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Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain
Brain Research, 368 (1986) 193-196 Elsevier
193
BRE 21410
Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain WILLIAM E. HEYDORN, G. JOSEPH CREED, KHANH Q. NGUYEN and DAVID M. JACOBOWITZ Laboratory of Clinical Science, NIMH, Bethesda, MD 20892 (U.S.A.)
(Accepted November 5th, 1985) Key words: serotonin - - 5,7-dihydroxytryptamine- - central nervous system protein - two-dimensional gel electrophoresis - - computerized scanning densitometry
Proteins which are apparently regulated in concentration in two different areas of the rat brain by the indole neurotransmitter serotonin were identified using two-dimensional gel electrophoresis combined with computerized scanning densitometry. Reduction in central serotonin levels produced a decrease in the concentration of 3 different proteins (2 in the parietal cortex, 1 in the hippocampus). Two proteins, both in the hippocampus, were elevated in concentration following serotonin depletion. These results demonstrate that there exist in the brain a limited number of proteins whose concentration is influenced by serotonin.
Two-dimensional gel electrophoresis (2DE) coupled with highly sensitive silver stains and computerized scanning d e n s i t o m e t r y makes it possible to separate and quantitate a large n u m b e r of proteins from an extremely small tissue sample. Using this m e t h o d ology, H e y d o r n et al.S studied the proteins present within microdissected tissue samples t a k e n from discrete nuclei and other areas of the rat brain. Their results showed that there were few qualitative differences in protein patterns that could be detected among the brain areas studied. H o w e v e r , m a r k e d regional differences in the relative concentration of individual proteins were noted, with as many as 10% of the proteins that were e x a m i n e d varying by m o r e than 10-fold between brain areas. Recently, efforts in this area have focused on identifying the subsets of proteins present in the central nervous system (CNS) and visible on two-dimensional gels that are regulated in concentration by the classical neurotransmitters. Previous studies have shown that the disruption of the cholinergic input to cortex and hippocampus results in an alteration in the concentration of a limited n u m b e r of proteins in both of these brain areas 10. In contrast, destruction of the noradrenergic system in brain by either an electrolyt-
ic lesion of the locus coeruleus or neonatal administration of 6 - h y d r o x y d o p a m i n e produces a much more complex pattern of protein changes, with different brain areas exhibiting changes in different proteins 1~. The present data expands upon this previous work by reporting on the effects of intraventricular administration of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) on the concentration of individual proteins in the parietal cortex and the hippocampus. These results d e m o n s t r a t e that serotonin depletion, like n o r e p i n e p h r i n e depletion, produces changes in the a p p a r e n t concentration of different proteins in different brain areas. Male S p r a g u e - D a w l e y rats (Zivic-Miller L a b o r a tories, Allison Park, P A ) were used for these studies. On the day of the e x p e r i m e n t , rats were divided into 2 groups. O n e group of animals received an intraperitoneal injection of d e s m e t h y l i m i p r a m i n e (25 mg free base/kg body weight), while control rats received 0.9% NaCI. Previous studies have shown that the norepinephrine-depleting effect of high doses of 5,7-DHT can be p r e v e n t e d by p r e t r e a t m e n t with desmethylimipramine~. In addition, a single dose of desmethylimipramine has been shown to have no effect on the concentration of individual proteins in rat
Correspondence: W.E. Heydorn, Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892, U.S.A.
