections from Vp or Vo to these autonomic relay areas have not been reported. Trigeminal projections to the NTS originate mainly from Vc and are more extensive than those from Vi 21'26. Further, examination of more rostral portions of Vsp did not reveal projections from Vp or from Vo neurons to the NTS in the rat (C.B. Saper, personal communication). The present results extend those of previous studies 7'8 to suggest that local release of glutamate within the Vi or the Vc portions of the Vsp exerts a significant influence on adrenomedullary secretion and on autonomic function in the cat. The evoked responses are consistent with those that would be expected to accompany stimulation of trigeminal nociceptors, however, the relationship between glutaminergic input to the Vsp and the integration of noxious trigeminal sensory information is not well defined. Although the most rostral portions of the trigeminal brain stem complex may also contribute to the integration of trigeminal nociceptive input, the present results indicate that glutaminergic input to Vp and Vo neurons does not have a consistent influence on adrenal and autonomic function.
Acknowledgements. The author wishes to thank A.P. Benetti and D.F. Bereiter for their expert technical assistance. This study was supported in part by Grant NS26137 from the National Institutes of Health. REFERENCES 1 Allison, D.J. and Powis, D.A., Adrenal catecholamine secretion during stimulation of the nasal mucous membrane in the rabbit, J. Physiol., 217 (1971) 327-339.
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23 Kumada, M., Dampney, R.A.L. and Reis, D.J., The trigeminal depressor response: A novel vasodepressor response originating from the trigeminal system, Brain Res., 119 (1977) 305-326. 24 Marfurt, C.F., The central projections of trigeminal primary afferent neurons in the cat as determined by the tranganglionic transport of horseradish peroxidase, J. Comp. Neurol., 203 (1981) 785-798. 25 Matsuo, R., Yamamoto, T., Yoshitaka, K. and Morimoto, T., Neural substrates for reflex salivation induced by taste, mechanical, and thermal stimulation of the oral region in decerebrate rats, Jpn. J. Physiol., 39 (1989) 349-357. 26 Menetrey, D. and Basbaum, A.I., Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation, J. Comp. Neurol., 255 (1987) 439-450. 27 Nagano, S., Myers, J.A. and Hall, R.D., Representation of the cornea in the brain stem of the rat, Exp. Neurol., 49 (1975) 653-670. 28 Panneton, W.M., Trigeminal mediation of the diving response in the muskrat, Brain Res., 560 (1991) 321-325. 29 Panneton, W.M. and Burton, H., Corneal and periocular representation within the trigeminal sensory complex in the cat studied with transganglionic transport of horseradish peroxidase, J. Comp. Neurol., 199 (1981) 327-344. 30 Panneton, W.M. and Burton, H., Projections from the paratrigeminal nucleus and the medullary and spinal dorsal horns to the peribrachial area in the cat, Neuroscience, 15 (1985) 779-797. 31 Pickoff-Matuk, J.F., Rosenfeld, J.P. and Broton, J.G., Lesions of the mid-spinal trigeminal complex are effective in producing perioral thermal hypoalgesia, Brain Res., 382 (1986) 291-298. 32 Salt, T.E. and Hill, R.G., Pharmacological differentiation between responses of rat medullary dorsal horn neurons to noxious mechanical and noxious thermal cutaneous stimuli, Brain Res., 263 (1983) 167-171. 33 Sessle, B.J., The neurobiology of facial and dental pain: Present knowledge, future directions, J. Dent. Res., 66 (1987) 962-981. 34 Sessle, B.J. and Greenwood, L.F., Inputs to trigeminal brain stem neurones from facial, oral, tooth pulp and pharyngolaryngeal tissues. I. Responses to innocuous and noxious stimuli, Brain Res., 117 (1976) 211-226. 35 Shigemoto, R., Nakanishi, S. and Mizuno, N., Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: An in situ hybridization study in adult and developing rat, J. Comp. Neurol., 322 (1992) 121-135. 36 Shigenaga, Y., Okamoto, T., Nishimori, T., Suemune, S., Nasution, I.D., Chen, I.C., Tsuru, K., Yoshida, A., Tabuchi, K., Hosoi, M. and Tsuru, H., Oral and facial representation in the trigeminal principal and rostral spinal nuclei of the cat, J. Comp. Neurol., 244 (1986) 1-18. 37 Shigenaga, Y., Suemune, S., Nishimura, M., Nishimori, T., Sato, H., Ishidori, H., Yoshida, A., Tsuru, K., Tsuiki, Y., Dakeoka, Y., Nasution, I.D. and Hosoi, M., Topographic representation of lower and upper teeth within the trigeminal sensory nuclei of adult cat as demonstrated by the transganglionic transport of horseradish peroxidase, J. Comp. Neurol., 251 (1986) 299-316. 38 Sjoqvist, O., Studies on pain conduction in the trigeminal nerve. A contribution to the surgical treatment of facial pain, Acta Psychiat. Neurol. Scand., 1, Suppl. 17 (1938) 1-139. 39 Takemura, M., Sugimoto, T. and Shigenaga, Y., Difference in central projection of primary afferents innervating facial and intraoral structures in the rat, Exp. Neurol., 111 (1991) 324-331. 40 Tallaksen-Greene, S.J., Young, A.B., Penny, J.B. and Beitz, A.J., Excitatory amino acid binding sites in the trigeminal principal sensory and spinal trigeminal nuclei of the rat, Neurosci. Lett., 141 (1992) 79-83. 41 Vyklicky, L., Keller, O., Jastreboff, P., Vyklicky, L.J. and Butkhuzi, S.M., Spinal trigeminal tractotomy and nociceptive reactions evoked by tooth pulp stimulation in the cat, J. Physiol. (Paris), 73 (1977) 379-386. 42 Wiberg, M., Westman, J. and Blomqvist, A., The projection to the mesencephalon from the sensory trigeminal nuclei. An anatomical study in the cat, Brain Res., 399 (1986) 51-68.
