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Regional effects of 6-hydroxydopamine on catecholamine containing neurones in rat brain and spinal cord
364
SHORT COMMUNICATIONS
Regional effects of 6-hydroxydopamine on catecholamine containing neurones in rat brain and spinal cord In previous studies we have shown that the injection of 6-hydroxydopamine (6-OHDA) into the cerebrospinal fluid of the rat brain leads to a long lasting depletion of noradrenaline (NA) and dopamine (DM) from the whole brain s. This was accompanied by a loss of tyrosine hydroxylase activity and aH-NA uptake sites in hyp0thalamus and striatum, suggesting that 6-OHDA induces degenerative changes in catecholamine-containing nerve terminals in the CNS s. We have now examined in more detail the effects of intraventricular injections of 6-OHDA on endogenous catecholamines, tyrosine hydroxylase activity and 8H-NA uptake in various regions of the rat CNS. The results show that such treatment causes a widespread loss of these components from all areas of the brain, and from spinal cord. Adult male albino Wistar rats received two intraventricular injections of 6OHDA (250 #g/dose) with a 48 h interval between doses, as described previously8. The animals were killed 16-25 days later and their brains removed and dissected according to the procedure of Glowinski and Iversen4. For catecholamine assays, brain tissue from 3-5 animals was pooled and NA or DM extracted by ion-exchange chromatography and assayed fluorimetrically8. Tyrosine hydroxylase activity was assayed by a modification of the method of McGeer et al.5, 8. The incubation contained in a final volume of 0.11 ml: potassium phosphate buffer pH 6.0, 0.4 M; NSD 1055, 0.25 mM; reduced pteridine cofactor (DMPH4), 1 mM; L-tyrosine (side chain, 2,3-3H), 20 #M; Triton X-100, 1 ~o; fl-mercaptoethanol, 0.4 M; 1:4 isotonic sucrose homogenate of brain, 50 #1. The samples were incubated for 15 min at 37°C, and the 8H-DOPA formed was isolated by adsorption and elution from alumina as described by McGeer et al. 5. This method was found to give higher values than that of McGeer et al. 5 which we had previously used. This is presumably due to release of particle-bound enzyme by Triton X-100, and the addition of cofactors. (-)-aH-NA uptake was measured in samples of brain homogenates incubated with the labelled amine (0.05 #M) for 5 min as described by Snyder and Coyle6. Values for control animals are presented in Table I, and the results obtained from 6-OHDA treated animals are expressed as percentages of the mean control values in Fig. 1. Endogenous NA concentrations were severely depressed in all brain regions (3-35 ~ of control) and in the spinal cord (10 ~ of control). The depletion of NA was most severe in cerebral cortex, where the NA concentration was reduced by more than 95 ~ . Despite the large amount of cortical tissue available for assay, the amounts of NA remaining in this region were barely detectable by the fluorimetric assay procedure used. The region least affected was the medulla-pons, in which the NA concentration remained at 34.5 ~o of the control value. Whereas NA in this region accounted for an average of 15 7oo of the total NA content of control rat brain, in treated animals almost 50~o of the remaining NA was present in the medulla-pons region. Endogenous dopamine could only be assayed reliably in the striatum of control animals, and in this area the endogenous dopamine was reduced by almost 95 ~ after 6-OHDA treatment. Brain Research, 24 (1970) 364--367
SHORT COMMUNICATIONS
365
TABLE I REGIONAL DISTRIBUTION OF ENDOGENOUS CATECHOLAMINES, TYROSINE HYDROXYLASE ACTIVITY AND 3H-NORADRENALINE UPTAKE IN CONTROL RAT BRAIN AND SPINAL CORD
Values are means and S.E.M. for 4-6 experiments, involvinganalysis of tissue from individual animals (tyrosine hydroxylase and [SH]noradrenaline uptake) or of pooled tissue from 3-5 rats (endogenous catecholamine determinations).
