Brain Research, 121 (1977) 113-120
113
@ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
FACTORS C O N T R I B U T I N G TO T H E M O D U L A T I O N OF N O R E P I N E P H RINE U P T A K E BY SYNAPTOSOMES F R O M MOUSE BRAIN CORTEX
BEATRIZ MOISSET The Jackson Laboratory, Bar Harbor, Maine 04609 (U.S.A.)
(Accepted May 28th, 1976)
SUMMARY The mode of inheritance of the synaptosomal mechanism for uptake of norepinephrine (NE) was studied in two inbred strains of mice, BALB/cBy and C57BL/6By, along with the reciprocal F1 hybrids and 7 recombinant inbred strains, CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ and CXBK. All these strains were also tested in the open field as a measure of response to mild stress, since stress had been shown to affect the kinetic constants of synaptosomal uptake. The two parental strains show a significant difference in Km for NE uptake similar to that previously reported between BALB/cJ and C57BI_/10J, and no significant difference in Vma:,. The F1 hybrids resemble C57BL/6By, and the recombinant inbred strains show no significant differences from either parent with only minor exceptions. This makes further genetic analysis impossible with the data available at this time. A high positive correlation exists between Km and Vma,, (r = 0.89). The affinity for NE uptake and the number of uptake sites available seem to be modulated in a coordinated fashion. When the data on Km for all strains tested are pooled, a bimodal distribution is apparent. There are two populations with means of 2.25 and 4.03 × 10-7 M, respectively. Analysis of open field ambulation enables the strains to be divided into a high (C57BL/6By, BXCF1, CXBF1, CXBD, CXBE, and CXBK) and a low group (BALB/ cBy, CXBG, CXBH, CXBI and CXBJ). There is a significant negative interstrain correlation (r = 0.87) between open field ambulation and Km for NE uptake. If we take open field ambulation as an index of reactivity to stress (high ambulation-low reactivity and vice versa), then we can regroup the data on NE uptake into two categories: 78 ~ of the mice from the highly reactive strains presents high Km for NE uptake, while only 35 ~ of the non-reactive mice show high Kin. The bimodal distribution is apparent in both cases and the means of both high Km groups are
114 identical; the same is true of the means for both low K,,~ groups of strains of mice. It appears that Km for NE uptake does not vary along a continuum but it presents two discrete values. This would be suggestive of the existence of two distinct conformational states for the presumably proteinic uptake site. Stress presumably causes a switch from the high affinity confol mation to the low affinity conformation. Thus a higher percentage of individuals fi'om strains highly reactive to stress shows low affinity for NE uptake.
INTRODUCTION The neurotransmitter norepinephrine (NE} is taken up by the synaptlc membrane of noradrenergic nerve endings by an active transport mechanism which is saturable, requires sodium and potassium and is temperature dependentL The maximal rate of uptake of NE at infinitely high concentrations of substrate (Vm~xl can be regarded as an estimate of the total number of available uptake sites at a given time. The substrate concentration at which 1/2 of the maximal velocity is attained, apparent Michaelis constant (Kin) is an estimate of the affinity of the noradrenergic uptake site for the substrate. These two kinetic constants o f NE uptake can be determined in vitro in tissue slices 10 or in crude synaptosomal preparations 5 from brain tissue. It has been shown that fight stress and electroconvutsive shock cause a decrease in affinity (higher Kin) and an increase in the number ot availaNe uptake sites (higher Vm~x) of N E uptake by the synaptosomal preparation from brain cortex in mice ~,6.11 A strain of mice, BALB/cJ, is highly emotional as determined by the open field test (low ambulation and high defecation), while C57BL/10J is regarded as having low emotionality according to this test 8,4. These two strains differ in the quality of NE uptake by synaptosomal preparations from 3 brain regions (cortex, diencephalonmesencephalon and pons-medulla) with BALB/cJ presenting a higher Km than C57BL, 10J in all these tissues 9. In order to analyze the genetic determination of this strain difference in NE uptake as well as the interaction between N E uptake by cortical synaptosomes and emotionality or response to stress, 7 recombinant strains, along with the tw~ parental strains and the two reciprocal crosses, were studied. Recombinant inbred (RI) strains are bred by crossing two inbred strains and then by brother and sister mating from the F2 for at least 20 generations: at this point more than 99 ~ homozygosity is attained, and each recombinant strain carries some genes from each parental strain in different combinations. Such a battery of recombinant strains can be regarded as a replicable recombinant population, and thus it is very useful for a quick genetic analysis which often yields information on genetic linkage and models for genetic determination 1. All the strains were also tested in the open field for 3 min as a measure of their response to the mild stress of a novel environment.
