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Effects of bilateral lesion of the locus coeruleus and of neonatal administration of 6-hydroxydopamine on the concentration of individual proteins in rat brain
Brain Research, 367 (1986) 31-38
31
Elsevier BRE 11486
Effects of Bilateral Lesion of the Locus Coeruleus and of Neonatal Administration of 6-Hydroxydopamine on the Concentration of Individual Proteins in Rat Brain WILLIAM E. HEYDORN 1, KHANH Q. NGUYEN 1, G. JOSEPH CREED 1, RICHARD M. KOSTRZEWA2and DAVID M. JACOBOWlTZ1
1Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 3D-48, Bethesda, MD 20892 and 2Department of Pharmacology, East Tennessee State University, Quillen-Dishner College of Medicine, Johnson Oty, TN37614 (U.S.A.) (Accepted July 2nd, 1985)
Key words: locus coeruleus lesion - - neonatal 6-hydroxydopamine - - central nervous system protein - two-dimensional gel electrophoresis - - norepinephrine - - scanning densitometry
The role that norepinephrine plays in regulating the concentration of different proteins in the parietal cortex, hippocampus and cerebellum was assessed by investigating the effects of either a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine. Two weeks after lesioning the locus coeruleus, the concentration of two different proteins was elevated in the hippocampus; a third protein was reduced in concentration in this brain area as a result of the lesion. Three proteins were affected in concentration in the cerebellum after the locus coeruleus lesion - - two were elevated in concentration and one was reduced in concentration. No proteins were altered in concentration in the parietal cortex as a result of the lesion. Seventy days after neonatal treatment with 6hydroxydopamine, a total of 6 proteins were found to be changed. Four of these (one in the hippocampus and 3 in the parietal cortex) were reduced in concentration while two proteins (both in the cerebellum) were elevated in concentration after neonatal treatment with the catecholamine neurotoxin. There was little overlap between those proteins affected in concentration by the bilateral lesion of the locus coeruleus and those changed by neonatal treatment with 6-hydroxydopamine. These results suggest that the concentration of a number of different proteins may, under normal physiologicalconditions, be regulated in vivo by norepinephrine in the brain.
INTRODUCTION The locus coeruleus (LC) is a relatively small pontine structure located adjacent to the fourth ventricle 1. Biochemical as well as histochemical studies of this structure have revealed that the LC is the source of one of the m a j o r ascending norepinephrine (NE) pathways in the central nervous system5,16. Therefore, a relatively simple and efficient method of altering levels of NE in discrete areas of the adult rat brain is by electrolytically or chemically destroying the LC. In animals receiving such a treatment, cortical levels of NE have been shown to be significantly reduced in comparison to those measured in untreated control animals3.16,18. In addition, biochemical as well as behavioral indices of noradrenergic input to various brain areas have b e e n shown to be altered in rats in which the LC has been destroyed3,7,19. A second method of altering catecholamine levels
in the central nervous system of adult rats is by the neonatal administration of the catecholamine neurotoxin 6-hydroxydopamine ( 6 - O H D A ) . This compound is capable of penetrating the immature b l o o d - b r a i n barrier of n e w b o r n rat pups z6 and irreversibly halting the rostral development of the central noradrenergic system 2. Interestingly, if drug treatment is limited to the first 3 days of life, an apparent hyperinnervation occurs caudally towards the cerebellum and brainstem15,28. Thus, the rostral catecholamine denervation that occurs after neonatal 6O H D A differs qualitatively from that obtained after an LC lesion in that the cortical and hippocampal formations of adult rats receiving an LC lesion have an existing catecholamine innervation removed, while those rats treated neonatally with 6 - O H D A have essentially never had a catecholamine input to the cortex or hippocampus. Additionally, catecholamine levels are decreased in the cerebellum after an LC le-
Correspondence: W.E. Heydorn, Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892, U.S.A.
32 sion while being elevated after neonatal treatment on days 1-3 of life with 6-OHDA. The present study investigates the effect of an electrolytic lesion of the LC and the neonatal administration of 6-OHDA for 3 days on the concentration of discrete proteins in the parietal cortex, hippocampus and cerebellum of adult rats. This was accomplished by using the combination of two-dimensional gel electrophoresis (2DE) and computerized scanning densitometry. The present report demonstrates that an alteration in noradrenergic neurotransmission in the cortex, hippocampus and cerebellum produces changes in the apparent concentration of a number of different proteins. Interestingly, most of the proteins that were affected by an electrolytic lesion of the LC in adult rats were different from those altered by neonatal treatment with 6-OHDA. MATERIALS AND METHODS
I Animals Locus coeruleus lesion. Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing between 350 and 375 g were used for all experiments. On the day of surgery, animals were anesthetized with a mixture of halothane (2-4%) and 95% O : - 5 % CO 2. Radiofrequency lesions were then placed bilaterally in the LC using a Grass LM 4 lesion maker. Initial studies indicated that the combination of dual electrodes and the use of two electrode placements, rather than one, resulted in a more complete lesioning of the LC with less damage to surrounding neuronal tissue. Thus, with the incisor bar set at -3.0 mm, the coordinates of the lesion were: anteriorposterior + 0.2 mm and -0.8 mm from the interaural line at zero, lateral + 1.3 mm, depth 6.4 mm below dura. Lesions were made using coated electrodes with an exposed tip 1.0 mm long and 0.15 mm in diameter. The current used for all lesions was 15 mA delivered for 10 s. Sham animals were treated identically except that the electrodes were inserted to a depth of 2.0 mm below dura and no current was applied. After surgery, animals were allowed to survive for 14 days. This time period has been shown to be sufficient to produce both a reduction in central NE stores and a maximal change in other indices of catecholamine function in the central nervous system 7. 6-Hydroxydopamine treatment. Timed-pregnant
Sprague-Dawley rats were received on day 14 of gestation and housed individually in plastic cages on a 12 h light-dark cycle (lights on at 06.00 h). Within 24 h of birth, each pup received a subcutaneous injection of either 6-hydroxydopamine (100/~g) or vehicle (0.9% NaC1 containing 0.1% ascorbic acid). Injections were repeated on days 2 and 3 so that each pup received a total of 3 injections. Pups were weaned at 30 days of age, and were killed by decapitation on day 70.