! c)4
brain `). One hour later, animals were anesthetized with Chloropent anesthesia (0.2-0.3 rnlll()t)g bodx weight). Animals pretreated with desmethylimipramine received an injection of 5,7-DHT (200 ,ug of free base in 20 !H of ().2(D: ascorbic acid dissolved in saline) into the lateral ventricle. Control animals received an equal amount of vehicle. Animals were then allowed to recover from anesthesia. Eight days later, all rats were killed by decapitation. and their brains quickly removed. A piece of tissue was removed from the occipital cortex, weighed and frozen for subsequent analysis of serotonin content according to the procedure of Chiueh el al. <5. The remaining brain tissue was frozen in powdered dry ice. Brains were then cut into 300 !zm thick coronal sections using a cryostat. Tissue samples were dissected from these frozen sections using the micropunch technique of Palkovits >. Four punches (1000 # m each) were taken from the hippocampus at coordinate A 3430 and from the parietal cortex at coordinate A 6860 (ref. 14). These tissue samples (approximately 0.15 mg of protein each) were immediately placed into 30ul of sample detergent solution s consisting of fi-mercaptoethanol, glycerol, ampholytes and Nonidet Pa,, briefly sonicated and then frozen. 2DE was performed according to the procedure of O'Farrel115 with minor modifications for neuronal tissueS. lsoelectric focusing in the first dimension was accomplished using tube gels t27.5 × 2.5 mm in size made of 4.1% acrylamide, 8.9 M urea and 2% ampholytes (4:1 ratio of 5/7 and 3/10). In the second dimension, proteins were separated by mass using slab gels 140 x 110 x 0.8 mm in size made of 10% acrylamide, 0.1% sodium dodecyl sulfate and 0.375 M Tris, pH 8.8. Gels were stained with silver ~ and the optical density of individual protein spots determined by computerized scanning densitometry 7. All data were analyzed using a two-tailed Student's/-test. Intraventricular injection of 5,7-DHT produced, as expected, a marked reduction in central serotonin levels. In control rats, the tissue concentration of serotonin (expressed as pg/mg wet weight _+ S.E.M.) averaged 292.9 _+ 9.6 (n = 8). In rats treated with 5,7-DHT, serotonin levels were reduced 86% to 41.0 _+ 9.9 (n = 12; P < 0,001 compared to control). Having established that central serotonin levels were reduced following intraventricular administration of 5,7-DHT, tissue samples from the parietal
cortex and the hippocampus ~crc lelllO~.cci 1~)I ;lll;.I.t'~ sis bv 2DE. A representative gel generated t~r lhcsc studies is shown in Fig. 1. For orientatiol~ purposes, the ordinate and the abscissa are marked ior molecular weight and isoelectric point (pit, rc,,pecllv¢lx Over 200 proteins were sufficiently well resolved thai accurate densitometric measurements coukl i~e made. In addition, on this figure are noted the Iocalion and permanent indexing numbers." ~: ~,f the 5 proteins found to be altered m concentration m either the parietal cortex or the hippocampus tiHlowmg intraventricular administration of 5,7-DHT. As can be seen in Table I, two proteins were reduced m concentration in the parietal cortex bv 5.7-DHT. In the hippocampus, the serotonergic neurotoxin elevated the concentration of two different proteins, while reducing the concentration of a single protein. There was no overlap in the proteins found to be altered in the two brain areas examined. These results add to the list of proteins present m discrete areas of the rat brain that are apparently regulated in concentration by one of the classical neurotransmitters. The pattern of changes noted here is qualitatively similar to that obtained after depletion of norepinephrine concentrations in the CNS in that there was little overlap in the protein changes in the different brain areas following norepinephrme depletion l~. In contrast, ablation of the cholinergic input to cortex and hippocampus by electrolytic lesions of the nucleus of the tractus diagonalis produced changes in the concentration of the same limited number of proteins in both of these brain areas > . Thus, it appears that acetylcholine regulates the concentration of a subset of proteins in more than one brain area. In contrast, the effects of both serotonm and norepinephrine are somewhat more complex, with the pattern of protein changes obtained dependent upon the brain area examined. The one protein found to be reduced in concentration in the hippocampus of rats receiving an intraventricular injection of 5,7-DHT was protein no. 102 (see Table I). Previous studies have shown that the amount of this protein present in hippocampus is also influenced by norepinephrine II. Lesioning of the locus coeruleus in adult rats produced no change in the concentration of this protein. However, if neonatal rat pups are treated with 6-hydroxydopamine, the concentration of protein no, 102 in the hippocampus
195
94.0o-J
t 0
67.0
x I-"1" (.9
43.0
LU
< ..J
30.0
LU ..J ..........
.........