162 43 Wilcox, G.L., Excitatory neurotransmitters and pain. In M.R. Bond, J.E. Charlton and C.J. Woolf (Eds.), Proceedings of the Vlth World Congress on Pain, Elsevier, New York, 1991, pp. 97-117. 44 Winer, B.J., Statistical Principles in Experimental Design, 2nd edn., McGraw-Hill, New York, 1971, pp. 514-603.
45 Young, R.F., Effect of trigeminal tractotomy on dental sensation in humans, J. Neurosurg., 56 (1982) 812-818. 46 Young, R.F., Oleson, T.D. and Perryman, K.M., Effect of trigeminal tractotomy on behavioral response to dental pulp stimulation in the monkey, J. Neurosurg., 55 (1981) 420-430.
155
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19192
Microinjections of glutamate within trigeminal subnucleus interpolaris alters adrenal and autonomic function in the cat David A. Bereiter Departments of Neuroscience and Surgery Brown University/Rhode Island Hospital, Providence, RI 02903 (USA) (Accepted 20 April 1993)
Key words." Adrenal catecholamine; Autonomic control; Excitatory amino acid; Spinal trigeminal nucleus
The influence of rostral portions of the trigeminal sensory complex on adrenal and autonomic function was assessed by microinjections of L-glutamate (500 or 5 mM, 100 nl) directed at subnucleus interpolaris (Vi) or at the nucleus principalis/subnucleus oralis level (Vp/Vo) in chloralose-anesthetized cats. Microinjections of glutamate (500 mM) within Vi evoked prompt (by + 1 min) dose-related increases in the adrenal secretion of epinephrine (+11.4+2.5 ng/min, P<0.001), adrenal blood flow (+0.19+0.06 ml/min, P<0.05), mean arterial pressure (+6.6_+3.0 mmHg, P < 0.025) and heart rate (+8.0+2.7 beats/min, P < 0.01, n = 16). Microinjections of lower doses of L-glutamate (5 mM, n = 7) within Vi had no effect. Microinjections of 500 mM glutamate within V p / V o (n = 15) or within the spinal trigeminal tract (n = 13) had no consistent effect on adrenal or autonomic function. Plasma concentrations of ACTH were not altered significantly by glutamate regardless of dose or of the site of injection. The results suggest that local release of glutamate within Vi, but not within Vp/Vo, influences adrenal and autonomic function. Together with previous results obtained after injections of glutamate within subnucleus caudalis, these data indicate that glutaminergic input to both Vi and to more caudal portions of the spinal trigeminal nucleus contribute to the control of autonomic function such as that which often accompanies trigeminal nociception.