Tissue preparation After decapitation, the brain was rapidly removed from the skull and placed into a brain block. The forebrain was separated from the hindbrain just caudal to the superior colliculus and both sections were frozen with powdered dry-ice. Subsequently, brains were frozen onto microtome specimen plates and sectioned in a cryostat maintained at -7 °C. Transverse sections (300 /~m thick) were cut from all brains. In addition, in those animals receiving an electrolytic lesion of the LC, thin sections (50/~m thick) were taken through the lesion site to verify the extent of tissue destruction. Tissue samples (approximately 40/zg of protein) were then microdissected from the frozen 300/~m thick coronal sections using the micropunch technique of Palkovits 24. The coordinates for the tissue dissection were as follows: parietal cortex A6860, hippocampus A343017, cerebellum P2800-P340025.
Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was performed according to the procedure of O'Farrel123 with minor modifications for neuronal tissue 8. Proteins were visualized using a highly sensitive silver stain as previously described 9.
Quantitative analysis of proteins on two-dimension gels Quantitative analysis of optical density values for individual protein spots was performed according to the method of Goldman et al. 6. Because the final density of each protein spot appearing on the gels is related to both its degree of staining and its size on the gel (in square millimeters), the units for density are optical density x square millimeters6. At the conclusion of the analysis, optical density measurements
33 were normalized (by using approximately 30 constantly appearing non-saturating protein spots) to correct for day-to-day differences in the amount of protein entering each gel and variations in the density of the silver stain among individual gels 6. All data were then analyzed using a two-tailed Student's ttest. A P value of < 0.01 was selected as the criterion for significance. Analysis o f norepinephrine levels in individual brain areas
The content of NE in the parietal cortex, hippocampus and cerebellum was assessed using the radiometric-enzymatic assay of Coyle and Henry 4. Protein content of individual tissue samples was assayed according to the procedure of Lowry et al. 21. Catecholamine concentrations are expressed as ng NE/mg protein. RESULTS Examination of the 50/~m thin sections taken from LC lesioned rats revealed that the major cluster of cell bodies in the LC was destroyed in all lesioned brains studied. Fig. 1 is a transverse thin section stained with 0.1% thionin showing the midpoint of an electrolytic lesion of the LC in a representative animal. Only those animals demonstrating complete bilateral destruction of the LC were included in this study. The effect of an electrolytic lesion of the LC and the effect of neonatal treatment with 6 - O H D A on NE levels in the parietal cortex, hippocampus and cerebellum are summarized in Table I. Two weeks after ablating the LC, levels of NE were reduced 93% in the parietal cortex, 80% in the hippocampus
Fig. 1. Transverse thin section (50 pm) showing the midpoint of the electrolytic lesion of the LC in a representative lesioned animal. Only those animals demonstrating complete bilateral destruction of the LC were included in this study.
and 79% in the cerebellum, consistent with previous observations ~6. Secenty days after neonatal treatment with 6 - O H D A , norepinephrine levels were reduced 93% in the parietal cortex and 91% in the hippocampus. In contrast, NE levels were elevated 165% in the cerebellum, again consistent with reports that neonatal treatment with 6 - O H D A during the first 3 days of life produces an elevation of NE content in the cerebellum of adult rats15. 2s. Having thus demonstrated that destruction of the LC and neonatal treatment with 6 - O H D A produced the expected changes in NE content in the parietal cortex, hippocampus and cerebellum, tissue punches from each of these brain regions were removed for analysis of proteins by 2DE. A photograph of a representative gel obtained when proteins within these tissue samples are separated by 2DE is shown in Fig.
TABLE I Effect of a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine on the concentration of norepinephrine in different areas of the rat brain
Data show ,X _+ S.E.M. Units are ng of norepinephrine/mg protein. Number of observations shown in parentheses. In all cases, the lesioned or drug-treated group is significantly different from the corresponding control group (P < 0.001, two-tailed Student's t-test). Neonatal 6-hydroxydopamine
Hippocampus Parietal cortex Cerebellum
Locus coeruleus lesion
Control
Experimental
Control
Experimental
3.17 _+0.40 (12) 2.84 + 0.40 (10) 1.48 + 0.13 (11)
0.27 _+0.08 (12) 0.20 + 0.07 (12) 3.92 _+0.44 (11)
3.20 + 0.37 (8) 1.65 + 0.34 (8) 1.73 + 0.29 (8)
0.63 _+0.10 (10) 0.12 _+0.08 (10) 0.38 + 0.04 (10)
34
94.0--
,-?, 0
67.0
--
43.0
--
I.-
x I1" (9
l.U rr ....I ¢0 3 0 . 0 -ILl _.1 0
o 48
20.1 14.4
5.'0
610
710
pl Fig. 2. Representative gel obtained when tissue samples of rat brain were electrophoresed in two dimensions and then stained with silver. This particular gel was generated using tissue obtained from the parietal cortex, but is also representative of that seen using tissue from either the hippocampus or the cerebellum. For orientation purposes, the ordinate and the abscissa are marked for molecular weight and pI, respectively. In addition, each of the proteins found to be altered by either a bilateral LC lesion or by neonat~fl treatment with 6-OHDA, along with their permanent indexing numbers for rat brains.14, are indicated.
2. F o r orientation purposes, the ordinate and the abscissa are m a r k e d for molecular weight and isoelectric point (pI), respectively. As can be seen from this figure, only proteins within a limited pI range (5.0-7.0) and molecular weight range (100,000-10,000 daltons) were s e p a r a t e d under the conditions utilized. In spite of this, a large n u m b e r of distinct protein spots were well resolved on our twodimensional gels. Of the protein spots that were visible, 140 were judged to be sufficiently well resolved from both adjacent spots and the surrounding b a c k g r o u n d so that accurate densitometric m e a s u r e m e n t s could be made. Of these, a total of 11 different proteins were found to be altered in concentration in at least one of the brain areas examined as a result of either an elec-
trolytic lesion of the LC or neonatal treatment with 6O H D A . The location of each of these 11 proteins, along with their p e r m a n e n t indexing numbers for rat brainS: 4, are indicated in Fig. 2. In the hippocampus, a total of 4 different proteins were altered in concentration by one of the manipulations employed in this experiment (Table II). Of these 4 proteins, only one (no. 102) was affected by neonatal treatment with 6 - O H D A . This protein, with a molecular weight of 26,000 daltons and a pI of 5.0, was reduced 34% in concentration as a result of the catecholamine neurotoxin. Bilateral LC lesion elevated the concentration of two different proteins (nos. 8, 72) in the hippocampus between 26% and 30% over that obtained in corresponding control animals. A single protein (no. 48) was reduced 16% in
35 d e n s i t y in t h e h i p p o c a m p u s a f t e r t h e L C lesion. Neonatal treatment with 6-OHDA
p l 5.8) was r e d u c e d 3 1 % a n d p r o t e i n no. 19 ( m o l . w t .
