- Email: [email protected]
Biochemical evidence for simultaneous activation of multiple locus coeruleus efferents
Life Sciences, Vol. 26, pp. 1373-1378 Printed in the U.S.A.
Pergamon Press
BIOCHEMICAL EVIDENCE FOR SIMULTANEOUS ACTIVATION OF MULTIPLE LOCUS COERULEUS EPFERENTS J. N. Crawley*, J. W. Haas and R. H. Roth Departments of Psychiatry and Pharmacology Yale University School of Medicine New Haven, CT 06510 (Received in final form February 20, 1980) SurunarZ To test the hypothesis that all locus coeruleus projections are simultaneously activated when the locus coeruleus cells fire, the norepinephrine metabolite 3-methoxy-4-hydroxyphenethyleneglycol was assayed in four regions of the central nervous system innervated by the locus coeruleus after three treatments designed to increase locus coeruleus firing in rats. Electrical stimulation of the locus coeruleus, intraperitoneal piperoxan treatment, and electric footshock all significantly increased MHPG levels in rat cerebral cortex, cerebellum, hippocampus, and spinal cord. The magnitude of ~IPG increase was greater after locus coeruleus stimulation than after footshock or piperoxan. No significant differences between increases in the above brain regions were found within each treatment group. Introduction The major n o r a d r e n e r g i c pathways i n the c e n t r a l n e r v o u s system a r i s e from the compact b i l a t e r a l l o c u s c o e r u l e u s n u c l e i (LC) ( 1 - 8 ) . While t h i s n o r a d r e n e r g i c c l u s t e r c o n t a i n s l e s s t h a n 2000 c e l l b o d i e s (6), h i s t o c h e m i c a l s t u d i e s s u g g e s t t h a t each c e l l may have thousands o f axon t e r m i n a l s and p r o j e c t to many brain regions , including cerebral c o r t e x , hippocampus, thalamus, limbic structures, hypothalamus, cerebellum, raphe nuclei, and spinal cord (I-8). A modulatory role for the LC has been postulated on the assumption that a single LC cell activates terminals in many or all of these regions (93. However, no definitive evidence exists to support the current notion that LC firing activates the multiple LC projections simultaneously and uniformly. The study reported here addresses this question through a biochemical technique, i.e. the analysis of neurotransmitter metabolism at several different terminal regions following three different treatments which activate the LC. Levels of the major brain norepinephrine (lqE) metabolite, 3-methoxy-4-hydroxyphenethyleneglycol (~PG) (11,123, have been shown to increase in cerebral cortex when the LC cell bodies are electrically stimulated (13-15), following piperoxan antagonism of inhibitory collaterals (17,18), and after footshock (19), and these different methods for activating the LC were therefore used in this study. The effects of these different methods for stimulating the LC in the production of MHPG in the cerebral cortex, hippocampus, cerebellum, and spinal cord, i.e. structures which are known to receive LC efferents (18), were determined. The percentage change of ~ P G in these different regions has been compared within and across treatments and the results are reported here.
*'Clinical Psychobiology Branch, Bldg. i0, Rm. 4S-239, Nl~i, Bethesda, MD 20205 0024-3205/80/171373-06502.00/0 Copyright (c) 1980 Pergamon Press Lid
1374
MHPG in Locus Coeruleus Terminal Regions
Vol. 26, No. 17, 1980
Methods Male Sprague-Dawley rats, 150-300 g were used for tile treatments described helow. Immediately after cessation of treatment, rats were guillotined, the brain and spinal cord removed, and dissected on ice into five regions: right and left cerebral cortical hemispheres, right and left hippocampus combined, total cerebellum, and thoracic and lumbar spinal cord. Tissue was frozen at -70°C until assay for MIIPG as previously described by gas chromatography-mass spectrometry (14,20). Control and treatment samples for each experiment were assayed together, with the glusalase blank value being subtracted from that of the tissue values. Locus coeruleus stimulation: The locus coeruleus of urethane anesthetized rats was stereotaxically and electrophysiologically located with a bipolar tungsten electrode as previously described (14,22). Unilateral stimulation was biphasic, each phase 200 p A in amplitude, 1 msec duration, at 20 Hz frequency, for 30 minutes. In a sham control group, the tungsten electrode was placed in the locus coeruleus for 30 minutes but no current was passed. Immediately after the end of the 30 minute stimulation period, the rat was sacrificed and brain regions dissected as described above.
