308
Brain Research, 528 (1990) 308-310
Elsevier
BRES 24296
Short Communications
The effect of aging on the
D1
dopamine receptors in human frontal cortex
Jacques de Keyser!, Jean-Paul De Backer 2, Georges Vauquelin 2 and Guy Ebinger 1 1Department of Neurology, Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels (Belgium) and eProtein Chemistry Laboratory, Vrije Universiteit Brussel, St. Genesius Rode (Belgium)
(Accepted 12 June 1990) Key words: Dopaminergic; Dopamine receptor, D1; Aging; Cerebral cortex; Human brain
Dopaminergic pathways to the cerebral cortex may be involved in cognitive function. We examined the effect of aging on the D1 dopamine receptors, and their high-agonist affinity (RH) sites, in postmortem human frontal cortex (n = 32; age range, 19-88 years). With aging, there was a significant decrease in the densities of the D1 dopamine receptors, and their RH sites, in human frontal cortex. The age-related reduction of cortical dopaminergic neurotransmission might contribute to the decline in cognitive abilities of elderly persons. Similar to rodents 8, the human cerebral cortex receives a widespread dopaminergic innervation from the midbrain 4'9. Dopamine receptors in human cerebral cortex are mainly of the DI type 3'5. The D 1 receptors in human frontal cortex exhibit similar affinities for antagonists of different chemical classes to those in the striatum 6. They are also composed of high affinity (RH) and low affinity (RL) sites for dopamine 6. However, in contrast to striatum and amygdala, the R H sites of the D 1 receptors in human frontal cortex are not converted into RL sites in the presence of GTP, and their activation by dopamine is not associated with a stimulation of the adenylate cyclase enzyme 6'7. For adenylate cyclase-coupled receptors, the RH sites are believed to represent receptors that interact with the guanine nucleotide binding protein G s 11. In analogy with the cyclase-coupled D~ receptors, it is tempting to assume that the GTP-resistant RH-sites of the D 1 receptors in human frontal cortex may also represent the physiologically relevant receptors. The 'normal' human aging process is associated with a decline in cognitive abilities, especially of memory ~4. With aging, there is a reduction in cortical acetylcholine and norepinephrine, which are neurotransmitters that are thought to play an important role in memory and learning 14. Studies in animals and in patients with Parkinson's disease suggest that dopaminergic projections to the cerebral cortex may also be implicated in cognitive function ~°'12'a7'18. However, the dopamine content in human neocortical regions does not significantly decrease
with age 1. In the present study, we assessed the effect of aging on the density of the I) 1 receptors, and their RH sites, in human frontal cortex. Brains were obtained at autopsy from 32 patients (aged 19-88 years; 12 females and 20 males), who had died without evidence of neurologic or psychiatric disease. The brains were stored at -80 °C. Postmortem delay ranged from 3 to 24 h (mean 13 + 4 h). None of the patients had received dopamine agonists or antipsychotics. Frontal cortex (area 9) was dissected at 4 °C, and subsequently homogenized in 20 mM Tris-HCl (pH 7.4)/120 mM NaCI/2 mM MgC12. The homogenates were centrifuged in a Sorvall centrifuge at 30,000 g for 20 min. The membranes were washed twice with repeated centrifugations at 30,000 g for 15 min. The supernatant was discarded and the pellet resuspended in the same buffer containing 10% glycerol (vol/vol). Until the radioligand binding experiments, the samples were stored in liquid nitrogen. Protein concentrations were determined by the method of Lowry et al. 13. Binding assays with [3H]SCH 23390 ((5)-(+)-8-chloro2,3,4,5-tetrahydro-3-methyl-5-phenyl-l-N-3°benzazepine; 85 Ci/mmol; Amersham) were done in duplicate in 0.5 ml of 20 mM Tris-HCl (pH 7.4)/120 mM NaC1/2 mM MgC12, containing 0.6 mg/ml of membrane protein. Saturation bindings were performed with 6 concentrations of [3H]SCH 23390 ranging from 0.5 to 6 nM, and 2 nM of [3H]SCH 23390 was used for competition binding experiments with dopamine (performed in the presence of 10 /~M pargyline). Spiperone (100 nM) was included in the assays to prevent binding to 5-HT2 serotonin receptors.