affected the
47,000, pI 5.8) was r e d u c e d 2 0 % in a d u l t a n i m a l s
c o n c e n t r a t i o n o f 3 d i f f e r e n t p r o t e i n s in t h e p a r i e t a l
t r e a t e d at b i r t h w i t h t h e n e u r o t o x i n . In c o n t r a s t , n o
c o r t e x ( T a b l e III). P r o t e i n no. 2 ( m o l . w t . 65,000, pI
p r o t e i n s w e r e f o u n d t o b e a f f e c t e d in t h e p a r i e t a l cor-
5.8) was r e d u c e d 2 7 % , p r o t e i n n o . 3 ( m o l . w t . 64,000,
tex a f t e r a b i l a t e r a l l e s i o n o f t h e L C .
TABLE II
Proteins affected in concentration in the hippocampus after either neonatal treatment with 6-hydroxydopamine or a bilateral lesion o f the locus coeruleus Protein number corresponds to the numbering system of Heydorn et al. 8 and Jacobowitz and Heydorn TM. Data show ,X + S.E.M. Units are optical density x mm 2. Neonatal 6-hydroxydopamine: control, n = 8, experimental, n = 8; locus coeruleus lesion: control, n = 12, experimental, n = 10.
Protein number
M r ( x lO -3)
pl
Control
Experimental
% Change
Neonatal 6-hydroxydopamine 102 26
5.0
0.901 + 0.067
0.602 + 0.071"
-34
Locus coeruleus lesion 8 62 48 22 72 57
6.1 5.3 6.0
0.324 + 0.013 0.654 + 0.021 0.475 _+ 0.020
0.422 + 0.015"** 0.547 + 0.022** 0.601 + 0.035*
+30 -16 +26
* P < 0.01; ** P < 0.005; *** P < 0.001 compared to value obtained in corresponding control group.
TABLE III
Proteins affected in concentration in the parietal cortex after neonatal treatment with 6-hydroxydopamine No proteins were found to be altered in concentration in the parietal cortex after a bilateral lesion of the locus coeruleus. Protein number corresponds to the numbering system of Heydorn et al.8 and Jacobowitz and Heydorn 14. Data show X + S.E.M. Units are optical density x mm2. n = 8 for control and experimental groups.
Protein number
M, ( x lO-3)
pl
Control
Experimental
% Change
2 3 19
65 64 47
5.8 5.8 5.8
1.736 + 0.098 0.751 + 0.067 1.281 + 0.049
1.272 + 0.075** 0.518 + 0.032* 1.015 + 0.031"**
-27 -31 -20
* P < 0.01; ** P < 0.005; *** P < 0.001 compared to value obtained in corresponding control group.
TABLE IV
Proteins affected in concentration in the cerebellum after either neonatal treatment with 6-hydroxydopamine or a bilateral lesion o f the locus coeruleus Protein number corresponds to the numbering system of Heydorn et al. s and Jacobowitz and Heydorn t4. Data show X + S.E.M. Units are optical density x mm2. Neonatal 6-hydroxydopamine: control, n = 9, experimental, n = 8; locus coeruleus lesion: control, n = 8, experimental, n = 9.
Protein number
pl
Control
Experimental
% Control
Neonatal 6-hydroxydopamine 31 50 72 57
6.6 6.0
0.816 + 0.036 0.161 __+0.026
1.061 + 0.067** 0.270 + 0.018"*
+30 +68
Locus coeruleus lesion 4 67 37 36 110 30
5.9 6.7 6.3
0.319 + 0.018 0.231 + 0.019 0.054 + 0.009
0.239 + 0.011"* 0.317 + 0.013"* 0.086 + 0.005*
-25 +37 +59
M r ( X lO -3)
* P < 0.01 ; ** P < 0.005 compared to value obtained in corresponding control group.
3~ In the cerebellum. 5 different proteins were altered in concentration after either neonatal treatment with 6-OHDA or a bilateral LC lesion (Table IV). Of these, only a single protein (no. 4) was reduced in concentration (--25%), and this occurred after the LC lesion. Two other proteins (nos. 37, 110) were elevated in concentration in the cerebellum after bilateral LC lesioning. In addition, two proteins (nos. 31, 72) were also elevated in concentration by 30% and 68%, respectively, in adult rats treated neonatally with 6-OHDA.