Footshock: A NRS/LVE Shock G e n e r a t o r / S c r a m b l e r , Model S C S - 0 0 4 , Tech S e r vice, Inc., Beltsville, MD, was u s e d t o d e l i v e r f o o t s h o c k . Constant current o f 2 mA, c a l i b r a t e d b y t h e m e t h o d o f D a v i s ( 2 3 ) , was d e l i v e r e d a c r o s s p a r a l l e l b a r s s p a c e d a t 2 cm i n t e r v a l s , o n e s h o c k e v e r y 10 s e c o n d s , 100 msec d u r a t i o n per shock (19,21,24-26). Unanesthetized controls were placed in the apparatus f o r 30 m i n u t e s w i t h o u t a p p l i c a t i o n of footshock current. Rats were sacrificed i m m e d i a t e l y a f t e r t h e 30 m i n u t e f o o t s h o c k s e s s i o n . Piperoxan: 5 mg/kg Piperoxan-HCl (Rhone-Poulenc, France) in saline was administered intraperitoneally. Unanesthetized rats were kept in quiet home cages for 30 minutes until sacrifice. Results Significant increases were found in ~IPG levels in all central nervous system tissue investigated after each of the three types of treatment. Table I
TABLE I NPG
Levels After Locus Coeruleus
Region
Stimulation
Total NIPG (ng/g)
Sham (4) 2.7
Stimulated
(4)
Cerebral Cortex
98.1 +
llippocampus
92.4 + 12.2
158.8 + 25.5*
Cerebellum
76.9 + 10.3
161.6 + 24.5**
Spinal Cord
77.0 +
156.2 +
7.3
175.9 + 13.3"**
Results are expressed as mean + S.E.M. Values in parentheses of animals per group. *p < .05; **p < .01; ***p < .005.
7.9***
represent number
Vol. 26, No. 17, 1980
MHPG in Locus Coeruleus Terminal Regions
1375
shows the increases after locus coeruleus stimulation. There was a mean increase of 85% among all the regions, with each region increasing significantly at least below p < .05. MHPG levels in the cerebral cortex hemisphere contralateral to stimulation were 126.6 + 10.3 ng/g. Table I| shows that piperoxan produced a mean increase of 59% across central nervous system regions, with the highest increase in the cerebellum. All increases were significant at p < .005 by t-test analysis. In Table IIl, footshock produced a mean increase of 52% with the lowest increase in the cerebral cortex. All increases were significant at least below p < .05 by t-test analysis.
TABLE I I MHPG Levels After Piperoxan
Total ~{PG
Region
(ng/g) Saline
(5)
Piperoxan (S) 5 mg/kg i.p.
Cerebral Cortex
94.8 + 5.7
149.9 + 9.1"**
}~ppocampus
69.4 + 4.1
105.4 + 7.8***
Cerebellum
68.7 + 4.3
117.2 + 8.2***
Spinal Cord
62.4 + 3.5
100.7 + 4.7***
Results are expressed as mean + S.E.M. of animals per group. ***p < ?005.
Values in parentheses
represent number
TABLE III MHPG Levels After Footshock Region
Total ~IPG
(ng/g) Control
(5)
Footshocked
(5)
Cerebral Cortex
59.0 + 3.0
80.6 + 1.5"**
llippocampus
81.1 + 4.5
98.0 + 6.4*
Cerebellum
52.5 + 9.3
82.1 + 5.3*
Spinal Cord
69.9 + 5.7
109.1 + 8.9***
Results are expressed as mean + S.E.M. Values in parentheses of animals per group. ***p < ~005. *p < .05.
represent number
Figure 1 illustrates the magnitude of N~IPG increase in the four central nervous system regions after the three treatments. Electrical stimulation produced larger increases in all regions. Variability was seen in the percentage increase in MHPG in regions within a given treatment. Analysis of variance
1376
MHPG in Locus Coeruleus Termlnal Regions
Vol. 26, No. 17, 1980
using two factor repeated measures (27) showed no significant differences in brain regional increases due to LC stimulation or to footshock, but a small difference (p < .05) in brain regional increases due to piperoxane, where the increase in cerebellum was higher than in spinal cord or hippocampus.
I
I00
1 Cerebral Cortex Hippocompus Cerebellum
F77~ Spinol Cord
e. .,I- 8 0 E ////
z
.:+:.:.:+:. :::::::::::::::
////
::::::::::::::: ::::::::::::::: ::::::::::::::: +:+:+:+: :::::::::::::::: ::::::::::::::;:
tu 6 0
:.:,:+:.:+:.
z
40
N
.:.:.:.:.:+:, ::::::::::::::: ::::::::::::::: ::::::::::::::: z+:.:-:,:-: :::::::::::;::: :::::::::::::::
////t
i / i / /
20
i/i//
::::::;:;:::::; ::::;::::::::::: :::::::::::::::: :.:,:+:,:.:.: ::::::::::::::::
///~
¢//1
LC STIMULATED 20 Hz, ;~00 FA
FOOTSHOCK
PIPEROXAN
2 mA
5 mg/kg
FIG. 1 Regional increases in total ~IPG in rat central nervous system. Percentage increases were calculated for three types of treatments which influence central noradrenergic function: (a) locus coeruleus stimulation as compared to sham operated controls; (b) electric footshock as compared to apparatus sham controls; and (c) acute piperoxan administration as compared to saline controls.