03; 20. 1-14.4-5.0
6.0
7.0
pl Fig. 1. Representative gel generated when proteins contained within tissue samples taken from either the parietal cortex or the hippocampus are separated by two-dimensional gel electrophoresis and stained with silver. 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 intraventricular administration of 5,7-dihydroxytryptamine, along with their permanent indexing numbers for rat brain s,12 are indicated.
o f t h e r e s u l t i n g a d u l t rats is significantly less t h a n t h a t
102 in t h e h i p p o c a m p u s m a y be r e g u l a t e d by b o t h
o f c o n t r o P 1. S u c h a f i n d i n g , c o m b i n e d w i t h t h e p r e s -
n o r e p i n e p h r i n e a n d s e r o t o n i n . S u c h a f i n d i n g is o f in-
e n t r e s u l t s , s u g g e s t s t h a t t h e a m o u n t o f p r o t e i n no.
t e r e s t in light o f r e c e n t s t u d i e s w h i c h h a v e i n d i c a t e d
TABLE I Effect o f 5, 7-dihydroxytryptamine administration on the relative concentration o f individual proteins in different regions ~f the rat brain Protein number"
pl
Molecular weight (× 10 --7)
Control (n = 8)
5, 7-DHT (n = 9)
q# Change
(A) Parietal cortex 1 199
5.6 5.2
94 26
0.156 _+ 0.010 b 0.217 _+ 0.012
0.121 +_ 0.0(19"* 0.158 _+ 0.018"*
-22 -27
(B) Hippocampus 102 129 156
5.0 6.6 6.4
26 62 62
0.455 + 0.029 0.222 _+ 0.019 0.071 + 0.005
0.348 + 0.038* 0.276 + 0.016" 0.096 -+ 0.006***
24 +24 +35
* P < 0.05, ** P < 0.025, *** P < 0.005 when compared with corresponding control value. " Corresponds to the numbering system of Heydorn et al.K and Jacobowitz and Heydorn ~2. b Units are optical density × millimeters squared 7.
that there may exist a reciprocal functional relationship between certain noradrenergic aim serotonergic axons in the CNS. For example, it has beel~ shown
that the Dx-dopamine receptor has ,~ n~tccula~ weight of 94,000 daltons i. a ~alue idcntic:d it> tb,at
/#adrenergic receptors that occurs after repeated
calculated for protein no. 1 !see Tahlc It suggest-, that this protein may be related to the 1) -dopammc receptor. The present data also suggest that tt'~c a m o u n t of protein no. 1 present in the parietal cortex
administration of certain antidepressant compounds 3,13. Thus, the similarity of the effects of neo-
is regulated by the level of serotonin m that brain area. Such data indicate that the serot~mcrgic sy',-
natal treatment with 6-hydroxydopamine and intraventricular injection of 5,7-DHT on the concentra-
tern, in addition to influencing noradrencrgic neurons in the CNS, may interact with d o p a m m e recep-
tion of protein no. 102 in the hippocampus may be a reflection of the interaction between the noradrenergic
tors in different brain regions, In summary, there appear to exist at least 5 prote-
and the serotonergic systems in the CNS.
ins within the CNS which are regulated m concentra-
Examination of Table I reveals that protein no. 1 is reduced in concentration, following 5 , 7 - D H T administration. This protein was also found to be reduced in
tion by serotonin. Future studies will be aimed at
lhat an intact central serotonergic input is necessar\ to demonstrate the usual reduction m the n u m b e r of
concentration in the nucleus accumbens of rats fed a diet low in iron (Youdim et al., manuscript m preparation). Biochemical studies done in brain tissue taken from rats deficient in iron has revealed a decrease in the n u m b e r of D2-dopamine binding sites, with no change in a-adrenergic, fl-adrenergic, muscarinic or benzodiazepine binding sites e. The recent finding 1 Amlaiky, N. and Caron, M.G., Photoaffinity labeling of the D2-dopamine receptor using a novel high-affinity radioiodinated probe, J, Biol. Chem., 260 (1985) 1983-1986. 2 Ashkenazi, R., Ben-Shachar, D. and Youdim, M.B.H., Nutritional iron and dopamine binding sites in the rat brain, Pharrnacol. Biochern. Behav.. 