Brain region
Endogenous ?CA (l~g/g)
Hypothalamus Medulla-pons Mid-brain Cortex Hippocampus Cerebellum Spinal cord Striatum
1.31 ± 0.109 0.48 ± 0.028 0.42 4- 0.065 0.29 4- 0.024 0.25 ± 0.006 0.21 4- 0.035 0.25 ± 0.028 7.56 ± 0.460 (dopamine)
Tyrosine hydroxylase (nmoles DOPAIg/h)
6.5 4- 0.80 0.9 ± 0.25 1.4 ± 0.23 0.3 ± 0.05 0.5 4- 0.09 0.3 ± 0.05 3.4 ± 0.58 18.5 4- 2.47
[aH]Noradrenaline uptake (concentration ratio) o 6.0 -4-0.43 3.1 ± 0.69 4.3 ± 0.47 4.9 ± 0.57 4.5 ± 0.56 2.9 ± 0.45 2.9 ± 0.25 19.7 ± 2.54
i--j= ENDOGENOUS
CATECHOLAMINE
100
J~=
TYROSINE
B=
H3-NA
HYDROXYLASE
UPTAKE
90
'°!ii iii 7o
>e i
60
i ,
X i
-X i
t
O U i
-:3: I
ttl U i
~. ~ i
~-i
. . . . . . . . . . . .
50
000 4O
Fig. 1. Effects of 6-hydroxydopamine treatment on catecholamines (endogenous dopamine in striatum, N A all other regions), tyrosine hydroxylase and 8 H - N A uptake in various regions of rat CNS. Each value is expressed as a percentage of control (cf Table I), and is mean ± S.E. of 4-6 experiments. ~ = value too l o w to be accurately determined. A l l values are significantly lower than those of the corresponding controls (Table I).
T y r o s i n e hydroxylase activity was also significantly reduced in all regions o f the b r a i n a n d in the spinal cord; the loss o f enzyme activity varying from almost 90 in spinal cord to 50 % in m e d u l l a - p o n s , which was again the region least affected
Brain Research, 24 (1970) 364-367
366
SHORT COMMUNICATIONS
TABLE II EFFECTS OF
6-OHDA
TREATMENT ON TYROSINE HYDROXYLASE AND
aH-NA
UPTAKE IN RAT STRIATUM,
HYPOTHALAMUS AND MEDULLA-PONS COMPARISON OF RESULTS OBTAINED BY DIFFERENT METHODS
Comparison of values in 6-OHDA treated rats using two different methods for assay of tyrosine hydroxylase; and measurements of (-)-aH-NA uptake by homogenates (present results) or slices9 of various brain regions. Each value is expressed as percent of the corresponding control value, and is mean ± S.E. for 4-6 animals. Percent of control values
Hypothalamus Striatum Medulla-pons
Tyrosine hydroxylase
SaH] Noradrenaline uptake
Present results
Results of Uretsky and Iversen 8
Present results
Results of Uretsky et al. 9
30.5 ± 4.72 18.9 ± 2.70 49.4 ± 11.7
28.8 ± 3.20 25.0 ± 2.01 not measured
11.4 ± 3.1 29.7 ± 4.1 24.7 ± 7.5
15.5 ± 2.8 32.9 ± 7.5 46.0 ± 6.3*
* P < 0.05.
by the drug treatment. The levels of tyrosine hydroxylase activity in the cerebellum of treated animals were too low to be accurately measured. The uptake of aH-NA by homogenates of the various brain regions was also significantly depressed in 6 - O H D A treated animals (Fig. 1); the reduction varying from 9 0 ~ in mid-brain to 70 ~ in striatum. The results obtained for tyrosine hydroxylase and ZH-NA uptake in hypothalamus, striatum and medulla-pons in the present study are compared (Table II) with those previously obtained using slightly different methods. In general there is good agreement between these results, although 3H-NA uptake into slices of the medulla-pons region was less severely reduced by 6 - O H D A than the uptake of 3H-NA by synaptosomes prepared from this region (present results). We believe that this result, together with the relatively large amounts of endogenous N A and tyrosine hydroxylase remaining in this brain region may reflect the resistance of the cell body regions of NA-containing neurones to the destructive effects of 6-OHDA. The medullapons region contains most of the cell bodies of the NA-containing neurones in the brain 3. In slices the uptake of aH-NA may occur into both the terminals and cell body regions of surviving NA-neurones, whereas in homogenates 3H-NA uptake is measured only into nerve terminals, which are known from other studies to be more severely affected by 6 - O H D A treatment 7. This interpretation is also in accord with morphological evidence which suggests that the perikarya of NA-neurones in the rat brain survive 6 - O H D A treatment1, 2. In summary, our results confirm that 6 - O H D A has a long lasting effect on catecholamine neurones in the rat CNS. The present findings show that the drug, when injected into the lateral ventricle of the brain, affects such neurones in all parts of the brain and has a profound effect also on those in the spinal cord, suggesting Brain Research, 24 (1970) 364-367
367
SHORT COMMUNICATIONS
that intraventricularly injected 6 - O H D A distributes widely t h r o u g h o u t the CNS. N. J. Uretsky is a U.S. Public Health Service Post-doctoral Fellow.