Discussion Each of tile three qualitatively different treatments significantly increased t~iPG in all regions studied. Locus coeruleus stimulation at 20 Hz produced the largest ~{PG increase, possibly due to direct external control of firing rate overshadowing inhibitory feedback loops. Treatments that do not directly drive LC firing would be more subject to the many buffering influences on LC firing (16). Despite these differences in tlle magnitude of the ~{PG increase as a function of the type of treatment, the increase of MHPG in the four brain regions was relatively constant within each of the different methods used for LC activation. These results support the stated hypothesis that the LC activates its multiple projections simultaneously. ~te
relatively small degree of variability in ~JPG increase in the brain
Vol. 26, No. 17, 1980
MHPG in Locus Coeruleus Terminal Regions
1377
r e g i o n s may be a t t r i b u t a b l e s i m p l y t o t h e small sample s i z e s o f t h e s e t r e a t m e n t groups. A l t e r n a t i v e l y , l o c a l f e e d b a c k l o o p s have been p o s t u l a t e d t o r e g u l a t e amount o f t r a n s m i t t e r r e l e a s e d p e r i m p u ls e by p r e s y n a p t i c i n h i b i t i o n a t t h e terminals (28,29). P o s s i b l y , d i f f e r e n t b r a i n r e g i o n s have more o r l e s s o f such p r e s y n a p t i c i n h i b i t i o n , r e s u l t i n g i n t h e v a r i a b l e p r o f i l e o f r e g i o n a l MHPG changes t o d i f f e r e n t s t i m u l i . In t h e s p e c i f i c case o f s p i n a l cord v a r i a b i l i t y , t h e r e may be a more d i r e c t e x p l a n a t i o n , t h e s e v e r a l s o u r c e s o f s p i n a l cord MttPG: LC p r o j e c t i o n s ( 3 0 - 3 4 ) , A1 n o r a d r e n e r g i c p r o j e c t i o n s ( 1 ) , and C1 e p i nephrine projections (35,36). S p i n a l cord NtPG i s , t h e r e f o r e , l e s s c o m p l e t e l y de pen d en t on LC f i r i n g r a t e s than c e r e b e l l u m , c e r e b r a l c o r t e x , and hippocampus, @~ose s o l e n o r a d r e n e r g i c i n n e r v a t i o n a r i s e s from t h e LC ( 1 - 8 ) . ~ l e e x p e r i m e n t s d e s c r i b e d h e r e do n o t a d d r e s s the q u e s t i o n o f t o p o g r a p h i c o r g a n i z a t i o n o f t h e LC c e l l b o d i e s . One e l e c t r o p h y s i o l o g i c a l r e p o r t , t h a t t h e hippocampal c e l l r e s p o n s e t o LC s t i m u l a t i o n i s most marked when t h e e l e c t r o d e i s p l a c e d i n t h e d o r s a l and a n t e r i o r r e g i o n s o f the LC n u c l e u s (10), s u g g e s t s t h a t t h e c e l l s do n o t e q u a l l y p r o j e c t t o a l l t e r m i n a l r e g i o n s , ttistochemical s t u d i e s s u g g e s t t h a t t h e LC c l u s t e r c o n t a i n s s u b p o p u l a t i o n s w i t h t o p o g r a p h i c a l efferent organization (33,37,38). As a l l t h r e e t r e a t m e n t s o f t h e p r e s e n t st u d y were d e s i g n e d t o a c t i v a t e t h e e n t i r e LC n u c l e u s , t h i s i s s u e must be examined through o t h e r approaches. In c o n c l u s i o n , t r e a t m e n t s t h a t a c t i v a t e t h e l o c u s c o e r u l e u s w i l l i n c r e a s e n o r a d r e n e r g i c m e t a b o l i t e l e v e l s s i m u l t a n e o u s l y i n many and p o s s i b l y a l l o f t h e c e n t r a l n e r v o u s system r e g i o n s i n n e r v a t e d by t h e LC. This e v i d e n c e s u p p o r t s t h e h y p o t h e s i s o f s i m u l t a n e o u s a c t i v a t i o n o f d i f f u s e e f f e r e n t s , and t h e p r o posed m o d u l a t o r y r o l e o f t h e LC. I t a l s o s u g g e s t s t h a t b i o c h e m i c a l a n a l y s i s o f n o r a d r e n e r g i c m e t a b o l i t e l e v e l s i n one r e g i o n i n n e r v a t e d by t h e LC may be s u f f i c i e n t t o a c c u r a t e l y r e f l e c t t h e LC r e s p o n s e a t many o f i t s t e r m i n a l regions. Acknowledgements This work was supported in part by USPIIS grants NI-14092, NS-I0174, ~ 24393, MH-25642, and MII-14276.
References i. 2. 3. 4. S. 6. 7. 8. 9. I0. 11.
12. 13. 14.