17 (Suppl. 1) (1982) 43-47. 3 Brunello, N., Barbaccia, M,L., Chuang, D. and Costa. E., Down-regulation of fi-adrenergic receptors following repeated injections of desmethylimipraminc: permissive role of serotonergic axons, Neuropharmacology, 21 (1982) 1145-1149. 4 Chiueh, (7.('., Zukowska-Grojcc, Z.. Kirk, K.L. and Kopin, I.J., 6-Fluorocatecholamines as false adrenergic neurotransmitters, J. Pharrnacol. Exp. Ther., 225 (1983) 529-533. 5 Chiueh, C.(,, Markey, S.P., Burns, R.S., Johannessen, J.N., Pert, A. and Kopin, I.J., Neurochemical and behavioral effects of systemic and intranigral administration of Nmethyl-4-phenyl-l,2,3,6-tetrahydropyridine in the rat, Eur. J. Pharrnacol.. 100 (1984) 189-194, 6 Gerson, S. and Baldessarini, R.J., Selective destruction of serotonin terminals in rat forebrain by high doses of 5,7-dihydroxytryptamine, Brain Research, 85 (1975) 140-145. 7 Goldman, D., Merril, C.R., Polinsky, R.J. and Ebert, M H . . Lymphyocyte proteins in Huntington's disease: quantitative analysis by use of two-dimensional electrophoresis and computerized densitometry, Clin. (7hem., 28 (1982) 1021-1025. 8 Heydorn, W.E., Creed, G.J., Goldman, D., Kanter, D., Merril, CR. and Jacobowitz, D., Mapping and quantitalion of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis. J Neuro-
learning more about these and the other proteins which are apparently regulated in concentration by the classical neurotransmitters. The authors would like to thank Dr. Chuang C. Chiueh for determining serotonin tissue concentrations for this study. The expert secretarial assistance of Mrs. Lois Brown is also gratefully acknowledged.
sci., 3 (1983) 2597-261)6. 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 electrophoresis, J. Pharrnacol. Exp. Ther., 229 (1984) 622-628. 10 Heydorn, W.E., Nguyen, K.Q., Creed, G.J, and Jacob()witz, 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. 11 Heydorn, W.E., Nguyen, K.Q., Creed, G.J., Kostrzewa, R.M. and Jacobowitz, D.M., 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, in press. 12 Jacobowitz, D.M. and Heydorn, W.E., Two-dimensional gel electrophoresis used in neurobiological studies of proteins in discrete areas of the rat brain, Clin. Chern., 30 (1984) 1996-2002. 13 Janowsky, A., Okada, F., Manier, D.H., Applegate, C.D., Sulser, F. and Steranka, L.R,, Role of serotonergic input in the regulation of the fl-adrenergic receptor-coupled adenylate cyclase system, Science, 218 (1982) 900-901. 14 K6nig, J.F.R. and Klippel, R.A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stern, Williams and Wilkins, Baltimore, 1963.
15 O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250 (1975) 4007-402t. 16 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450.
193
BRE 21410
Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain WILLIAM E. HEYDORN, G. JOSEPH CREED, KHANH Q. NGUYEN and DAVID M. JACOBOWITZ Laboratory of Clinical Science, NIMH, Bethesda, MD 20892 (U.S.A.)
(Accepted November 5th, 1985) Key words: serotonin - - 5,7-dihydroxytryptamine- - central nervous system protein - two-dimensional gel electrophoresis - - computerized scanning densitometry
Proteins which are apparently regulated in concentration in two different areas of the rat brain by the indole neurotransmitter serotonin were identified using two-dimensional gel electrophoresis combined with computerized scanning densitometry. Reduction in central serotonin levels produced a decrease in the concentration of 3 different proteins (2 in the parietal cortex, 1 in the hippocampus). Two proteins, both in the hippocampus, were elevated in concentration following serotonin depletion. These results demonstrate that there exist in the brain a limited number of proteins whose concentration is influenced by serotonin.