Department of Pharmacology, University of Cambridge, Cambridge (Great Britain)
L. L. IVERSEN N. J. URETSKY
1 BARTHOLINI,G., RICHARDS, J. G., AND PLETSCHER, A., Dissociation between biochemical and ultrastructural effects of 6-hydroxydopamine in rat brain, Experientia (Basel), 26 (1970) 142-144. 2 BLOOM,F. E., ALGERI,S., GROPETTI,A., REVUELTA,A., AND COSTA,E., Lesions of central norepinephrine terminals with 6-OH-dopamine: biochemistry and fine structure, Science, 166 (1969) 1284-1286. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoaminc-containingneurons in the central nervous system, Acta physiol, scand., 62, Suppl. 232 (1964). 4 GLOWINSKI,J., AND IVERSEN, L. L., Regional studies of catecholamines in the rat brain. I. The disposition of Ha-norepinephrine, H3-dopamine and Ha-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 5 MCGEER, E. G., GIBSON, S., AND MCGEER, P. L., Some characteristics of brain tyrosine hydroxylase, Canad. J. Biochem., 45 (1967) 1557-1563. 6 SNYDER, S. H., AND COYLE, J. T., Regional differences in Ha-norepinephrine and Ha-dopamine uptake into rat brain homogenates, J. Pharmacol. exp. Ther., 165 (1969) 78-86. 7 THOENEN,H., ANDTRANZER,J. P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 261 (1968) 271-288. 8 URETSKY,N. J., AND IVERSEN,L. L., Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neuroehem., 17 (1970) 269-278. 9 URETSKY,N. J., SIMMONDS,m. A., AND IVERSEN,L. L., Changes in the retention and metabolism of Ha-L-norepinephrine in rat brain in vivo after 6-hydroxydopamine pretreatment, J. Pharmacol. exp. Ther., in press. (Accepted September 18th, 1970)
Brain Research, 24 (1970) 364-367
SHORT COMMUNICATIONS
Regional effects of 6-hydroxydopamine on catecholamine containing neurones in rat brain and spinal cord In previous studies we have shown that the injection of 6-hydroxydopamine (6-OHDA) into the cerebrospinal fluid of the rat brain leads to a long lasting depletion of noradrenaline (NA) and dopamine (DM) from the whole brain s. This was accompanied by a loss of tyrosine hydroxylase activity and aH-NA uptake sites in hyp0thalamus and striatum, suggesting that 6-OHDA induces degenerative changes in catecholamine-containing nerve terminals in the CNS s. We have now examined in more detail the effects of intraventricular injections of 6-OHDA on endogenous catecholamines, tyrosine hydroxylase activity and 8H-NA uptake in various regions of the rat CNS. The results show that such treatment causes a widespread loss of these components from all areas of the brain, and from spinal cord. Adult male albino Wistar rats received two intraventricular injections of 6OHDA (250 #g/dose) with a 48 h interval between doses, as described previously8. The animals were killed 16-25 days later and their brains removed and dissected according to the procedure of Glowinski and Iversen4. For catecholamine assays, brain tissue from 3-5 animals was pooled and NA or DM extracted by ion-exchange chromatography and assayed fluorimetrically8. Tyrosine hydroxylase activity was assayed by a modification of the method of McGeer et al.5, 8. The incubation contained in a final volume of 0.11 ml: potassium phosphate buffer pH 6.0, 0.4 M; NSD 1055, 0.25 mM; reduced pteridine cofactor (DMPH4), 1 mM; L-tyrosine (side chain, 2,3-3H), 20 #M; Triton X-100, 1 ~o; fl-mercaptoethanol, 0.4 M; 1:4 isotonic sucrose homogenate of brain, 50 #1. The samples were incubated for 15 min at 37°C, and the 8H-DOPA formed was isolated by adsorption and elution from alumina as described by McGeer et al. 5. This method was found to give higher values than that of McGeer et al. 5 which we had previously used. This is presumably due to release of particle-bound enzyme by Triton X-100, and the addition of cofactors. (-)-aH-NA uptake was measured in samples of brain homogenates incubated with the labelled amine (0.05 #M) for 5 min as