A. DAHLSTROM and K. FUXE, ACTA Physiological Scandanavica 6__44Suppl. 247 1-85 (1965). U. UNGERSTEDT, ACTA Physiological Scandanavica82 Suppl. 367 1-76 (1971). A. LINDVALL and A. BJORKLUND, ACTA Scandanavica Suppl. 412 1-48 (1974). V. PICKEL, M. SEGAL and F. E. BLOOM, J. Comp. Neur. 15S 15-42 (1974). M. PALKOVITS and b. M. JACOBOWITZ, J. Comp. Neur. 157 29-42 {1974). L. W. SWANSON, Brain Res. 110 39-56 (1976). B. E. JONES and R. Y. MOORE, Brain Res. 127 23-53 (1977) D. M. BOWDEN, b. C. GERMANand W. D. POYNTER, Brain Res. 145 2S7-276 (1978). S. NAKAMURA, J . P h y s i o l . 267 641-658 (1977). M. SEGAL and F. E. BLOOM, B r a i n Res. 72 9 9 - i 1 4 (1974). S. M. SCHANBERG, J . J . SCHILDKRAUT, G. R. BREESE and I . J. KOPIN, Biochem. Pharmacol. 17 247-254 (1968). J. P. ADER, F. A. J. MUSKIET, H. J. JEURING and J. KORF, J. Neurochem. 30 1213-1216 (1978). J. KORF, G. K. AGHAJANIAN and R. II. ROTll, Europ. J. Pharmacol. 21305-310 (1973). J . N. CRAWLEY, S. E. IIATTOX, J . W. MAAS and R. If. ROTH, B r a i n Res. 141
1378
MHPG in Locus Coeruleus Terminal Regions
Vol. 26, No. 17, 1980
17.
380-384 (1978). J. N. CRAWLEY, J. W. HAAS and R. H. ROTII, Brain Res., in press (1979). G. K. AGHAJANIAN, In: Essays in Neurochemistry and Neuropharmacology, M. B. H. YOUDIM, W. LOVENBERG, D. R. SHARP,AN and J. R. LAGNADO, Eds., pp. 1-22, John Wiley and Sons, New York (1978). G. K. AGHAJANIAN, J. M. CEDARBAU~4, and R. Y. WANG, Brain Res. 136 570-
18. 19.
J. ~4. CEDARBAUM and G. K. AG}IAJANIAN, Life Sci. 23 1383-1392 (1978). J. KORF, G. K. AGHAJANIAN and R. H. ROTH, Neuropharmacol. 12 933-938
20.
J. W. MAAS, S. E. }LATTOX, D. H. LANDIS and R. H. ROI]I, Brain Res. 118 167-
15. 16.
s77 {1977). (1973). 173 (1976). 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35, 36. 37. 38°
E. A. STONE, Life Sci. 16 1725-1730 (1975). Y. HUANG and J. W. MAAS, Brain Res. i15 91-97 (1976). M. DAVIS and D. I. ASTRACHAN, J. Exp. Psychol. 4 95-103 (1978). J. ROSSIER, R. GUILLEMIN, and F. BLOOM, Europ. ~. Pharmacol. 48 465-466 (1978). S. LAVIELLE, J. P. TASSIN, A. M. THIERRY, G. BLANC, D. IIERVE, C. BARTHELEMY and J. GLOWINSKI, Brain Res. 168 585-594 (1978). S. LEVINE, J. MADDEN, R. L. CONNER, J. R. MOSKAL and D. C. ANDERSON, Physiolo Behav. i0 467-471 (1973). B. J. WINER, S t a t i s t i c a l P r i n c i p l e s i n Experimental Design, McGraw-Hill, New York (1962). S. Z. LANGER, Br. J. Pharmacol. 60 481-497 (1977). K. STARKE and K. P. ALT~IANN, Neuropharmacol. 12 339-347 (1973). L. NYGREN and 1. OLSON, Brain Res. 132 85-93 (1977). M. B. HANCOCKand C. L. FOUGEROUSSE, Brain Res. B u l l . 1 229-234 (1976). J. N. CRAWLEY, R. H. ROTH and J. W. MAAS, Brain Res. 166 180-184 (1979). J. P. ADER, F. POSTEMAand J. KORF, J. Neural T r a n s . , 45(3) 159-173 (1979). Jo W. CO~ISSIONG, S. O. }IELLSTRO~4, N. H. NEFF, Br ai n Res. 148 207-213 (1978). J . L. REID, J . A. ZIVIN and I. J . KOPIN, J . Neurochem. 26 629-631 (1976). T. HOKFELT, K. FUXE, M. GOLDSTEIN and O. JOHANSSON, Br ai n Res. 66 235-251 (1974). K. SATOH, H. TOHYAMA, K. YAMAMOTO, T. SAKUMOTO, and N. SHIMIZU, Exp. Brain Res. 30 175-186 (1977). S. E. LOUGHLIN, S. L. FOOTE, and F. E. BLOOM, Soc. For Neurosciences Abstract #1136 (1979) o
Pergamon Press
BIOCHEMICAL EVIDENCE FOR SIMULTANEOUS ACTIVATION OF MULTIPLE LOCUS COERULEUS EPFERENTS J. N. Crawley*, J. W. Haas and R. H. Roth Departments of Psychiatry and Pharmacology Yale University School of Medicine New Haven, CT 06510 (Received in final form February 20, 1980) SurunarZ To test the hypothesis that all locus coeruleus projections are simultaneously activated when the locus coeruleus cells fire, the norepinephrine metabolite 3-methoxy-4-hydroxyphenethyleneglycol was assayed in four regions of the central nervous system innervated by the locus coeruleus after three treatments designed to increase