DISCUSSION These results suggest that the apparent concentration of a number of different proteins in the central nervous system of the rat are regulated by the catecholamine neurotransmitter norepinephrine. Specifically, two weeks after electrolytically ablating the locus coeruleus, the concentration of 6 different proteins were found to be altered in either the hippocampus or the cerebellum. Of these 6 proteins, only two (nos. 4, 48) showed a decrease as a result of the lesion, while the 4 others (nos. 8, 37, 72, 110) showed an increase in concentration as a result of the lesion. No proteins were altered in the parietal cortex as a result of the lesion. In addition, 6 proteins were found to be altered in concentration in adult rats treated neonatally with 6-hydroxydopamine. Of these 6, only 2 proteins (both in the cerebellum) showed an increase in concentration as a result of the neurotoxin. The other 4 proteins (one in the hippocampus, 3 in the parietal cortex) were decreased in concentration in rats treated neonatally with the neurotoxin. It is unclear why most of the proteins affected in concentration by electrolytic lesions of the LC do not overlap with those affected in concentration by neonatal administration of 6-OHDA, even though both treatments produced the expected large changes in central levels of NE (see Table I). The reason for these differences may be related to the fact that adult animals received an LC lesion while only newborn pups received 6-OHDA. The central catecholamine system of the rat is immature at birth, and develops over the first 3-4 weeks of life 2°. Thus, in those neonatal pups receiving 6-OHDA, essentially no catecholamine innervation had ever existed in the cortex
and hippocampus. This suggests that the proleins found to be altered in concentration in adult rats treated with 6-OHDA require a catecholamme input during the neonatal development period for normal concentrations of these proteins to develop. If the NE input to cortex and hippocampus is removed in adulthood, the concentration of most of these proteins in brain is apparently unaffected. In contrast, removing the noradrenergic input to cortex and cerebellum in adulthood by electrolytically ablating the LC results in an alteration in the concentration of 6 different proteins. This suggests that these proteins are, under normal circumstances, regulated m concentration by NE in such a manner that removing the innervation results in an alteration in protein concentration. However, if the noradrenergic innervation to the cortex is not permitted to occur (by administering 6-OHDA to neonates), then the concentration of most of these proteins in adulthood does not differ between control and drug-treated animals. It may be that, in the neurotoxin-treated neonatal animals, another neurotransmitter system assumes control in regulating the concentration of these proteins. Further research into the overall biochemical effects of 6-OHDA will be necessary to determine the validity of such a hypothesis. In addition to the finding that there was little overlap of those proteins changed after an LC lesion with those proteins changed after neonatal 6-OHDA, it was also interesting that neonatal treatment with 6O H D A affected different proteins in the hippocampus, parietal cortex and cerebellum. Similarly, lesioning the LC had different effects in the hippocampus than in the cerebellum, with no effect in the parietal cortex. These results suggest that t h e regulation of the concentration of individual proteins in brain by NE is a complex matter. Under the most simplistic of conditions, one would expect that an LC lesion would alter the concentration of one or more proteins in all brain areas that have been affected by the electrolytic lesion. Similarly, neonatal treatment with 6-OHDA should have similar effects on brain areas that have been deprived of a noradrenergic innervation, and these effects would be opposite to those obtained in areas that are hyperinnervated. This, however, was not the case in the present study. The data presented here suggest that there is no single group of proteins in the CNS that are visible on
37 two-dimensional gels under the present experimental conditions that are regulated in concentration in all brain areas by NE. This may be because such proteins do not exist in brain. However, it may be that such proteins do exist in the CNS but were not visualized and analyzed in the present experiment. This may be because their isoelectric points and/or subunit molecular weights did not fall into the range selected for the present study. Additionally, it is possible that such proteins exist within these physico-chemical constraints, but are not visible on our two-dimensional gels because they exist in brain at a concentration below the sensitivity of the silver stain used to visualize proteins in the present experiment. In either event, as the data presented here suggest, no single group of proteins analyzed in the present experiment is regulated in a simplistic manner by NE. One question that remains to be addressed is the identity of the proteins found to be altered in concentration in the present experiment. To date, we have positively identified 2 of the 11 proteins found to change in the present experiment. One of these is protein no. 19, which appears to be the fl-isozyme of cytosolic (soluble) glutamic oxaloacetic transaminase (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)11, the enzyme responsible for catalyzing the interconversion of L-aspartate and a-ketoglutarate to oxaloacetate and glutamate. To date, there exist no reports demonstrating possible interrelationships between NE and this enzyme in brain. However, as the present data suggest, NE may play a role in regulating the concentration of this enzyme in the parietal cortex. A second protein positively identified and found to be elevated in the cerebellum after neonatal treatment with 6-OHDA is protein no. 31, which has been identified as the non-neuronal form of the enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11)10. There exist at least two possible explanations for this finding. First, it may be that more nonneuronal elements were obtained from cerebellar tissue of 6-OHDA-treated rats than from corresponding control rats. Such a hypothesis seems unlikely since no change was detected in the concentration of glial fibrillary acidic protein TM in the cerebellum after neonatal treatment with 6-OHDA. Second, it is possible that 6-OHDA retarded the natural conversion of non-neuronal enolase to neuron-specific enolase
that occurs in rats during cerebellar development22,27, resulting in a greater amount of nonneuronal enolase being detected in rats treated neonatally with the catecholamine neurotoxin. Such an argument is strengthened by the recent finding that enolase levels in adipose tissue may be regulated by catecholamines 29. The results presented in this report add to the list of proteins visible on two-dimensional gels generated using rat brain tissues that are apparently regulated in concentration by a specific neurotransmitter. More importantly, the present results suggest that different neurotransmitters, in addition to quantitatively affecting unique sets of proteins, also have qualitatively distinct effects on discrete proteins in the central nervous system. For example, it has been shown that electrolytic ablation of the nucleus of the tractus diagonalis (nucleus basalis), which provides the primary cholinergic innervation to the occipital cortex and the hippocampus 13, results in an alteration in the concentration of 4 different proteins in both of these brain areas12.Thus, it appears that acetylcholine influences the concentration of a discrete number of proteins in multiple areas of the central nervous system. In contrast, as the present results demonstrate, NE regulates the concentration of different proteins in different brain areas. In addition, there appears to be no overlap between those proteins regulated in concentration by the catecholamines and those regulated by acetylcholine 12. This information on neurotransmitter regulation of protein concentrations should prove valuable when attempting to interpret the effects of various pharmacologic investigations on the apparent concentration of individual proteins visible on two-dimensional gels. Future studies will be aimed at isolating and characterizing these proteins found to be regulated by specific neurotransmitters in an attempt to compile a database which can then be used in the study of specific neurobiological problems. ACKNOWLEDGEMENTS The expert secretarial assistance of Mrs. Lois Brown is gratefully acknowledged. William E. Heydorn was a Research Associate, Pharmacology-Toxicology Program, National Institute of General Medical Science at the time this research was performed.