INTRODUCTION The contribution of rostral portions of the trigeminal sensory complex in mediating the discriminative and reflex aspects of orofacial pain has remained controversial. Early clinical reports that surgical interruption of the descending spinal trigeminal tract caused a selective loss of facial pain and thermal sensation 38 suggested that trigeminal subnucleus caudalis (Vc) was necessary for trigeminal pain. More recent results, however, indicate that trigeminal tractotomy at the level of the obex does not abolish pain reactions evoked by perioral or intraoral stimuli in humans 45 or in animals 16'31'41'46. Also, considerable anatomical 2'24'29'36'37'39 and electrophysiological evidence 4,12,17,19,20,27,34 indicates that input from tooth pulp and corneal nerves are processed within rostral portions of the trigeminal brain stem complex. Although the mechanisms that underlie the sensory-discriminative aspects of trigeminal nociception have received much attention, stimulation of
trigeminal nociceptors also evoke autonomic reflex or affective responses 6'33. However, the anatomical pathways and the neurochemical mechanisms that underlie the changes in autonomic function evoked by trigeminal nociceptors are not well defined. Early tractotomy studies 18 suggested that afferent projections to the most rostral portions of the trigeminal brain stem complex alone were not sufficient to support the autonomic responses to corneal stimulation. Excitatory amino acid transmitters are well associated with the integration of nociceptive input within the spinal cord (see ref. 43). Similarly, anatomical 3'44'~5'22'35'4° and physiological32 evidence suggests that glutamate may function as a neurotransmitter within the spinal trigeminal system. In previous studies 7'8, microinjections of glutamate within Vc evoked adrenal and autonomic responses similar qualitatively to those seen after noxious thermal stimulation of the corneal surface in the anesthetized cat 6. The purpose of the present study was to determine if local release of L-glutamate, by microinjection, within rostral portions of the trigeminal sensory complex af-
Correspondence." D.A. Bereiter, Brown University/Rhode Island Hospital, Division of Surgical Research, Neuroendocrine Laboratory, Providence, RI 02903, USA. Fax: (1) (401) 444-8052.
156 fects adrenal secretory or autonomic function in the cat. MATERIALS AND METHODS
General methods Adult cats of either sex (2.9-5.9 kg) were deprived of food overnight but were given free access to water. After an initial tranquilizing dose of ketamine-HCl (30 mg/kg, i.m.) to permit insertion of a cephalic vein catheter, anesthesia was achieved by a-chloralose (75 mg/kg, i.v.) given as a full dose prior to surgery. Supplemental doses of chloralose (7.5 mg/kg, i.v.) were given every 60-90 min. Catheters were placed in the descending aorta (via the femoral artery) to monitor arterial pressure, in the cephalic vein for the infusion of normal saline during blood sampling and for the administration of all drugs, and in the femoral artery for the collection of peripheral blood samples. Adrenal venous blood was sampled directly from a catheter placed in the left lumboadrenal vein. After tracheostomy, animals were respired artificially (15 strokes per min) with O2-supplemented room air and expiratory CO 2 was monitored continuously and maintained within normal range (3.0-4.5%). Heart rate was monitored from a standard 3-lead electrocardiogram. Animals were paralyzed with gallamine triethiodide (5 mg/kg, i.v.) after completion of all surgical procedures and supplemental doses (3 mg/kg, i.v.) were given hourly. Depth of anesthesia was determined before surgery and prior to gallamine administration by pupillary constriction and loss of withdrawal reflexes. Body temperature was kept at 38°C with a heating blanket. All surgical incisions were infiltrated with 2% lidocaine jelly.
Microinjection technique After placement of all catheters, the animal was secured in a stereotaxic frame (Kopf, Tujunga, CA), the muscles overlying the occipital bone were reflected and a portion of the bone was removed with a dental drill. Microinjections were delivered via a 28 gauge stainless steel cannula or from a glass micropipette positioned stereotaxically above the targeted brain stem site at a 40 ° angle off vertical. The injection cannula was filled with either a high (500 mM) or low (5 mM) concentration of L-glutamate (pH 8.0 in artificial cerebrospinal fluid plus 2% fast green dye) and delivered in a 100 nl volume over approximately 1 min. The patency of the injection cannula was confirmed upon removal from the brain at the end of the 10 min sampling period. A bolus injection of chloralose (25 mg/kg) was given at the end of the experiment and the animal was perfused through the heart with normal saline followed by 10% buffered formalin. The sites of microinjection were identified in transverse sections (40/zm) by the deposit of dye and mapped onto a series of brain stem outlines adapted from Berman t°.
Experimental design A total of 38 cats each received one to three microinjections of glutamate directed at the nucleus principalis (Vp), subnucleus oralis (Vo) or at subnucleus interpolaris (Vi). Each microinjection was delivered in a total volume of 100 nl ipsilateral to the adrenal vein catheter on the left side of the brain stem. Peripheral arterial (0.5-1.0 ml) and adrenal venous (0.4-1.0 ml) blood samples were collected over 20-60 s at - 5 and 0 min (prestimulus controls) and at 1, 3, 6 and 10 min after the onset of each microinjection. The volume of adrenal venous blood collected per unit time permitted an estimate of total adrenal blood flow. The timing for the first postinjection adrenal venous sample began 10-30 s prior to + 1 min depending on the flow rate for each animal. At least 30 min elapsed between subsequent microinjections.