locus coeruleus firing in rats. Electrical stimulation of the locus coeruleus, intraperitoneal piperoxan treatment, and electric footshock all significantly increased MHPG levels in rat cerebral cortex, cerebellum, hippocampus, and spinal cord. The magnitude of ~IPG increase was greater after locus coeruleus stimulation than after footshock or piperoxan. No significant differences between increases in the above brain regions were found within each treatment group. Introduction The major n o r a d r e n e r g i c pathways i n the c e n t r a l n e r v o u s system a r i s e from the compact b i l a t e r a l l o c u s c o e r u l e u s n u c l e i (LC) ( 1 - 8 ) . While t h i s n o r a d r e n e r g i c c l u s t e r c o n t a i n s l e s s t h a n 2000 c e l l b o d i e s (6), h i s t o c h e m i c a l s t u d i e s s u g g e s t t h a t each c e l l may have thousands o f axon t e r m i n a l s and p r o j e c t to many brain regions , including cerebral c o r t e x , hippocampus, thalamus, limbic structures, hypothalamus, cerebellum, raphe nuclei, and spinal cord (I-8). A modulatory role for the LC has been postulated on the assumption that a single LC cell activates terminals in many or all of these regions (93. However, no definitive evidence exists to support the current notion that LC firing activates the multiple LC projections simultaneously and uniformly. The study reported here addresses this question through a biochemical technique, i.e. the analysis of neurotransmitter metabolism at several different terminal regions following three different treatments which activate the LC. Levels of the major brain norepinephrine (lqE) metabolite, 3-methoxy-4-hydroxyphenethyleneglycol (~PG) (11,123, have been shown to increase in cerebral cortex when the LC cell bodies are electrically stimulated (13-15), following piperoxan antagonism of inhibitory collaterals (17,18), and after footshock (19), and these different methods for activating the LC were therefore used in this study. The effects of these different methods for stimulating the LC in the production of MHPG in the cerebral cortex, hippocampus, cerebellum, and spinal cord, i.e. structures which are known to receive LC efferents (18), were determined. The percentage change of ~ P G in these different regions has been compared within and across treatments and the results are reported here.
*'Clinical Psychobiology Branch, Bldg. i0, Rm. 4S-239, Nl~i, Bethesda, MD 20205 0024-3205/80/171373-06502.00/0 Copyright (c) 1980 Pergamon Press Lid
1374
MHPG in Locus Coeruleus Terminal Regions
Vol. 26, No. 17, 1980
Methods Male Sprague-Dawley rats, 150-300 g were used for tile treatments described helow. Immediately after cessation of treatment, rats were guillotined, the brain and spinal cord removed, and dissected on ice into five regions: right and left cerebral cortical hemispheres, right and left hippocampus combined, total cerebellum, and thoracic and lumbar spinal cord. Tissue was frozen at -70°C until assay for MIIPG as previously described by gas chromatography-mass spectrometry (14,20). Control and treatment samples for each experiment were assayed together, with the glusalase blank value being subtracted from that of the tissue values. Locus coeruleus stimulation: The locus coeruleus of urethane anesthetized rats was stereotaxically and electrophysiologically located with a bipolar tungsten electrode as previously described (14,22). Unilateral stimulation was biphasic, each phase 200 p A in amplitude, 1 msec duration, at 20 Hz frequency, for 30 minutes. In a sham control group, the tungsten electrode was placed in the locus coeruleus for 30 minutes but no current was passed. Immediately after the end of the 30 minute stimulation period, the rat was sacrificed and brain regions dissected as described above.