38 REFERENCES 1 Amaral, D.G. and Sinnamon, H.M., The locus coeruleus: neurobiology of a central noradrenergic nucleus, Progr. Neurobiol., 9 (1977) 147-196. 2 Angeletti, P. and Levi-Montalcini, R., Sympathetic nerve cell destruction in newborn mammals by 6-hydroxydopamine, Proc. Natl. Acad. Sci. U.S.A., 65 (1970) 114-121. 3 Anlezark, G., Crow. T. and Greenway, A., Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions, Science, 181 (1973) 682-684. 4 Coyle, J.T. and Henry, D., Catecholamines in fetal and newborn rat brain, J. Neurochem., 21 (1973) 61-67. 5 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62, Suppl., 232 (1964) 1-55. 6 Goldman, D., Merril, C.R., Polinsky, R.J. and Ebert, M.H., Lymphocyte proteins in Huntington's disease: quantitative analysis by use of two-dimensional electrophoresis and computerized densitometry, Clin. Chem.. 28 (1982) 102t- 1025. 7 Harik, S., Duckrow, B., LaManna, J., Rosenthal, M., Sharma, V. and Banerjee, S., Cerebral compensation for chronic noradrenergic denervation induced by locus coeruleus lesion: recovery of receptor binding, isoproterenol-induced adenylate cyclase activity and oxidative metabolism, J. Neurosci., 1 (1981) 641-649. 8 Heydorn, W.E., Creed, G.J., Goldman, D., Kanter, D., Merril, C.R. and Jacobowitz, D., Mapping and quantitation of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis, J. Neurosci., 3 (1983) 2597-2606. 9 Heydorn, W.E., Creed, G.J. and Jacobowitz, D.M., The effect of desmethylimipramine and reserpine on the concentration of specific proteins in the parietal cortex and the hippocampus of rats as analyzed by two-dimensional gel clectrophoresis, J. Pharmacol. Exp. Ther., 229 (1984) 622-628. 10 Heydorn, W.E., Creed, G.J., Marangos, P.J. and Jacobowitz, D.M., Identification of neuron-specific enolase and non-neuronal enolase in human and rat brain on two-dimensional polyacrylamide gels, J. Neurochem., 44 (1985) 201-209. 11 Heydorn, W.E., Creed, G.J., Wada, H. and Jacobowitz, D.M., Immunological evidence for the existence of two subforms of soluble glutamic oxaloacetic transaminase (sGOT) in human and rat brain, Neurochem. Int., 7 (1985) 833-841. 12 Heydorn, W.E., Nguyen, K.Q., Creed, G.J. and Jacobowitz, D.M., Effect of reduction of cholinergic input on the concentration of specific proteins in different cortical regions of the rat brain, Brain Research. 339 (1985) 209-218. 13 Jacobowitz, D.M. and Creed, G.J., Cholinergic projection sites of the nucleus of tractus diagonalis, Brain Res. Bull.,
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31
Elsevier BRE 11486
Effects of Bilateral Lesion of the Locus Coeruleus and of Neonatal Administration of 6-Hydroxydopamine on the Concentration of Individual Proteins in Rat Brain WILLIAM E. HEYDORN 1, KHANH Q. NGUYEN 1, G. JOSEPH CREED 1, RICHARD M. KOSTRZEWA2and DAVID M. JACOBOWlTZ1
1Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 3D-48, Bethesda, MD 20892 and 2Department of Pharmacology, East Tennessee State University, Quillen-Dishner College of Medicine, Johnson Oty, TN37614 (U.S.A.) (Accepted July 2nd, 1985)
Key words: locus coeruleus lesion - - neonatal 6-hydroxydopamine - - central nervous system protein - two-dimensional gel electrophoresis - - norepinephrine - - scanning densitometry
The role that norepinephrine plays in regulating the concentration of different proteins in the parietal cortex, hippocampus and cerebellum was assessed by investigating the effects of either a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine. Two weeks after lesioning the locus coeruleus, the concentration of two different proteins was elevated in the hippocampus; a third protein was reduced in concentration in this brain area as a result of the lesion. Three proteins were affected in concentration in the cerebellum after the locus coeruleus lesion - - two were elevated in concentration and one was reduced in concentration. No proteins were altered in concentration in the parietal cortex as a result of the lesion. Seventy days after neonatal treatment with 6hydroxydopamine, a total of 6 proteins were found to be changed. Four of these (one in the hippocampus and 3 in the parietal cortex) were reduced in concentration while two proteins (both in the cerebellum) were elevated in concentration after neonatal treatment with the catecholamine neurotoxin. There was little overlap between those proteins affected in concentration by the bilateral lesion of the locus coeruleus and those changed by neonatal treatment with 6-hydroxydopamine. These results suggest that the concentration of a number of different proteins may, under normal physiologicalconditions, be regulated in vivo by norepinephrine in the brain.
INTRODUCTION The locus coeruleus (LC) is a relatively small pontine structure located adjacent to the fourth ventricle 1. Biochemical as well as histochemical studies of this structure have revealed that the LC is the source of one of the m a j o r ascending norepinephrine (NE) pathways in the central nervous system5,16. Therefore, a relatively simple and efficient method of altering levels of NE in discrete areas of the adult rat brain is by electrolytically or chemically destroying the LC. In animals receiving such a treatment, cortical levels of NE have been shown to be significantly reduced in comparison to those measured in untreated control animals3.16,18. In addition, biochemical as well as behavioral indices of noradrenergic input to various brain areas have b e e n shown to be altered in rats in which the LC has been destroyed3,7,19. A second method of altering catecholamine levels
in the central nervous system of adult rats is by the neonatal administration of the catecholamine neurotoxin 6-hydroxydopamine ( 6 - O H D A ) . This compound is capable of penetrating the immature b l o o d - b r a i n barrier of n e w b o r n rat pups z6 and irreversibly halting the rostral development of the central noradrenergic system 2. Interestingly, if drug treatment is limited to the first 3 days of life, an apparent hyperinnervation occurs caudally towards the cerebellum and brainstem15,28. Thus, the rostral catecholamine denervation that occurs after neonatal 6O H D A differs qualitatively from that obtained after an LC lesion in that the cortical and hippocampal formations of adult rats receiving an LC lesion have an existing catecholamine innervation removed, while those rats treated neonatally with 6 - O H D A have essentially never had a catecholamine input to the cortex or hippocampus. Additionally, catecholamine levels are decreased in the cerebellum after an LC le-
Correspondence: W.E. Heydorn, Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892, U.S.A.
32 sion while being elevated after neonatal treatment on days 1-3 of life with 6-OHDA. The present study investigates the effect of an electrolytic lesion of the LC and the neonatal administration of 6-OHDA for 3 days on the concentration of discrete proteins in the parietal cortex, hippocampus and cerebellum of adult rats. This was accomplished by using the combination of two-dimensional gel electrophoresis (2DE) and computerized scanning densitometry. The present report demonstrates that an alteration in noradrenergic neurotransmission in the cortex, hippocampus and cerebellum produces changes in the apparent concentration of a number of different proteins. Interestingly, most of the proteins that were affected by an electrolytic lesion of the LC in adult rats were different from those altered by neonatal treatment with 6-OHDA. MATERIALS AND METHODS
I Animals Locus coeruleus lesion. Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing between 350 and 375 g were used for all experiments. On the day of surgery, animals were anesthetized with a mixture of halothane (2-4%) and 95% O : - 5 % CO 2. Radiofrequency lesions were then placed bilaterally in the LC using a Grass LM 4 lesion maker. Initial studies indicated that the combination of dual electrodes and the use of two electrode placements, rather than one, resulted in a more complete lesioning of the LC with less damage to surrounding neuronal tissue. Thus, with the incisor bar set at -3.0 mm, the coordinates of the lesion were: anteriorposterior + 0.2 mm and -0.8 mm from the interaural line at zero, lateral + 1.3 mm, depth 6.4 mm below dura. Lesions were made using coated electrodes with an exposed tip 1.0 mm long and 0.15 mm in diameter. The current used for all lesions was 15 mA delivered for 10 s. Sham animals were treated identically except that the electrodes were inserted to a depth of 2.0 mm below dura and no current was applied. After surgery, animals were allowed to survive for 14 days. This time period has been shown to be sufficient to produce both a reduction in central NE stores and a maximal change in other indices of catecholamine function in the central nervous system 7. 6-Hydroxydopamine treatment. Timed-pregnant
Sprague-Dawley rats were received on day 14 of gestation and housed individually in plastic cages on a 12 h light-dark cycle (lights on at 06.00 h). Within 24 h of birth, each pup received a subcutaneous injection of either 6-hydroxydopamine (100/~g) or vehicle (0.9% NaC1 containing 0.1% ascorbic acid). Injections were repeated on days 2 and 3 so that each pup received a total of 3 injections. Pups were weaned at 30 days of age, and were killed by decapitation on day 70.