Two-dimensional gel electrophoresis (2DE) coupled with highly sensitive silver stains and computerized scanning d e n s i t o m e t r y makes it possible to separate and quantitate a large n u m b e r of proteins from an extremely small tissue sample. Using this m e t h o d ology, H e y d o r n et al.S studied the proteins present within microdissected tissue samples t a k e n from discrete nuclei and other areas of the rat brain. Their results showed that there were few qualitative differences in protein patterns that could be detected among the brain areas studied. H o w e v e r , m a r k e d regional differences in the relative concentration of individual proteins were noted, with as many as 10% of the proteins that were e x a m i n e d varying by m o r e than 10-fold between brain areas. Recently, efforts in this area have focused on identifying the subsets of proteins present in the central nervous system (CNS) and visible on two-dimensional gels that are regulated in concentration by the classical neurotransmitters. Previous studies have shown that the disruption of the cholinergic input to cortex and hippocampus results in an alteration in the concentration of a limited n u m b e r of proteins in both of these brain areas 10. In contrast, destruction of the noradrenergic system in brain by either an electrolyt-
ic lesion of the locus coeruleus or neonatal administration of 6 - h y d r o x y d o p a m i n e produces a much more complex pattern of protein changes, with different brain areas exhibiting changes in different proteins 1~. The present data expands upon this previous work by reporting on the effects of intraventricular administration of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) on the concentration of individual proteins in the parietal cortex and the hippocampus. These results d e m o n s t r a t e that serotonin depletion, like n o r e p i n e p h r i n e depletion, produces changes in the a p p a r e n t concentration of different proteins in different brain areas. Male S p r a g u e - D a w l e y rats (Zivic-Miller L a b o r a tories, Allison Park, P A ) were used for these studies. On the day of the e x p e r i m e n t , rats were divided into 2 groups. O n e group of animals received an intraperitoneal injection of d e s m e t h y l i m i p r a m i n e (25 mg free base/kg body weight), while control rats received 0.9% NaCI. Previous studies have shown that the norepinephrine-depleting effect of high doses of 5,7-DHT can be p r e v e n t e d by p r e t r e a t m e n t with desmethylimipramine~. In addition, a single dose of desmethylimipramine has been shown to have no effect on the concentration of individual proteins in rat
Correspondence: W.E. Heydorn, Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892, U.S.A.
! c)4
brain `). One hour later, animals were anesthetized with Chloropent anesthesia (0.2-0.3 rnlll()t)g bodx weight). Animals pretreated with desmethylimipramine received an injection of 5,7-DHT (200 ,ug of free base in 20 !H of ().2(D: ascorbic acid dissolved in saline) into the lateral ventricle. Control animals received an equal amount of vehicle. Animals were then allowed to recover from anesthesia. Eight days later, all rats were killed by decapitation. and their brains quickly removed. A piece of tissue was removed from the occipital cortex, weighed and frozen for subsequent analysis of serotonin content according to the procedure of Chiueh el al. <5. The remaining brain tissue was frozen in powdered dry ice. Brains were then cut into 300 !zm thick coronal sections using a cryostat. Tissue samples were dissected from these frozen sections using the micropunch technique of Palkovits >. Four punches (1000 # m each) were taken from the hippocampus at coordinate A 3430 and from the parietal cortex at coordinate A 6860 (ref. 14). These tissue samples (approximately 0.15 mg of protein each) were immediately placed into 30ul of sample detergent solution s consisting of fi-mercaptoethanol, glycerol, ampholytes and Nonidet Pa,, briefly sonicated and then frozen. 2DE was performed according to the procedure of O'Farrel115 with minor modifications for neuronal tissueS. lsoelectric focusing in the first dimension was accomplished using tube gels t27.5 × 2.5 mm in size made of 4.1% acrylamide, 8.9 M urea and 2% ampholytes (4:1 ratio of 5/7 and 3/10). In the second dimension, proteins were separated by mass using slab gels 140 x 110 x 0.8 mm in size made of 10% acrylamide, 0.1% sodium dodecyl sulfate and 0.375 M Tris, pH 8.8. Gels were stained with silver ~ and the optical density of individual protein spots determined by computerized scanning densitometry 7. All data were analyzed using a two-tailed Student's/-test. Intraventricular injection of 5,7-DHT produced, as expected, a marked reduction in central serotonin levels. In control rats, the tissue concentration of serotonin (expressed as pg/mg wet weight _+ S.E.M.) averaged 292.9 _+ 9.6 (n = 8). In rats treated with 5,7-DHT, serotonin levels were reduced 86% to 41.0 _+ 9.9 (n = 12; P < 0,001 compared to control). Having established that central serotonin levels were reduced following intraventricular administration of 5,7-DHT, tissue samples from the parietal