Footshock: A NRS/LVE Shock G e n e r a t o r / S c r a m b l e r , Model S C S - 0 0 4 , Tech S e r vice, Inc., Beltsville, MD, was u s e d t o d e l i v e r f o o t s h o c k . Constant current o f 2 mA, c a l i b r a t e d b y t h e m e t h o d o f D a v i s ( 2 3 ) , was d e l i v e r e d a c r o s s p a r a l l e l b a r s s p a c e d a t 2 cm i n t e r v a l s , o n e s h o c k e v e r y 10 s e c o n d s , 100 msec d u r a t i o n per shock (19,21,24-26). Unanesthetized controls were placed in the apparatus f o r 30 m i n u t e s w i t h o u t a p p l i c a t i o n of footshock current. Rats were sacrificed i m m e d i a t e l y a f t e r t h e 30 m i n u t e f o o t s h o c k s e s s i o n . Piperoxan: 5 mg/kg Piperoxan-HCl (Rhone-Poulenc, France) in saline was administered intraperitoneally. Unanesthetized rats were kept in quiet home cages for 30 minutes until sacrifice. Results Significant increases were found in ~IPG levels in all central nervous system tissue investigated after each of the three types of treatment. Table I
TABLE I NPG
Levels After Locus Coeruleus
Region
Stimulation
Total NIPG (ng/g)
Sham (4) 2.7
Stimulated
(4)
Cerebral Cortex
98.1 +
llippocampus
92.4 + 12.2
158.8 + 25.5*
Cerebellum
76.9 + 10.3
161.6 + 24.5**
Spinal Cord
77.0 +
156.2 +
7.3
175.9 + 13.3"**
Results are expressed as mean + S.E.M. Values in parentheses of animals per group. *p < .05; **p < .01; ***p < .005.
7.9***
represent number
Vol. 26, No. 17, 1980
MHPG in Locus Coeruleus Terminal Regions
1375
shows the increases after locus coeruleus stimulation. There was a mean increase of 85% among all the regions, with each region increasing significantly at least below p < .05. MHPG levels in the cerebral cortex hemisphere contralateral to stimulation were 126.6 + 10.3 ng/g. Table I| shows that piperoxan produced a mean increase of 59% across central nervous system regions, with the highest increase in the cerebellum. All increases were significant at p < .005 by t-test analysis. In Table IIl, footshock produced a mean increase of 52% with the lowest increase in the cerebral cortex. All increases were significant at least below p < .05 by t-test analysis.
TABLE I I MHPG Levels After Piperoxan
Total ~{PG
Region
(ng/g) Saline
(5)
Piperoxan (S) 5 mg/kg i.p.
Cerebral Cortex
94.8 + 5.7
149.9 + 9.1"**
}~ppocampus
69.4 + 4.1
105.4 + 7.8***
Cerebellum
68.7 + 4.3
117.2 + 8.2***
Spinal Cord
62.4 + 3.5
100.7 + 4.7***
Results are expressed as mean + S.E.M. of animals per group. ***p < ?005.
Values in parentheses
represent number
TABLE III MHPG Levels After Footshock Region
Total ~IPG
(ng/g) Control
(5)
Footshocked
(5)
Cerebral Cortex
59.0 + 3.0
80.6 + 1.5"**
llippocampus
81.1 + 4.5
98.0 + 6.4*
Cerebellum
52.5 + 9.3
82.1 + 5.3*
Spinal Cord
69.9 + 5.7
109.1 + 8.9***
Results are expressed as mean + S.E.M. Values in parentheses of animals per group. ***p < ~005. *p < .05.
represent number
Figure 1 illustrates the magnitude of N~IPG increase in the four central nervous system regions after the three treatments. Electrical stimulation produced larger increases in all regions. Variability was seen in the percentage increase in MHPG in regions within a given treatment. Analysis of variance
1376
MHPG in Locus Coeruleus Termlnal Regions
Vol. 26, No. 17, 1980
using two factor repeated measures (27) showed no significant differences in brain regional increases due to LC stimulation or to footshock, but a small difference (p < .05) in brain regional increases due to piperoxane, where the increase in cerebellum was higher than in spinal cord or hippocampus.
I
I00
1 Cerebral Cortex Hippocompus Cerebellum
F77~ Spinol Cord
e. .,I- 8 0 E ////
z
.:+:.:.:+:. :::::::::::::::
////
::::::::::::::: ::::::::::::::: ::::::::::::::: +:+:+:+: :::::::::::::::: ::::::::::::::;:
tu 6 0
:.:,:+:.:+:.
z
40
N
.:.:.:.:.:+:, ::::::::::::::: ::::::::::::::: ::::::::::::::: z+:.:-:,:-: :::::::::::;::: :::::::::::::::
////t
i / i / /
20
i/i//
::::::;:;:::::; ::::;::::::::::: :::::::::::::::: :.:,:+:,:.:.: ::::::::::::::::
///~
¢//1
LC STIMULATED 20 Hz, ;~00 FA
FOOTSHOCK
PIPEROXAN
2 mA
5 mg/kg
FIG. 1 Regional increases in total ~IPG in rat central nervous system. Percentage increases were calculated for three types of treatments which influence central noradrenergic function: (a) locus coeruleus stimulation as compared to sham operated controls; (b) electric footshock as compared to apparatus sham controls; and (c) acute piperoxan administration as compared to saline controls.