Tissue preparation After decapitation, the brain was rapidly removed from the skull and placed into a brain block. The forebrain was separated from the hindbrain just caudal to the superior colliculus and both sections were frozen with powdered dry-ice. Subsequently, brains were frozen onto microtome specimen plates and sectioned in a cryostat maintained at -7 °C. Transverse sections (300 /~m thick) were cut from all brains. In addition, in those animals receiving an electrolytic lesion of the LC, thin sections (50/~m thick) were taken through the lesion site to verify the extent of tissue destruction. Tissue samples (approximately 40/zg of protein) were then microdissected from the frozen 300/~m thick coronal sections using the micropunch technique of Palkovits 24. The coordinates for the tissue dissection were as follows: parietal cortex A6860, hippocampus A343017, cerebellum P2800-P340025.
Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was performed according to the procedure of O'Farrel123 with minor modifications for neuronal tissue 8. Proteins were visualized using a highly sensitive silver stain as previously described 9.
Quantitative analysis of proteins on two-dimension gels Quantitative analysis of optical density values for individual protein spots was performed according to the method of Goldman et al. 6. Because the final density of each protein spot appearing on the gels is related to both its degree of staining and its size on the gel (in square millimeters), the units for density are optical density x square millimeters6. At the conclusion of the analysis, optical density measurements
33 were normalized (by using approximately 30 constantly appearing non-saturating protein spots) to correct for day-to-day differences in the amount of protein entering each gel and variations in the density of the silver stain among individual gels 6. All data were then analyzed using a two-tailed Student's ttest. A P value of < 0.01 was selected as the criterion for significance. Analysis o f norepinephrine levels in individual brain areas
The content of NE in the parietal cortex, hippocampus and cerebellum was assessed using the radiometric-enzymatic assay of Coyle and Henry 4. Protein content of individual tissue samples was assayed according to the procedure of Lowry et al. 21. Catecholamine concentrations are expressed as ng NE/mg protein. RESULTS Examination of the 50/~m thin sections taken from LC lesioned rats revealed that the major cluster of cell bodies in the LC was destroyed in all lesioned brains studied. Fig. 1 is a transverse thin section stained with 0.1% thionin showing the midpoint of an electrolytic lesion of the LC in a representative animal. Only those animals demonstrating complete bilateral destruction of the LC were included in this study. The effect of an electrolytic lesion of the LC and the effect of neonatal treatment with 6 - O H D A on NE levels in the parietal cortex, hippocampus and cerebellum are summarized in Table I. Two weeks after ablating the LC, levels of NE were reduced 93% in the parietal cortex, 80% in the hippocampus
Fig. 1. Transverse thin section (50 pm) showing the midpoint of the electrolytic lesion of the LC in a representative lesioned animal. Only those animals demonstrating complete bilateral destruction of the LC were included in this study.
and 79% in the cerebellum, consistent with previous observations ~6. Secenty days after neonatal treatment with 6 - O H D A , norepinephrine levels were reduced 93% in the parietal cortex and 91% in the hippocampus. In contrast, NE levels were elevated 165% in the cerebellum, again consistent with reports that neonatal treatment with 6 - O H D A during the first 3 days of life produces an elevation of NE content in the cerebellum of adult rats15. 2s. Having thus demonstrated that destruction of the LC and neonatal treatment with 6 - O H D A produced the expected changes in NE content in the parietal cortex, hippocampus and cerebellum, tissue punches from each of these brain regions were removed for analysis of proteins by 2DE. A photograph of a representative gel obtained when proteins within these tissue samples are separated by 2DE is shown in Fig.
TABLE I Effect of a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine on the concentration of norepinephrine in different areas of the rat brain
Data show ,X _+ S.E.M. Units are ng of norepinephrine/mg protein. Number of observations shown in parentheses. In all cases, the lesioned or drug-treated group is significantly different from the corresponding control group (P < 0.001, two-tailed Student's t-test). Neonatal 6-hydroxydopamine
Hippocampus Parietal cortex Cerebellum
Locus coeruleus lesion
Control
Experimental
Control
Experimental
3.17 _+0.40 (12) 2.84 + 0.40 (10) 1.48 + 0.13 (11)
0.27 _+0.08 (12) 0.20 + 0.07 (12) 3.92 _+0.44 (11)
3.20 + 0.37 (8) 1.65 + 0.34 (8) 1.73 + 0.29 (8)
0.63 _+0.10 (10) 0.12 _+0.08 (10) 0.38 + 0.04 (10)
34
94.0--
,-?, 0
67.0
--
43.0
--
I.-
x I1" (9
l.U rr ....I ¢0 3 0 . 0 -ILl _.1 0
o 48
20.1 14.4
5.'0
610
710
pl Fig. 2. Representative gel obtained when tissue samples of rat brain were electrophoresed in two dimensions and then stained with silver. This particular gel was generated using tissue obtained from the parietal cortex, but is also representative of that seen using tissue from either the hippocampus or the cerebellum. For orientation purposes, the ordinate and the abscissa are marked for molecular weight and pI, respectively. In addition, each of the proteins found to be altered by either a bilateral LC lesion or by neonat~fl treatment with 6-OHDA, along with their permanent indexing numbers for rat brains.14, are indicated.