electrochemical detection as described previously 6. The intra-assay and interassay coefficients of variation for a cat plasma pool containing 2.29 ng/extraction volume of norepinephrine were 2.25 and 5.82%, respectively; and for a plasma pool containing 2.45 ng/extraction volume of epinephrine were 2.78 and 4.49%, respectively; and for a plasma pool containing 2.24 ng/extraction volume of dopamine were 4.38 and 4.72%, respectively. The adrenal secretory rate for each catecholamine species was calculated by the formula: [adrenal plasma concentration ( n g / m l ) - p e r i p h e r a l plasma concentration (ng/ml)]×adrenal plasma flow ( m l / m i n ) = ng/min for epinephrine, norepinephrine and dopamine. Plasma adrenocorticotropin (ACTH) was determined by direct radioimmunoassay 6 using an antibody generated against a conjugate of ACTH 1-24 that reacts completely with ACTH 1-39 and ACTH 1-24, but not with either the N-terminal end (ACTH 1-13) or with the C-terminal end (ACTH 18-39) fragments of the AC~H molecule. Charcoal-stripped cat plasma was added to the ACTH 1-39 standards (Bachem, Torrance, CA) to control for matrix effects. Intra-assay and interassay coefficients of variation for a cat plasma pool with an ACTH concentration of 101 pg/ml were 4.9% and 14.6%, respectively.
Statistical analyses The responses were analyzed after first grouping the anatomical location of each site of microinjection according to the description of Berman 1°. The statistical analyses included five treatment groups: (a) microinjections of 500 mM glutamate at sites within Vp or Vo, transverse planes P 4.0-P 7.1 (n = 15); (b) microinjections of 500 mM glutamate within Vi, planes P 10.8-P 13.5 (n = 16); (c) microinjections of 500 mM glutamate within the spinal trigeminal tract (Vtr), predominantly at planes P 10.8-P 13.5 (n = 13); (d) microinjections of 5 mM glutamate within the boundaries of Vi (n = 7); and (e) microinjections of 500 mM glutamate at sites located outside of these trigeminal brain stem regions (n = 14). As a point of reference in the transverse plane, the obex level of the cat brain stem would correspond approximately to a plane of P 14.5 of the atlas of Berman 1°. Thus, all sites of microinjection were at least 1 mm rostral to the obex. All of the data included in these analyses were collected from animals where the mean arterial pressure remained above 70 mmHg and the patency of the injection cannula was verified at the end of the experiment. The adrenal secretion of catecholamines, peripheral plasma concentrations of catecholamines and ACTH, adrenal blood flow, adrenal vascular conductance, mean arterial pressure, and heart rate responses to injections of L-glutamate were assessed by two-way analysis of variance (location and time) corrected for repeated measures 44. The analyses compared the absolute values to the prestimulus value (mean of - 5 and 0 min values) for each variable and individual comparisons used the Newman-Keuls test after analysis of variance. Adrenal catecholamine secretory rates and plasma concentrations of ACTH were assessed after logarithmic transformation to reduce the error variance. In addition to these group comparisons, each microinjection site was encoded on the brain stem outlines (see Fig. 2) for the evoked change in adrenal secretion of epinephrine. The 95% confidence limit for prestimulus ( - 5 and 0 min) variability was calculated across all sites of injection of glutamate (n = 65) as an absolute difference (za = 2.33 ng/min) and as a percentage difference (A% = 52.4%). The symbols on the brain stem outlines reflect the peak (maximum) change in secretion of epinephrine that was seen during the 10 min postinjection sampling period when compared against these 95% confidence limits. All data in the text and in the figures represent the mean + S.E.M. Spearman rank correlation (r s) analysis was used to assess possible relationships between response variables.
Biochemical determinations
RESULTS
Peripheral arterial and adrenal venous blood samples were collected on ice in tubes containing EDTA (30 /zl/ml whole blood). After centrifugation the plasma was stored at - 60°C for subsequent analyses. The plasma concentrations of catecholamines were determined from 100/xl of plasma after alumina extraction by HPLC with
Adrenal secretion of catecholamines Microinjections of the high dose (500 mM) of glutamate within Vi evoked a significant (P < 0.001) in-
157
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Fig. 1. Dose-related effects of microinjections of L-glutamate within subnucleus interpolaris on the adrenal secretion of epinephrine (top panel) and on the adrenal epinephrine/norepinephrine secretory ratio. Symbols: closed circles = high dose (500 mM, 100 nl, n = 16); open circles = low dose (5 mM, 100 nl, n = 7). * P < 0.05, ** P < 0.01 versus prestimulus value; a p < 0.05, b p < 0.01 versus low dose group; shaded bar on abscissa indicates period of microinjection.