cortex and the hippocampus ~crc lelllO~.cci 1~)I ;lll;.I.t'~ sis bv 2DE. A representative gel generated t~r lhcsc studies is shown in Fig. 1. For orientatiol~ purposes, the ordinate and the abscissa are marked ior molecular weight and isoelectric point (pit, rc,,pecllv¢lx Over 200 proteins were sufficiently well resolved thai accurate densitometric measurements coukl i~e made. In addition, on this figure are noted the Iocalion and permanent indexing numbers." ~: ~,f the 5 proteins found to be altered m concentration m either the parietal cortex or the hippocampus tiHlowmg intraventricular administration of 5,7-DHT. As can be seen in Table I, two proteins were reduced m concentration in the parietal cortex bv 5.7-DHT. In the hippocampus, the serotonergic neurotoxin elevated the concentration of two different proteins, while reducing the concentration of a single protein. There was no overlap in the proteins found to be altered in the two brain areas examined. These results add to the list of proteins present m discrete areas of the rat brain that are apparently regulated in concentration by one of the classical neurotransmitters. The pattern of changes noted here is qualitatively similar to that obtained after depletion of norepinephrine concentrations in the CNS in that there was little overlap in the protein changes in the different brain areas following norepinephrme depletion l~. In contrast, ablation of the cholinergic input to cortex and hippocampus by electrolytic lesions of the nucleus of the tractus diagonalis produced changes in the concentration of the same limited number of proteins in both of these brain areas > . Thus, it appears that acetylcholine regulates the concentration of a subset of proteins in more than one brain area. In contrast, the effects of both serotonm and norepinephrine are somewhat more complex, with the pattern of protein changes obtained dependent upon the brain area examined. The one protein found to be reduced in concentration in the hippocampus of rats receiving an intraventricular injection of 5,7-DHT was protein no. 102 (see Table I). Previous studies have shown that the amount of this protein present in hippocampus is also influenced by norepinephrine II. Lesioning of the locus coeruleus in adult rats produced no change in the concentration of this protein. However, if neonatal rat pups are treated with 6-hydroxydopamine, the concentration of protein no, 102 in the hippocampus
195
94.0o-J
t 0
67.0
x I-"1" (.9
43.0
LU
< ..J
30.0
LU ..J ..........
.........
03; 20. 1-14.4-5.0
6.0
7.0
pl Fig. 1. Representative gel generated when proteins contained within tissue samples taken from either the parietal cortex or the hippocampus are separated by two-dimensional gel electrophoresis and stained with silver. 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 intraventricular administration of 5,7-dihydroxytryptamine, along with their permanent indexing numbers for rat brain s,12 are indicated.
o f t h e r e s u l t i n g a d u l t rats is significantly less t h a n t h a t
102 in t h e h i p p o c a m p u s m a y be r e g u l a t e d by b o t h
o f c o n t r o P 1. S u c h a f i n d i n g , c o m b i n e d w i t h t h e p r e s -
n o r e p i n e p h r i n e a n d s e r o t o n i n . S u c h a f i n d i n g is o f in-
e n t r e s u l t s , s u g g e s t s t h a t t h e a m o u n t o f p r o t e i n no.
t e r e s t in light o f r e c e n t s t u d i e s w h i c h h a v e i n d i c a t e d
TABLE I Effect o f 5, 7-dihydroxytryptamine administration on the relative concentration o f individual proteins in different regions ~f the rat brain Protein number"
pl
Molecular weight (× 10 --7)
Control (n = 8)
5, 7-DHT (n = 9)
q# Change
(A) Parietal cortex 1 199
5.6 5.2
94 26
0.156 _+ 0.010 b 0.217 _+ 0.012
0.121 +_ 0.0(19"* 0.158 _+ 0.018"*
-22 -27
(B) Hippocampus 102 129 156
5.0 6.6 6.4
26 62 62
0.455 + 0.029 0.222 _+ 0.019 0.071 + 0.005
0.348 + 0.038* 0.276 + 0.016" 0.096 -+ 0.006***
24 +24 +35
* P < 0.05, ** P < 0.025, *** P < 0.005 when compared with corresponding control value. " Corresponds to the numbering system of Heydorn et al.K and Jacobowitz and Heydorn ~2. b Units are optical density × millimeters squared 7.