described by Snyder and Coyle6. Values for control animals are presented in Table I, and the results obtained from 6-OHDA treated animals are expressed as percentages of the mean control values in Fig. 1. Endogenous NA concentrations were severely depressed in all brain regions (3-35 ~ of control) and in the spinal cord (10 ~ of control). The depletion of NA was most severe in cerebral cortex, where the NA concentration was reduced by more than 95 ~ . Despite the large amount of cortical tissue available for assay, the amounts of NA remaining in this region were barely detectable by the fluorimetric assay procedure used. The region least affected was the medulla-pons, in which the NA concentration remained at 34.5 ~o of the control value. Whereas NA in this region accounted for an average of 15 7oo of the total NA content of control rat brain, in treated animals almost 50~o of the remaining NA was present in the medulla-pons region. Endogenous dopamine could only be assayed reliably in the striatum of control animals, and in this area the endogenous dopamine was reduced by almost 95 ~ after 6-OHDA treatment. Brain Research, 24 (1970) 364--367
SHORT COMMUNICATIONS
365
TABLE I REGIONAL DISTRIBUTION OF ENDOGENOUS CATECHOLAMINES, TYROSINE HYDROXYLASE ACTIVITY AND 3H-NORADRENALINE UPTAKE IN CONTROL RAT BRAIN AND SPINAL CORD
Values are means and S.E.M. for 4-6 experiments, involvinganalysis of tissue from individual animals (tyrosine hydroxylase and [SH]noradrenaline uptake) or of pooled tissue from 3-5 rats (endogenous catecholamine determinations).
Brain region
Endogenous ?CA (l~g/g)
Hypothalamus Medulla-pons Mid-brain Cortex Hippocampus Cerebellum Spinal cord Striatum
1.31 ± 0.109 0.48 ± 0.028 0.42 4- 0.065 0.29 4- 0.024 0.25 ± 0.006 0.21 4- 0.035 0.25 ± 0.028 7.56 ± 0.460 (dopamine)
Tyrosine hydroxylase (nmoles DOPAIg/h)
6.5 4- 0.80 0.9 ± 0.25 1.4 ± 0.23 0.3 ± 0.05 0.5 4- 0.09 0.3 ± 0.05 3.4 ± 0.58 18.5 4- 2.47
[aH]Noradrenaline uptake (concentration ratio) o 6.0 -4-0.43 3.1 ± 0.69 4.3 ± 0.47 4.9 ± 0.57 4.5 ± 0.56 2.9 ± 0.45 2.9 ± 0.25 19.7 ± 2.54
i--j= ENDOGENOUS
CATECHOLAMINE
100
J~=
TYROSINE
B=
H3-NA
HYDROXYLASE
UPTAKE
90
'°!ii iii 7o
>e i
60
i ,
X i
-X i
t
O U i
-:3: I
ttl U i
~. ~ i
~-i
. . . . . . . . . . . .
50
000 4O
Fig. 1. Effects of 6-hydroxydopamine treatment on catecholamines (endogenous dopamine in striatum, N A all other regions), tyrosine hydroxylase and 8 H - N A uptake in various regions of rat CNS. Each value is expressed as a percentage of control (cf Table I), and is mean ± S.E. of 4-6 experiments. ~ = value too l o w to be accurately determined. A l l values are significantly lower than those of the corresponding controls (Table I).
T y r o s i n e hydroxylase activity was also significantly reduced in all regions o f the b r a i n a n d in the spinal cord; the loss o f enzyme activity varying from almost 90 in spinal cord to 50 % in m e d u l l a - p o n s , which was again the region least affected
Brain Research, 24 (1970) 364-367
366
SHORT COMMUNICATIONS
TABLE II EFFECTS OF
6-OHDA
TREATMENT ON TYROSINE HYDROXYLASE AND
aH-NA
UPTAKE IN RAT STRIATUM,
HYPOTHALAMUS AND MEDULLA-PONS COMPARISON OF RESULTS OBTAINED BY DIFFERENT METHODS
Comparison of values in 6-OHDA treated rats using two different methods for assay of tyrosine hydroxylase; and measurements of (-)-aH-NA uptake by homogenates (present results) or slices9 of various brain regions. Each value is expressed as percent of the corresponding control value, and is mean ± S.E. for 4-6 animals. Percent of control values
Hypothalamus Striatum Medulla-pons
Tyrosine hydroxylase
SaH] Noradrenaline uptake
Present results
Results of Uretsky and Iversen 8
Present results
Results of Uretsky et al. 9
30.5 ± 4.72 18.9 ± 2.70 49.4 ± 11.7
28.8 ± 3.20 25.0 ± 2.01 not measured
11.4 ± 3.1 29.7 ± 4.1 24.7 ± 7.5
15.5 ± 2.8 32.9 ± 7.5 46.0 ± 6.3*
* P < 0.05.