2. F o r orientation purposes, the ordinate and the abscissa are m a r k e d for molecular weight and isoelectric point (pI), respectively. As can be seen from this figure, only proteins within a limited pI range (5.0-7.0) and molecular weight range (100,000-10,000 daltons) were s e p a r a t e d under the conditions utilized. In spite of this, a large n u m b e r of distinct protein spots were well resolved on our twodimensional gels. Of the protein spots that were visible, 140 were judged to be sufficiently well resolved from both adjacent spots and the surrounding b a c k g r o u n d so that accurate densitometric m e a s u r e m e n t s could be made. Of these, a total of 11 different proteins were found to be altered in concentration in at least one of the brain areas examined as a result of either an elec-
trolytic lesion of the LC or neonatal treatment with 6O H D A . The location of each of these 11 proteins, along with their p e r m a n e n t indexing numbers for rat brainS: 4, are indicated in Fig. 2. In the hippocampus, a total of 4 different proteins were altered in concentration by one of the manipulations employed in this experiment (Table II). Of these 4 proteins, only one (no. 102) was affected by neonatal treatment with 6 - O H D A . This protein, with a molecular weight of 26,000 daltons and a pI of 5.0, was reduced 34% in concentration as a result of the catecholamine neurotoxin. Bilateral LC lesion elevated the concentration of two different proteins (nos. 8, 72) in the hippocampus between 26% and 30% over that obtained in corresponding control animals. A single protein (no. 48) was reduced 16% in
35 d e n s i t y in t h e h i p p o c a m p u s a f t e r t h e L C lesion. Neonatal treatment with 6-OHDA
p l 5.8) was r e d u c e d 3 1 % a n d p r o t e i n no. 19 ( m o l . w t .
affected the
47,000, pI 5.8) was r e d u c e d 2 0 % in a d u l t a n i m a l s
c o n c e n t r a t i o n o f 3 d i f f e r e n t p r o t e i n s in t h e p a r i e t a l
t r e a t e d at b i r t h w i t h t h e n e u r o t o x i n . In c o n t r a s t , n o
c o r t e x ( T a b l e III). P r o t e i n no. 2 ( m o l . w t . 65,000, pI
p r o t e i n s w e r e f o u n d t o b e a f f e c t e d in t h e p a r i e t a l cor-
5.8) was r e d u c e d 2 7 % , p r o t e i n n o . 3 ( m o l . w t . 64,000,
tex a f t e r a b i l a t e r a l l e s i o n o f t h e L C .
TABLE II
Proteins affected in concentration in the hippocampus after either neonatal treatment with 6-hydroxydopamine or a bilateral lesion o f the locus coeruleus Protein number corresponds to the numbering system of Heydorn et al. 8 and Jacobowitz and Heydorn TM. Data show ,X + S.E.M. Units are optical density x mm 2. Neonatal 6-hydroxydopamine: control, n = 8, experimental, n = 8; locus coeruleus lesion: control, n = 12, experimental, n = 10.
Protein number
M r ( x lO -3)
pl
Control
Experimental
% Change
Neonatal 6-hydroxydopamine 102 26
5.0
0.901 + 0.067
0.602 + 0.071"
-34
Locus coeruleus lesion 8 62 48 22 72 57
6.1 5.3 6.0
0.324 + 0.013 0.654 + 0.021 0.475 _+ 0.020
0.422 + 0.015"** 0.547 + 0.022** 0.601 + 0.035*
+30 -16 +26
* P < 0.01; ** P < 0.005; *** P < 0.001 compared to value obtained in corresponding control group.
TABLE III
Proteins affected in concentration in the parietal cortex after neonatal treatment with 6-hydroxydopamine No proteins were found to be altered in concentration in the parietal cortex after a bilateral lesion of the locus coeruleus. Protein number corresponds to the numbering system of Heydorn et al.8 and Jacobowitz and Heydorn 14. Data show X + S.E.M. Units are optical density x mm2. n = 8 for control and experimental groups.
Protein number
M, ( x lO-3)
pl
Control
Experimental
% Change
2 3 19
65 64 47
5.8 5.8 5.8
1.736 + 0.098 0.751 + 0.067 1.281 + 0.049
1.272 + 0.075** 0.518 + 0.032* 1.015 + 0.031"**
-27 -31 -20
* P < 0.01; ** P < 0.005; *** P < 0.001 compared to value obtained in corresponding control group.
TABLE IV
Proteins affected in concentration in the cerebellum after either neonatal treatment with 6-hydroxydopamine or a bilateral lesion o f the locus coeruleus Protein number corresponds to the numbering system of Heydorn et al. s and Jacobowitz and Heydorn t4. Data show X + S.E.M. Units are optical density x mm2. Neonatal 6-hydroxydopamine: control, n = 9, experimental, n = 8; locus coeruleus lesion: control, n = 8, experimental, n = 9.
Protein number
pl
Control
Experimental
% Control
Neonatal 6-hydroxydopamine 31 50 72 57
6.6 6.0
0.816 + 0.036 0.161 __+0.026
1.061 + 0.067** 0.270 + 0.018"*
+30 +68
Locus coeruleus lesion 4 67 37 36 110 30
5.9 6.7 6.3
0.319 + 0.018 0.231 + 0.019 0.054 + 0.009
0.239 + 0.011"* 0.317 + 0.013"* 0.086 + 0.005*
-25 +37 +59
M r ( X lO -3)
* P < 0.01 ; ** P < 0.005 compared to value obtained in corresponding control group.
3~ In the cerebellum. 5 different proteins were altered in concentration after either neonatal treatment with 6-OHDA or a bilateral LC lesion (Table IV). Of these, only a single protein (no. 4) was reduced in concentration (--25%), and this occurred after the LC lesion. Two other proteins (nos. 37, 110) were elevated in concentration in the cerebellum after bilateral LC lesioning. In addition, two proteins (nos. 31, 72) were also elevated in concentration by 30% and 68%, respectively, in adult rats treated neonatally with 6-OHDA.