that there may exist a reciprocal functional relationship between certain noradrenergic aim serotonergic axons in the CNS. For example, it has beel~ shown
that the Dx-dopamine receptor has ,~ n~tccula~ weight of 94,000 daltons i. a ~alue idcntic:d it> tb,at
/#adrenergic receptors that occurs after repeated
calculated for protein no. 1 !see Tahlc It suggest-, that this protein may be related to the 1) -dopammc receptor. The present data also suggest that tt'~c a m o u n t of protein no. 1 present in the parietal cortex
administration of certain antidepressant compounds 3,13. Thus, the similarity of the effects of neo-
is regulated by the level of serotonin m that brain area. Such data indicate that the serot~mcrgic sy',-
natal treatment with 6-hydroxydopamine and intraventricular injection of 5,7-DHT on the concentra-
tern, in addition to influencing noradrencrgic neurons in the CNS, may interact with d o p a m m e recep-
tion of protein no. 102 in the hippocampus may be a reflection of the interaction between the noradrenergic
tors in different brain regions, In summary, there appear to exist at least 5 prote-
and the serotonergic systems in the CNS.
ins within the CNS which are regulated m concentra-
Examination of Table I reveals that protein no. 1 is reduced in concentration, following 5 , 7 - D H T administration. This protein was also found to be reduced in
tion by serotonin. Future studies will be aimed at
lhat an intact central serotonergic input is necessar\ to demonstrate the usual reduction m the n u m b e r of
concentration in the nucleus accumbens of rats fed a diet low in iron (Youdim et al., manuscript m preparation). Biochemical studies done in brain tissue taken from rats deficient in iron has revealed a decrease in the n u m b e r of D2-dopamine binding sites, with no change in a-adrenergic, fl-adrenergic, muscarinic or benzodiazepine binding sites e. The recent finding 1 Amlaiky, N. and Caron, M.G., Photoaffinity labeling of the D2-dopamine receptor using a novel high-affinity radioiodinated probe, J, Biol. Chem., 260 (1985) 1983-1986. 2 Ashkenazi, R., Ben-Shachar, D. and Youdim, M.B.H., Nutritional iron and dopamine binding sites in the rat brain, Pharrnacol. Biochern. Behav.. 17 (Suppl. 1) (1982) 43-47. 3 Brunello, N., Barbaccia, M,L., Chuang, D. and Costa. E., Down-regulation of fi-adrenergic receptors following repeated injections of desmethylimipraminc: permissive role of serotonergic axons, Neuropharmacology, 21 (1982) 1145-1149. 4 Chiueh, (7.('., Zukowska-Grojcc, Z.. Kirk, K.L. and Kopin, I.J., 6-Fluorocatecholamines as false adrenergic neurotransmitters, J. Pharrnacol. Exp. Ther., 225 (1983) 529-533. 5 Chiueh, C.(,, Markey, S.P., Burns, R.S., Johannessen, J.N., Pert, A. and Kopin, I.J., Neurochemical and behavioral effects of systemic and intranigral administration of Nmethyl-4-phenyl-l,2,3,6-tetrahydropyridine in the rat, Eur. J. Pharrnacol.. 100 (1984) 189-194, 6 Gerson, S. and Baldessarini, R.J., Selective destruction of serotonin terminals in rat forebrain by high doses of 5,7-dihydroxytryptamine, Brain Research, 85 (1975) 140-145. 7 Goldman, D., Merril, C.R., Polinsky, R.J. and Ebert, M H . . Lymphyocyte proteins in Huntington's disease: quantitative analysis by use of two-dimensional electrophoresis and computerized densitometry, Clin. (7hem., 28 (1982) 1021-1025. 8 Heydorn, W.E., Creed, G.J., Goldman, D., Kanter, D., Merril, CR. and Jacobowitz, D., Mapping and quantitalion of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis. J Neuro-
learning more about these and the other proteins which are apparently regulated in concentration by the classical neurotransmitters. The authors would like to thank Dr. Chuang C. Chiueh for determining serotonin tissue concentrations for this study. The expert secretarial assistance of Mrs. Lois Brown is also gratefully acknowledged.
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