c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t was m a x i m a l by + 1 min, r e m a i n e d e l e v a t e d at + 6 min a n d r e t u r n e d t o w a r d s p r e s t i m u l u s v a l u e s by + 10 min. Mic r o i n j e c t i o n s o f t h e lower d o s e o f g l u t a m a t e (5 m M ) within Vi h a d no effect (Fig. 1, t o p panel). A l t h o u g h the mean prestimulus adrenal secretory rate of e p i n e p h r i n e was n u m e r i c a l l y l o w e r in t h e g r o u p o f cats t h a t r e c e i v e d t h e 5 m M d o s e o f g l u t a m a t e c o m p a r e d to t h e g r o u p t h a t r e c e i v e d 500 m M injections, this differe n c e was n o t significant. A n i n c r e a s e in t h e a d r e n a l s e c r e t i o n of e p i n e p h r i n e e v o k e d by Vi injections o f 500
m M g l u t a m a t e was a c o n s i s t e n t f i n d i n g as 15 o f t h e 16 sites d e m o n s t r a t e d a m a x i m u m i n c r e a s e t h a t e x c e e d e d t h e 95% c o n f i d e n c e limit for t h e v a r i a b i l i t y b e t w e e n t h e - 5 a n d 0 m i n p r e s t i m u l u s v a l u e s (Fig. 2, p l a n e s P 11.6 a n d P 13.5, + 1 . 0 - + 3 . 0 to obex). O f t h e seven i n j e c t i o n sites within Vi t h a t r e c e i v e d 5 m M g l u t a m a t e n o n e c a u s e d an i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t e x c e e d e d t h e 9 5 % c o n f i d e n c e limit for p r e s t i m u l u s v a r i a b i l i t y ( N o t e : t h e s e 7 sites a r e n o t e n c o d e d o n Fig. 2). A f t e r injections o f 500 m M glutam a t e within t h e V t r only 1 o f 13 sites c a u s e d an i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f e p i n e p h r i n e t h a t e x c e e d e d t h e 9 5 % c o n f i d e n c e limit for p r e s t i m u l u s variability. A l t h o u g h t h e m e a n g r o u p r e s p o n s e for all sites l o c a t e d w i t h i n V p / V o d i d n o t i n d i c a t e a signific a n t i n c r e a s e in t h e s e c r e t i o n o f e p i n e p h r i n e (see T a b l e I), several i n d i v i d u a l sites (5 o f 15) l o c a t e d m a i n l y within t h e d o r s o m e d i a l p o r t i o n o f V p / V o d i d r e s p o n d to 500 m M g l u t a m a t e with an i n c r e a s e in s e c r e t i o n (Fig. 2, p l a n e s P 4.6 a n d P 6.0). T h r e e sites w e r e l o c a t e d v e n t r a l to t h e spinal t r i g e m i n a l n u c l e u s at t h e level o f Vi (Fig. 2, p l a n e s P l l . 6 - P 13.5) in [he r e g i o n o f t h e ~audal v e n t r o l a t e r a l m e d u l l a r y A 1 g r o u p a n d collectively r e s p o n d e d to 500 m M g l u t a m a t e injections with a d e c r e a s e ( - 2 9 . 7 + 9 . 6 % ) in t h e a d r e n a l secretion o f e p i n e p h r i n e ( d a t a n o t shown). M i c r o i n j e c t i o n s o f 500 m M g l u t a m a t e within Vi c a u s e d a small b u t significant ( + 1.4 + 0.5 n g / m i n , P < 0.05) i n c r e a s e in t h e a d r e n a l s e c r e t i o n o f n o r e p i n e p h r i n e , w h e r e a s o t h e r t r e a t m e n t g r o u p s h a d no r e s p o n s e ( T a b l e I). C a l c u l a t i o n o f t h e a d r e n a l epinephrine/norepinephrine secretory ratio revealed t h a t 500 m M g l u t a m a t e i n j e c t i o n s w i t h i n Vi i n c r e a s e d t h e r a t i o f r o m 3.2 + 0.5 to a m a x i m u m o f 4.4 + 0.6 ( P < 0.001, n = 16) by + 6 min, i n d i c a t i n g a p r e f e r e n tial i n c r e a s e in t h e s e c r e t i o n o f e p i n e p h r i n e a b o v e t h a t o f n o r e p i n e p h r i n e (Fig. 1, l o w e r panel). T h e m e a n
TABLE I Summary of the adrenal and autonomic responses to microinjections of L-glutamate (500 raM, 100 nl) within the principal sensory nucleus / subnucleus oralis (Vp / Vo), the subnucleus interpolaris (Vi) or the spinal trigeminal tract (Vtr)
Peak = maximum value compared to prestimulus observed during the 10 min sampling period following microiniection. Adrenal Epi, adrenal secretion of epinephrine; adrenal NE, adrenal secretion of norepinephrine; ACTH, adrenocorticotropic hormone; ABF, adrenal blood flow; MAP, mean arterial pressure. Vp / Iio (n = 15) Prestimulus
AdrenalEpi(ng/min) AdrenalNE(ng/min) ACTH (pg/ml) ABF(ml/min) MAP(mmHg) Heart rate (beats/min)
5.3 _+ 1.1 4.1 + 0.8 97 +_21 0.70± 0.06 107 + 4 186 ± 5
* P < 0.05, * * P < 0.01 versus prestimulus value.