Discussion Each of tile three qualitatively different treatments significantly increased t~iPG in all regions studied. Locus coeruleus stimulation at 20 Hz produced the largest ~{PG increase, possibly due to direct external control of firing rate overshadowing inhibitory feedback loops. Treatments that do not directly drive LC firing would be more subject to the many buffering influences on LC firing (16). Despite these differences in tlle magnitude of the ~{PG increase as a function of the type of treatment, the increase of MHPG in the four brain regions was relatively constant within each of the different methods used for LC activation. These results support the stated hypothesis that the LC activates its multiple projections simultaneously. ~te
relatively small degree of variability in ~JPG increase in the brain
Vol. 26, No. 17, 1980
MHPG in Locus Coeruleus Terminal Regions
1377
r e g i o n s may be a t t r i b u t a b l e s i m p l y t o t h e small sample s i z e s o f t h e s e t r e a t m e n t groups. A l t e r n a t i v e l y , l o c a l f e e d b a c k l o o p s have been p o s t u l a t e d t o r e g u l a t e amount o f t r a n s m i t t e r r e l e a s e d p e r i m p u ls e by p r e s y n a p t i c i n h i b i t i o n a t t h e terminals (28,29). P o s s i b l y , d i f f e r e n t b r a i n r e g i o n s have more o r l e s s o f such p r e s y n a p t i c i n h i b i t i o n , r e s u l t i n g i n t h e v a r i a b l e p r o f i l e o f r e g i o n a l MHPG changes t o d i f f e r e n t s t i m u l i . In t h e s p e c i f i c case o f s p i n a l cord v a r i a b i l i t y , t h e r e may be a more d i r e c t e x p l a n a t i o n , t h e s e v e r a l s o u r c e s o f s p i n a l cord MttPG: LC p r o j e c t i o n s ( 3 0 - 3 4 ) , A1 n o r a d r e n e r g i c p r o j e c t i o n s ( 1 ) , and C1 e p i nephrine projections (35,36). S p i n a l cord NtPG i s , t h e r e f o r e , l e s s c o m p l e t e l y de pen d en t on LC f i r i n g r a t e s than c e r e b e l l u m , c e r e b r a l c o r t e x , and hippocampus, @~ose s o l e n o r a d r e n e r g i c i n n e r v a t i o n a r i s e s from t h e LC ( 1 - 8 ) . ~ l e e x p e r i m e n t s d e s c r i b e d h e r e do n o t a d d r e s s the q u e s t i o n o f t o p o g r a p h i c o r g a n i z a t i o n o f t h e LC c e l l b o d i e s . One e l e c t r o p h y s i o l o g i c a l r e p o r t , t h a t t h e hippocampal c e l l r e s p o n s e t o LC s t i m u l a t i o n i s most marked when t h e e l e c t r o d e i s p l a c e d i n t h e d o r s a l and a n t e r i o r r e g i o n s o f the LC n u c l e u s (10), s u g g e s t s t h a t t h e c e l l s do n o t e q u a l l y p r o j e c t t o a l l t e r m i n a l r e g i o n s , ttistochemical s t u d i e s s u g g e s t t h a t t h e LC c l u s t e r c o n t a i n s s u b p o p u l a t i o n s w i t h t o p o g r a p h i c a l efferent organization (33,37,38). As a l l t h r e e t r e a t m e n t s o f t h e p r e s e n t st u d y were d e s i g n e d t o a c t i v a t e t h e e n t i r e LC n u c l e u s , t h i s i s s u e must be examined through o t h e r approaches. In c o n c l u s i o n , t r e a t m e n t s t h a t a c t i v a t e t h e l o c u s c o e r u l e u s w i l l i n c r e a s e n o r a d r e n e r g i c m e t a b o l i t e l e v e l s s i m u l t a n e o u s l y i n many and p o s s i b l y a l l o f t h e c e n t r a l n e r v o u s system r e g i o n s i n n e r v a t e d by t h e LC. This e v i d e n c e s u p p o r t s t h e h y p o t h e s i s o f s i m u l t a n e o u s a c t i v a t i o n o f d i f f u s e e f f e r e n t s , and t h e p r o posed m o d u l a t o r y r o l e o f t h e LC. I t a l s o s u g g e s t s t h a t b i o c h e m i c a l a n a l y s i s o f n o r a d r e n e r g i c m e t a b o l i t e l e v e l s i n one r e g i o n i n n e r v a t e d by t h e LC may be s u f f i c i e n t t o a c c u r a t e l y r e f l e c t t h e LC r e s p o n s e a t many o f i t s t e r m i n a l regions. Acknowledgements This work was supported in part by USPIIS grants NI-14092, NS-I0174, ~ 24393, MH-25642, and MII-14276.
References i. 2. 3. 4. S. 6. 7. 8. 9. I0. 11.
12. 13. 14.