Correspondence: J. De Keyser, Department of Neurology, Academisch Ziekenhuis Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
309 ~I00 0 r~
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250"
=~ ;
200
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40
6'0
8'0
100
T
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-8
-'7
-6
-'5
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DOPAMINE CONCENTRATION(LOG M)
Fig. 1. Dopamine competition for the specific binding of [3H]SCH 23390 to D~ dopamine receptors in human cortex membranes. A representative curve is shown. The curve is computer-fitted best to a two-site model, distinguishing a high-affinity site (RH) and a low-affinity site (RL) for dopamine. RH = 20% (KH = 49 nM) and RL = 80% (KL = 11 pM).
"~
~:~- -
40
~.so
O
10 y=44.3317-0.2783x 0
• 0
After incubation for 20 min at 30 °C, 4 ml of ice-cold buffer was added, and the samples were rapidly filtered over W h a t m a n n GF/B glass fiber filters, under reduced pressure. Radioactivity remaining on the filters was measured by a Packard liquid scintillation spectrometer. Non-specific binding was determined in the presence of 10 p M (+)-butaclamol. Specific binding was calculated by subtracting non-specific binding from total binding. Bma x (equivalent to receptor density) and K d values were calculated by linear regression analysis of the Scatchard plots. The percentages of the R H and R L sites, and their K i values (respectively K H and KL) for dopamine, were determined from computer-assisted curve-fitting, as described by Minneman et al. 15. A representative dopamine/[aH]SCH 23390 competition curve in frontal cortex membranes is shown in Fig. 1. No correlation was found between the densities of the D 1 receptors and their R H sites, and postmortem delay, up to 24 h. This is for example illustrated in Fig. 2, for
A
c
200
2 E O E
100
C O
B C 8
00
o
• | 10
postmortem
00
• i 20
3O
delay (hours)
Fig. 2. Effect of postmortem delay on the densities of the D 1 dopamine receptors ( I ) and their RH sites (0) in human frontal cortex membranes from 7 individuals of the same age group (79-81 years).
| 20
-
i 40
r = 0 . 5 7 (p
Brain Research, 528 (1990) 308-310
Elsevier
BRES 24296
Short Communications
The effect of aging on the
D1
dopamine receptors in human frontal cortex
Jacques de Keyser!, Jean-Paul De Backer 2, Georges Vauquelin 2 and Guy Ebinger 1 1Department of Neurology, Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels (Belgium) and eProtein Chemistry Laboratory, Vrije Universiteit Brussel, St. Genesius Rode (Belgium)
(Accepted 12 June 1990) Key words: Dopaminergic; Dopamine receptor, D1; Aging; Cerebral cortex; Human brain
Dopaminergic pathways to the cerebral cortex may be involved in cognitive function. We examined the effect of aging on the D1 dopamine receptors, and their high-agonist affinity (RH) sites, in postmortem human frontal cortex (n = 32; age range, 19-88 years). With aging, there was a significant decrease in the densities of the D1 dopamine receptors, and their RH sites, in human frontal cortex. The age-related reduction of cortical dopaminergic neurotransmission might contribute to the decline in cognitive abilities of elderly persons. Similar to rodents 8, the human cerebral cortex receives a widespread dopaminergic innervation from the midbrain 4'9. Dopamine receptors in human cerebral cortex are mainly of the DI type 3'5. The D 1 receptors in human frontal cortex exhibit similar affinities for antagonists of different chemical classes to those in the striatum 6. They are also composed of high affinity (RH) and low affinity (RL) sites for dopamine 6. However, in contrast to striatum and amygdala, the R H sites of the D 1 receptors in human frontal cortex are not converted into RL sites in the presence of GTP, and their activation by dopamine is not associated with a stimulation of the adenylate cyclase enzyme 6'7. For adenylate cyclase-coupled receptors, the RH sites are believed to represent receptors that interact with the guanine nucleotide binding protein G s 11. In analogy with the cyclase-coupled D~ receptors, it is tempting to assume that the GTP-resistant RH-sites of the D 1 receptors in human frontal cortex may also represent the physiologically relevant receptors. The 'normal' human aging process is associated with a decline in cognitive abilities, especially of memory ~4. With aging, there is a reduction in cortical acetylcholine and norepinephrine, which are neurotransmitters that are thought to play an important role in memory and learning 14. Studies in animals and in patients with Parkinson's disease suggest that dopaminergic projections to the cerebral cortex may also be implicated in cognitive function ~°'12'a7'18. However, the dopamine content in human neocortical regions does not significantly decrease