DISCUSSION These results suggest that the apparent concentration of a number of different proteins in the central nervous system of the rat are regulated by the catecholamine neurotransmitter norepinephrine. Specifically, two weeks after electrolytically ablating the locus coeruleus, the concentration of 6 different proteins were found to be altered in either the hippocampus or the cerebellum. Of these 6 proteins, only two (nos. 4, 48) showed a decrease as a result of the lesion, while the 4 others (nos. 8, 37, 72, 110) showed an increase in concentration as a result of the lesion. No proteins were altered in the parietal cortex as a result of the lesion. In addition, 6 proteins were found to be altered in concentration in adult rats treated neonatally with 6-hydroxydopamine. Of these 6, only 2 proteins (both in the cerebellum) showed an increase in concentration as a result of the neurotoxin. The other 4 proteins (one in the hippocampus, 3 in the parietal cortex) were decreased in concentration in rats treated neonatally with the neurotoxin. It is unclear why most of the proteins affected in concentration by electrolytic lesions of the LC do not overlap with those affected in concentration by neonatal administration of 6-OHDA, even though both treatments produced the expected large changes in central levels of NE (see Table I). The reason for these differences may be related to the fact that adult animals received an LC lesion while only newborn pups received 6-OHDA. The central catecholamine system of the rat is immature at birth, and develops over the first 3-4 weeks of life 2°. Thus, in those neonatal pups receiving 6-OHDA, essentially no catecholamine innervation had ever existed in the cortex
and hippocampus. This suggests that the proleins found to be altered in concentration in adult rats treated with 6-OHDA require a catecholamme input during the neonatal development period for normal concentrations of these proteins to develop. If the NE input to cortex and hippocampus is removed in adulthood, the concentration of most of these proteins in brain is apparently unaffected. In contrast, removing the noradrenergic input to cortex and cerebellum in adulthood by electrolytically ablating the LC results in an alteration in the concentration of 6 different proteins. This suggests that these proteins are, under normal circumstances, regulated m concentration by NE in such a manner that removing the innervation results in an alteration in protein concentration. However, if the noradrenergic innervation to the cortex is not permitted to occur (by administering 6-OHDA to neonates), then the concentration of most of these proteins in adulthood does not differ between control and drug-treated animals. It may be that, in the neurotoxin-treated neonatal animals, another neurotransmitter system assumes control in regulating the concentration of these proteins. Further research into the overall biochemical effects of 6-OHDA will be necessary to determine the validity of such a hypothesis. In addition to the finding that there was little overlap of those proteins changed after an LC lesion with those proteins changed after neonatal 6-OHDA, it was also interesting that neonatal treatment with 6O H D A affected different proteins in the hippocampus, parietal cortex and cerebellum. Similarly, lesioning the LC had different effects in the hippocampus than in the cerebellum, with no effect in the parietal cortex. These results suggest that t h e regulation of the concentration of individual proteins in brain by NE is a complex matter. Under the most simplistic of conditions, one would expect that an LC lesion would alter the concentration of one or more proteins in all brain areas that have been affected by the electrolytic lesion. Similarly, neonatal treatment with 6-OHDA should have similar effects on brain areas that have been deprived of a noradrenergic innervation, and these effects would be opposite to those obtained in areas that are hyperinnervated. This, however, was not the case in the present study. The data presented here suggest that there is no single group of proteins in the CNS that are visible on
37 two-dimensional gels under the present experimental conditions that are regulated in concentration in all brain areas by NE. This may be because such proteins do not exist in brain. However, it may be that such proteins do exist in the CNS but were not visualized and analyzed in the present experiment. This may be because their isoelectric points and/or subunit molecular weights did not fall into the range selected for the present study. Additionally, it is possible that such proteins exist within these physico-chemical constraints, but are not visible on our two-dimensional gels because they exist in brain at a concentration below the sensitivity of the silver stain used to visualize proteins in the present experiment. In either event, as the data presented here suggest, no single group of proteins analyzed in the present experiment is regulated in a simplistic manner by NE. One question that remains to be addressed is the identity of the proteins found to be altered in concentration in the present experiment. To date, we have positively identified 2 of the 11 proteins found to change in the present experiment. One of these is protein no. 19, which appears to be the fl-isozyme of cytosolic (soluble) glutamic oxaloacetic transaminase (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)11, the enzyme responsible for catalyzing the interconversion of L-aspartate and a-ketoglutarate to oxaloacetate and glutamate. To date, there exist no reports demonstrating possible interrelationships between NE and this enzyme in brain. However, as the present data suggest, NE may play a role in regulating the concentration of this enzyme in the parietal cortex. A second protein positively identified and found to be elevated in the cerebellum after neonatal treatment with 6-OHDA is protein no. 31, which has been identified as the non-neuronal form of the enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11)10. There exist at least two possible explanations for this finding. First, it may be that more nonneuronal elements were obtained from cerebellar tissue of 6-OHDA-treated rats than from corresponding control rats. Such a hypothesis seems unlikely since no change was detected in the concentration of glial fibrillary acidic protein TM in the cerebellum after neonatal treatment with 6-OHDA. Second, it is possible that 6-OHDA retarded the natural conversion of non-neuronal enolase to neuron-specific enolase
that occurs in rats during cerebellar development22,27, resulting in a greater amount of nonneuronal enolase being detected in rats treated neonatally with the catecholamine neurotoxin. Such an argument is strengthened by the recent finding that enolase levels in adipose tissue may be regulated by catecholamines 29. The results presented in this report add to the list of proteins visible on two-dimensional gels generated using rat brain tissues that are apparently regulated in concentration by a specific neurotransmitter. More importantly, the present results suggest that different neurotransmitters, in addition to quantitatively affecting unique sets of proteins, also have qualitatively distinct effects on discrete proteins in the central nervous system. For example, it has been shown that electrolytic ablation of the nucleus of the tractus diagonalis (nucleus basalis), which provides the primary cholinergic innervation to the occipital cortex and the hippocampus 13, results in an alteration in the concentration of 4 different proteins in both of these brain areas12.Thus, it appears that acetylcholine influences the concentration of a discrete number of proteins in multiple areas of the central nervous system. In contrast, as the present results demonstrate, NE regulates the concentration of different proteins in different brain areas. In addition, there appears to be no overlap between those proteins regulated in concentration by the catecholamines and those regulated by acetylcholine 12. This information on neurotransmitter regulation of protein concentrations should prove valuable when attempting to interpret the effects of various pharmacologic investigations on the apparent concentration of individual proteins visible on two-dimensional gels. Future studies will be aimed at isolating and characterizing these proteins found to be regulated by specific neurotransmitters in an attempt to compile a database which can then be used in the study of specific neurobiological problems. ACKNOWLEDGEMENTS The expert secretarial assistance of Mrs. Lois Brown is gratefully acknowledged. William E. Heydorn was a Research Associate, Pharmacology-Toxicology Program, National Institute of General Medical Science at the time this research was performed.
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