A. DAHLSTROM and K. FUXE, ACTA Physiological Scandanavica 6__44Suppl. 247 1-85 (1965). U. UNGERSTEDT, ACTA Physiological Scandanavica82 Suppl. 367 1-76 (1971). A. LINDVALL and A. BJORKLUND, ACTA Scandanavica Suppl. 412 1-48 (1974). V. PICKEL, M. SEGAL and F. E. BLOOM, J. Comp. Neur. 15S 15-42 (1974). M. PALKOVITS and b. M. JACOBOWITZ, J. Comp. Neur. 157 29-42 {1974). L. W. SWANSON, Brain Res. 110 39-56 (1976). B. E. JONES and R. Y. MOORE, Brain Res. 127 23-53 (1977) D. M. BOWDEN, b. C. GERMANand W. D. POYNTER, Brain Res. 145 2S7-276 (1978). S. NAKAMURA, J . P h y s i o l . 267 641-658 (1977). M. SEGAL and F. E. BLOOM, B r a i n Res. 72 9 9 - i 1 4 (1974). S. M. SCHANBERG, J . J . SCHILDKRAUT, G. R. BREESE and I . J. KOPIN, Biochem. Pharmacol. 17 247-254 (1968). J. P. ADER, F. A. J. MUSKIET, H. J. JEURING and J. KORF, J. Neurochem. 30 1213-1216 (1978). J. KORF, G. K. AGHAJANIAN and R. II. ROTll, Europ. J. Pharmacol. 21305-310 (1973). J . N. CRAWLEY, S. E. IIATTOX, J . W. MAAS and R. If. ROTH, B r a i n Res. 141
1378
MHPG in Locus Coeruleus Terminal Regions
Vol. 26, No. 17, 1980
17.
380-384 (1978). J. N. CRAWLEY, J. W. HAAS and R. H. ROTII, Brain Res., in press (1979). G. K. AGHAJANIAN, In: Essays in Neurochemistry and Neuropharmacology, M. B. H. YOUDIM, W. LOVENBERG, D. R. SHARP,AN and J. R. LAGNADO, Eds., pp. 1-22, John Wiley and Sons, New York (1978). G. K. AGHAJANIAN, J. M. CEDARBAU~4, and R. Y. WANG, Brain Res. 136 570-
18. 19.
J. ~4. CEDARBAUM and G. K. AG}IAJANIAN, Life Sci. 23 1383-1392 (1978). J. KORF, G. K. AGHAJANIAN and R. H. ROTH, Neuropharmacol. 12 933-938
20.
J. W. MAAS, S. E. }LATTOX, D. H. LANDIS and R. H. ROI]I, Brain Res. 118 167-
15. 16.
s77 {1977). (1973). 173 (1976). 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35, 36. 37. 38°
E. A. STONE, Life Sci. 16 1725-1730 (1975). Y. HUANG and J. W. MAAS, Brain Res. i15 91-97 (1976). M. DAVIS and D. I. ASTRACHAN, J. Exp. Psychol. 4 95-103 (1978). J. ROSSIER, R. GUILLEMIN, and F. BLOOM, Europ. ~. Pharmacol. 48 465-466 (1978). S. LAVIELLE, J. P. TASSIN, A. M. THIERRY, G. BLANC, D. IIERVE, C. BARTHELEMY and J. GLOWINSKI, Brain Res. 168 585-594 (1978). S. LEVINE, J. MADDEN, R. L. CONNER, J. R. MOSKAL and D. C. ANDERSON, Physiolo Behav. i0 467-471 (1973). B. J. WINER, S t a t i s t i c a l P r i n c i p l e s i n Experimental Design, McGraw-Hill, New York (1962). S. Z. LANGER, Br. J. Pharmacol. 60 481-497 (1977). K. STARKE and K. P. ALT~IANN, Neuropharmacol. 12 339-347 (1973). L. NYGREN and 1. OLSON, Brain Res. 132 85-93 (1977). M. B. HANCOCKand C. L. FOUGEROUSSE, Brain Res. B u l l . 1 229-234 (1976). J. N. CRAWLEY, R. H. ROTH and J. W. MAAS, Brain Res. 166 180-184 (1979). J. P. ADER, F. POSTEMAand J. KORF, J. Neural T r a n s . , 45(3) 159-173 (1979). Jo W. CO~ISSIONG, S. O. }IELLSTRO~4, N. H. NEFF, Br ai n Res. 148 207-213 (1978). J . L. REID, J . A. ZIVIN and I. J . KOPIN, J . Neurochem. 26 629-631 (1976). T. HOKFELT, K. FUXE, M. GOLDSTEIN and O. JOHANSSON, Br ai n Res. 66 235-251 (1974). K. SATOH, H. TOHYAMA, K. YAMAMOTO, T. SAKUMOTO, and N. SHIMIZU, Exp. Brain Res. 30 175-186 (1977). S. E. LOUGHLIN, S. L. FOOTE, and F. E. BLOOM, Soc. For Neurosciences Abstract #1136 (1979) o