1,7 (n = 16) Peak
6.5 ± 1.5 5.5 + 1.1
157 + 61 0.75± 0.09 107 + 3 189 ± 6
Vtr (n = 13)
Prestimulus
Peak
Prestimulus
Peak
7.7 + 2.3 2.5 + 0.5 115 _+23 0.74± 0.07 108 ± 6 181 + 4
19.1 + 4.4** 3.9 + 0.6 * 99 + 12 0.93+ 0.1"* 115 ± 6 * 189 + 5 **
5.9 + 1.3 7.3 _+ 3.2 129 + 20 0.66± 0.09 103 ± 3 173 ± 6
6.7 + 2.0 8.7 ± 3.9 127 + 22 0.62± 0.1 103 ± 4 173 ± 7
158 ~_.
• Increase ]
.'.°:'z:'.;. J
"
kj ,
\" 0 n , . .
Fig. 2. Sites of microinjection of L-glutamate (500 mM, 100 nl) encoded for the evoked change in the adrenal secretion of epinephrine. A, increase; II, decrease; D, no change. 5M, trigeminal motor n.; 7N, facial nerve; CX, external cuneate n.; IO, inferior olivary n.; LRN, lateral reticular nucleus; NTS, n. tractus solitarius; SO, superior olivary n.; Vi, trigeminal subnucleus interpolaris; Vo, subnucleus oralis; Vp, principal trigeminal n.; Vtr, spinal trigeminal tract. Bar = 1.0 ram; numbers refer to anterior-posterior plane of section adapted from Berman z°.
prestimulus adrenal secretion of dopamine for all sites was 0.14_+ 0.03 ng/min (n = 65) and no significant responses to microinjections of glutamate were seen among the five treatment groups. The prestimulus peripheral arterial plasma concentrations of epinephrine, norepinephrine and dopamine averaged 0.05_+ 0.06 ng/ml, 0.39 _+0.03 n g / m l and 0.06 _+0.1 ng/ml (n = 65), respectively, and were not affected significantly by microinjections of glutamate (data not shown).
1.0 " ~"
0.9
N~
o.8 0.7
0.6
Cardiovascular responses
Microinjections of 500 mM glutamate within Vi evoked a sustained increase in total adrenal blood flow (P < 0.005), whereas 5 mM glutamate caused a smaller non-significant increase (Fig. 3, top panel). Analysis of variance revealed a significant (P < 0.025) interaction effect indicating a dose-related influence on total adrenal blood flow by injections within Vi. However, it was noted that even the low dose of glutamate evoked small increases in adrenal blood flow and in adrenal vascular conductance. In contrast to the sustained increase in total adrenal blood flow, mean arterial pressure (MAP) increased transiently after microinjections of 500 mM glutamate into Vi (+ 7.2 _+ 2.5 mmHg, P < 0.001) and returned towards prestimulus values by + 3 min (Fig. 3, middle panel). By 10 min after Vi injections of 500 mM glutamate, MAP fell below prestimulus values, whereas MAP remained below prestimulus values throughout the sampling period after 5 mM glutamate, a result that was consistent with the trigeminal depressor response 23. Correspondingly, the calculated adrenal vascular conductance (adrenal blood flow/mean arterial pressure, Fig. 3, bottom panel) showed a sustained increase (P < 0.001) after injec-
~r
,'... , ....
//,
'~ 125t ~ ~ 1 ~ 105
95',
•O 0.010 ~ 0.009 == F,
0.00a
O~
0.007
. . . . . . . . . . w
t
0.006 "o
< 0.005
I
0
Time(mln) 2
4
6
10
Fig. 3. Dose-related effects of microinjections of L-glutamate within subnucleus interpolaris on total adrenal blood flow (top panel), mean arterial pressure (MAP, middle panel), and on adrenal vascular conductance (lower panel), e, high dose (500 mM, 100 nl, n = 16); ©, low dose (5 mM, 100 nl, n = 7 ) . * P < 0 . 0 5 , * * P < 0 . 0 1 versus prestimulus value; shaded bar on abscissa indicates period of microinjection.