with age 1. In the present study, we assessed the effect of aging on the density of the I) 1 receptors, and their RH sites, in human frontal cortex. Brains were obtained at autopsy from 32 patients (aged 19-88 years; 12 females and 20 males), who had died without evidence of neurologic or psychiatric disease. The brains were stored at -80 °C. Postmortem delay ranged from 3 to 24 h (mean 13 + 4 h). None of the patients had received dopamine agonists or antipsychotics. Frontal cortex (area 9) was dissected at 4 °C, and subsequently homogenized in 20 mM Tris-HCl (pH 7.4)/120 mM NaCI/2 mM MgC12. The homogenates were centrifuged in a Sorvall centrifuge at 30,000 g for 20 min. The membranes were washed twice with repeated centrifugations at 30,000 g for 15 min. The supernatant was discarded and the pellet resuspended in the same buffer containing 10% glycerol (vol/vol). Until the radioligand binding experiments, the samples were stored in liquid nitrogen. Protein concentrations were determined by the method of Lowry et al. 13. Binding assays with [3H]SCH 23390 ((5)-(+)-8-chloro2,3,4,5-tetrahydro-3-methyl-5-phenyl-l-N-3°benzazepine; 85 Ci/mmol; Amersham) were done in duplicate in 0.5 ml of 20 mM Tris-HCl (pH 7.4)/120 mM NaC1/2 mM MgC12, containing 0.6 mg/ml of membrane protein. Saturation bindings were performed with 6 concentrations of [3H]SCH 23390 ranging from 0.5 to 6 nM, and 2 nM of [3H]SCH 23390 was used for competition binding experiments with dopamine (performed in the presence of 10 /~M pargyline). Spiperone (100 nM) was included in the assays to prevent binding to 5-HT2 serotonin receptors.
Correspondence: J. De Keyser, Department of Neurology, Academisch Ziekenhuis Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
309 ~I00 0 r~
z 8(
-~
250"
=~ ;
200
8
~ 150" o~
~ loo-
z ~z
m
50" y = 199.0762- 0.8739x r = 0.57 (p < 0.01)
~,, 2(
0
2'0
40
6'0
8'0
100
T
¥0 -9
-8
-'7
-6
-'5
-4
-3
50
DOPAMINE CONCENTRATION(LOG M)
Fig. 1. Dopamine competition for the specific binding of [3H]SCH 23390 to D~ dopamine receptors in human cortex membranes. A representative curve is shown. The curve is computer-fitted best to a two-site model, distinguishing a high-affinity site (RH) and a low-affinity site (RL) for dopamine. RH = 20% (KH = 49 nM) and RL = 80% (KL = 11 pM).
"~
~:~- -
40
~.so
O
10 y=44.3317-0.2783x 0
• 0
After incubation for 20 min at 30 °C, 4 ml of ice-cold buffer was added, and the samples were rapidly filtered over W h a t m a n n GF/B glass fiber filters, under reduced pressure. Radioactivity remaining on the filters was measured by a Packard liquid scintillation spectrometer. Non-specific binding was determined in the presence of 10 p M (+)-butaclamol. Specific binding was calculated by subtracting non-specific binding from total binding. Bma x (equivalent to receptor density) and K d values were calculated by linear regression analysis of the Scatchard plots. The percentages of the R H and R L sites, and their K i values (respectively K H and KL) for dopamine, were determined from computer-assisted curve-fitting, as described by Minneman et al. 15. A representative dopamine/[aH]SCH 23390 competition curve in frontal cortex membranes is shown in Fig. 1. No correlation was found between the densities of the D 1 receptors and their R H sites, and postmortem delay, up to 24 h. This is for example illustrated in Fig. 2, for
A
c
200
2 E O E
100
C O
B C 8
00
o
• | 10
postmortem
00
• i 20
3O
delay (hours)
Fig. 2. Effect of postmortem delay on the densities of the D 1 dopamine receptors ( I ) and their RH sites (0) in human frontal cortex membranes from 7 individuals of the same age group (79-81 years).
| 20
-
i 40
r = 0 . 5 7 (p