by the drug treatment. The levels of tyrosine hydroxylase activity in the cerebellum of treated animals were too low to be accurately measured. The uptake of aH-NA by homogenates of the various brain regions was also significantly depressed in 6 - O H D A treated animals (Fig. 1); the reduction varying from 9 0 ~ in mid-brain to 70 ~ in striatum. The results obtained for tyrosine hydroxylase and ZH-NA uptake in hypothalamus, striatum and medulla-pons in the present study are compared (Table II) with those previously obtained using slightly different methods. In general there is good agreement between these results, although 3H-NA uptake into slices of the medulla-pons region was less severely reduced by 6 - O H D A than the uptake of 3H-NA by synaptosomes prepared from this region (present results). We believe that this result, together with the relatively large amounts of endogenous N A and tyrosine hydroxylase remaining in this brain region may reflect the resistance of the cell body regions of NA-containing neurones to the destructive effects of 6-OHDA. The medullapons region contains most of the cell bodies of the NA-containing neurones in the brain 3. In slices the uptake of aH-NA may occur into both the terminals and cell body regions of surviving NA-neurones, whereas in homogenates 3H-NA uptake is measured only into nerve terminals, which are known from other studies to be more severely affected by 6 - O H D A treatment 7. This interpretation is also in accord with morphological evidence which suggests that the perikarya of NA-neurones in the rat brain survive 6 - O H D A treatment1, 2. In summary, our results confirm that 6 - O H D A has a long lasting effect on catecholamine neurones in the rat CNS. The present findings show that the drug, when injected into the lateral ventricle of the brain, affects such neurones in all parts of the brain and has a profound effect also on those in the spinal cord, suggesting Brain Research, 24 (1970) 364-367
367
SHORT COMMUNICATIONS
that intraventricularly injected 6 - O H D A distributes widely t h r o u g h o u t the CNS. N. J. Uretsky is a U.S. Public Health Service Post-doctoral Fellow.
Department of Pharmacology, University of Cambridge, Cambridge (Great Britain)
L. L. IVERSEN N. J. URETSKY
1 BARTHOLINI,G., RICHARDS, J. G., AND PLETSCHER, A., Dissociation between biochemical and ultrastructural effects of 6-hydroxydopamine in rat brain, Experientia (Basel), 26 (1970) 142-144. 2 BLOOM,F. E., ALGERI,S., GROPETTI,A., REVUELTA,A., AND COSTA,E., Lesions of central norepinephrine terminals with 6-OH-dopamine: biochemistry and fine structure, Science, 166 (1969) 1284-1286. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoaminc-containingneurons in the central nervous system, Acta physiol, scand., 62, Suppl. 232 (1964). 4 GLOWINSKI,J., AND IVERSEN, L. L., Regional studies of catecholamines in the rat brain. I. The disposition of Ha-norepinephrine, H3-dopamine and Ha-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 5 MCGEER, E. G., GIBSON, S., AND MCGEER, P. L., Some characteristics of brain tyrosine hydroxylase, Canad. J. Biochem., 45 (1967) 1557-1563. 6 SNYDER, S. H., AND COYLE, J. T., Regional differences in Ha-norepinephrine and Ha-dopamine uptake into rat brain homogenates, J. Pharmacol. exp. Ther., 165 (1969) 78-86. 7 THOENEN,H., ANDTRANZER,J. P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 261 (1968) 271-288. 8 URETSKY,N. J., AND IVERSEN,L. L., Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neuroehem., 17 (1970) 269-278. 9 URETSKY,N. J., SIMMONDS,m. A., AND IVERSEN,L. L., Changes in the retention and metabolism of Ha-L-norepinephrine in rat brain in vivo after 6-hydroxydopamine pretreatment, J. Pharmacol. exp. Ther., in press. (Accepted September 18th, 1970)
Brain Research, 24 (1970) 364-367