159 tions of 500 mM glutamate into Vi, whereas injections of 5 mM glutamate evoked smaller non-significant responses. Thus, the increase in total adrenal blood flow seen after injections of glutamate within Vi is likely due in part to active vasodilatation of the adrenal vascular bed and cannot be explained by the evoked increase in MAP. Heart rate increased ( + 8 . 0 + 2.7 beats/min, P < 0.001) by + 1 min after injection of 500 mM glutamate within Vi and returned towards prestimulus values by + 3 min. Neither heart rate nor the other cardiovascular variables were affected significantly by injections of 500 mM glutamate into V p / V o or into Vtr (Table I).
Arterial plasma concentrations of ACTH Mean arterial plasma concentrations of A C T H were not affected consistently by microinjections of 500 mM glutamate regardless of the site of injection (Table I), however, several individual sites did cause changes in plasma A C T H that exceeded the 95% confidence limits (A = 20.4 p g / m l , A% = 18.3%) for prestimulus variability. For example, 4 of 15 sites located within V p / V o , mainly in the dorsomedial portion, caused an increase in plasma A C T H and two sites caused a decrease in ACTH. After injection of 500 mM glutamate within Vi, 3 of 16 sites evoked an increase in plasma ACTH, but 7 of 16 sites caused a decrease in plasma ACTH. Among the 13 sites located within Vtr, three sites caused an increase in plasma A C T H and five sites caused a decrease in plasma ACTH.
Correlation analyses Spearman rank-order correlation analyses of the responses from the 16 sites of microinjection of 500 mM glutamate within Vi indicated that the magnitude of the evoked change in adrenal secretion of epinephrine was not correlated with that of total adrenal blood flow (r s = 0.185, P > 0.05), adrenal vascular conductance (r s = 0.175, P > 0.05), or with that of MAP (rs = 0.206, P > 0.05). Also, the magnitude of the evoked change in total adrenal blood flow was not correlated with that of MAP (r s = -0.156, P > 0.05) and the change in MAP was not correlated with the change in heart rate (r s = 0.196, P > 0.05). DISCUSSION The results indicated that microinjections of glutamate within Vi evoked significant dose-related increases in the adrenal secretion of catecholamines and in cardiovascular function, whereas injections of glutamate within the most rostral portions of the trigeminal sensory complex ( V p / V o ) had no consistent effects.
Although the volume of each injection was small (100 nl), uncertainty about the extent of the diffusion of the injected substance is always a concern for microinjection studies. It is not likely that the autonomic responses evoked by injections within Vi were due to the spread of glutamate to nearby structures since numerous injection sites were located laterally within the adjacent spinal trigeminal tract and were generally without effect. Glutamate injections within three sites located immediately ventral to Vi caused small decreases in the adrenal secretion of catecholamines in contrast to the significant increase in secretion evoked by sites within the adjacent Vi. In previous studies somewhat larger injection volumes (160 nl) were used, however, an apparent laminar organization was seen within Vc for glutamate-evoked adrenal and autonomic responses 7'8. Thus, the dose-related nature of the responses and the restricted anatomical localization of responsive sites suggested that the adrenal and autonomic responses evoked by microinjections of glutamate within Vi were the result of local action and were not due to widespread diffusion. Although the changes in adrenal and autonomic function evoked by microinjections of glutamate within Vi or Vc were similar qualitatively to those seen after noxious stimulation of trigeminal receptors 1'5'6, a role for glutamate as a neurotransmitter in trigeminal nociception has not been well established. Anatomical studies revealed that 2 0 - 3 0 % of trigeminal ganglion ceils in the rat were positive for glutamate-like immunoreactivity 22 and axon terminals containing glutamate-like immunoreactivity were found throughout the trigeminal brain stem complex ~4. At caudal levels of the Vsp glutamate-positive fibers and terminals were associated with tooth pulp nerves 3'~5. In receptor binding studies in the rat, the density of both N M D A (N-methyl-o-aspartate) and n o n - N M D A glutamate receptor subtypes within the Vsp had a rank-order of Vc > Vi > Vo 4°, a finding consistent with the order of magnitude of the autonomic responses evoked by microinjections of glutamate. It is not yet known if the adrenal and autonomic responses evoked by microinjections of glutamate within Vi or within Vc 7,s demonstrate a preference for one glutamate receptor subtype over another. Salt and Hill 32 have shown that iontophoretic application of a non-selective glutamate receptor antagonist within Vc blocked the neural responses to noxious mechanical but not to noxious thermal stimuli. The first systematic study of the Vsp and nociceptor-evoked autonomic reflex activity was reported by Gerard TM who noted increases in arterial pressure and pupillary dilatation that accompanied corneal stimula-
160 tion in the cat. Beginning at the level of the obex successively more rostral cuts of the descending spinal trigeminal tract were made until cornea-evoked autonomic reflex activity was abolished. Gerard noted that cornea-evoked autonomic activity persisted until the tract was lesioned at mid-Vi levels. Matsuo et al. 25 assessed the increase in volume of saliva secreted from the submandibular gland of the decerebrate rat after noxious thermal or mechanical stimulation of the perioral region. Lesions of Vi or of Vc greatly attenuated salivary flow, whereas lesions of the nucleus tractus solitarius (NTS) or of the parabrachial nuclei had no effect on the volume of saliva. Panneton 2s reported that injections of lidocaine within the Vsp at the level of the obex blocked the cardiorespiratory responses to nasal stimulation in the muskrat. In recent studies 9, we have found that the adrenal and autonomic responses evoked by chemical stimulation of the cornea were attenuated greatly by ipsilateral injections of lidocaine within the middle to caudal portions of Vi in the cat. Further, in this same study c-fos protein was localized immunocytochemically within ventrolateral portions of Vi and more caudally within Vc but was not detected in V p / V o regions. Electrophysiological studies 19'2° have revealed numerous nociceptive neurons within the middle and caudal regions of the Vi in the cat. Collectively, these results suggest that neurons within the Vi as well as within the Vc portions of Vsp contribute to the integration of sensory input relevant for trigeminal-evoked autonomic output. The present study also suggests that glutaminergic input to V p / V o regions has little influence on autonomic function. Although nociceptive input from perioral or intraoral structures clearly activates V p / V o neurons 12'17'34, this input may be mediated through non-glutaminergic pathways. Whereas the function of the nociceptive neurons within V p / V o is not known, it is also possible that these ceils mediate sensory-discriminative aspects of trigeminal nociception and that reflex autonomic adjustments to trigeminal nociceptive stimulation does not include a significant relay through the most rostral portions of the Vsp as originally suggested by Gerard TM. It is not yet known to what extent the central neural pathways that mediate the sensory-discriminative aspects of trigeminal input overlap with those that mediate the autonomic responses to noxious trigeminal stimuli. Although the adrenal and autonomic responses to Vi injections of glutamate were similar qualitatively to those seen after microinjections into Vc 7'8, significant differences also were noted. Glutamate injections within Vi did not affect plasma A C T H , whereas similar injections within laminae I - I I of Vc caused a reliable
increase 8. The adrenal secretion of catecholamines was accompanied by significant increases in total adrenal blood flow and in adrenal vascular conductance after injections of glutamate into Vi, whereas injections into laminae I - I I or V - V I of Vc had no effect total adrenal blood flow or on adrenal vascular conductance 7. The differences in adrenal and autonomic responses observed after injections of glutamate at different rostrocaudal sites within the trigeminal sensory complex suggests an underlying organization for control of autonomic function that cannot be explained simply by the density of glutamate receptor subtypes 4°. Also, the qualitative differences in endocrine and autonomic responses to injections of glutamate within Vi versus Vc argue against the notion that these results are due simply to widespread diffusion of glutamate between these two subnuclei. Efferent projections from Vi and from Vc to the hypothalamus, periaqueductal gray, parabrachial nuclei and to the NTS have been well described 11'13'2°'21'26'3°'42, whereas significant projections from Vp or Vo to these autonomic relay areas have not been reported. Trigeminal projections to the NTS originate mainly from Vc and are more extensive than those from Vi 21'26. Further, examination of more rostral portions of Vsp did not reveal projections from Vp or from Vo neurons to the NTS in the rat (C.B. Saper, personal communication). The present results extend those of previous studies 7'8 to suggest that local release of glutamate within the Vi or the Vc portions of the Vsp exerts a significant influence on adrenomedullary secretion and on autonomic function in the cat. The evoked responses are consistent with those that would be expected to accompany stimulation of trigeminal nociceptors, however, the relationship between glutaminergic input to the Vsp and the integration of noxious trigeminal sensory information is not well defined. Although the most rostral portions of the trigeminal brain stem complex may also contribute to the integration of trigeminal nociceptive input, the present results indicate that glutaminergic input to Vp and Vo neurons does not have a consistent influence on adrenal and autonomic function.
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