115 METHODS The subjects were male mice of the inbred strains BALB/cBy, C57BL/6By, their reciprocal crosses CXBFa and BXCF1, and 7 recombinant strains: CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ and CXBK. The mice were kept 4 or 5 per cage, only mice from the same litter were caged together to avoid the occurrence of fighting with its concurrent stress that would affect NE uptake. They were given food and water ad lib., the temperature was kept constant at about 22 °C, and the day-night cycle was 12 12 h. They were killed at 60-90 days of age. Care was taken to randomize the subjects of different strains for the daily assays. The tissue used was brain cortex. The dissecting technique and in vitro synaptosomal uptake method have been described elsewhere 5,9. The tissue samples were homogenized in 10 vol. of 0.25 M sucrose, centrifuged at 1000 × g for 10 rain and the supernatant fraction rich in synaptosomes was decanted. Aliquots of this fraction equivalent to 10 mg of original tissue were incubated for 5 rain at 37 °C in duplicate in the presence of 5 different concentrations (0.03, 0.04, 0.07, 0.l and 0.2 #M) of DL-[7-3H]norepinephrine (spec. act., 6.9 Ci/nmole, New England Nuclear) in Krebs-Ringer phosphate medium, pH 7.4 with ethylenediamine tetraacetic acid (EDTA, 0.05 mg/ml), ascorbic acid (0.2 mg/ml), glucose (2 mg/ml) and an MAO inhibitor, nialamide (15 #M). The concentration of tritiated NE was kept constant in all the media and carrier DL-norepinephrine hydrochloride (Calbiochem) dissolved in 1 m M hydrochloric acid containing ethylenediaminetetraacetic acid (0.05 mg/ml) was added to vary the concentration of NE in the media. The uptake of 0 °C was subtracted as a blank. The samples were centrifuged for 20 min at 40,000 × g and the pellets were rinsed with 0.9 ~ saline. They were extracted with a liquid scintillation fluid, Triton X-100-toluene (1:4, v/v) (with 5.5 g PPO and 125 mg POPOP per litter). Tritium was determined in an Isocap/300 liquid scintillation system at 60 °Jo efficiency. The double reciprocal plot of 1/net uptake velocity in #moles/g of original tissue/5 rain versus 1/tritiated NE in #moles gives lines which are determined by linear regression least squares analysis. From these lines individual values of K.m and Vmax were obtained. The open field apparatus is a box 74 c m x 74 cm of square surface with 23 cm high walls. On the inside, the floor and walls are covered with white plastic sheeting. The floor is divided into 25 equal squares by black lines. The subjects were male mice of the strains mentioned above, between 60 and 75 days of age. The mouse is placed in a corner square of the open field and allowed to explore it for 3 min. The number of squares crossed is the ambulation score. The results were analyzed by Student-Newman-Keuls multiple range test and Tukey's W estimate. RESULTS The kinetic constants Km and Vmax for synaptosomal uptake of NE in R | strains are shown in Tables I and II, ranked from highest to lowest. The asterisks
116 TABLE 1
Apparent Km for DL-~ 3H]norepinephrine uptake by cortical svnaptosornes o f mice Kinetic constants were determined for each mouse by incubating duplicate samples of cerebral cortex homogenates with 5 different concentrations of substrate. Km and Vmax were derived for each subject by c o m p u t e r analysis of Lineweaver-Burk plots of initial velocities of uptake. Subjects were male mice of the recombinant inbred strains, their progenitor strains and reciprocal Fa crosses. G r o u p s with non-overlapping asterisks (vertical comparison) are significantly different at least at the 0.05 level by S t u d e n t - N e w m a n - K e u l s multiple range test. N represents the n u m b e r of subjects. Tukey's W = 1.673
Strain
N
Km ( ~ 10 7 M)
~ S.E.M.
BALB/cBy CXBG CXBJ CXBI CXBH CXBE CXBK BXCFI CXBF1 C57BL/6By CXBD
8 10 8 9 9 8 8 8 11 8 10
4.43 3.87 3.81 3.36 3.21 2.97 2.96 2.95 2.90 2.80 2.51
0.536 0.278 0.327 0.381 0.24l 0.531 0.348 0.304 0.286 0.358 0.284
* * * * * * * * * *
* * * * * * * * * *
indicate non-significant differences by Student-Newman-Keuls test. C57BL/6By and BALB/cBy show a significant difference in Km by Student t-test ~t ~ 2.55, P < 0.025), with BALB/cBy having a higher Km than C57BL/6By. This agrees with the previous findings in the closely related strains BALB/cJ and C57BL/10J. One-way analysis of variance of all strains shows no significant difference in Km (F---- 2.55, P ~ 0.05), nor in Vmax (F 2.45. P > 0.05). The only strains that show significant difference in Km by Student-Newman-Keuls test are BALB/cBy T A B L E I1
Maximal velocity for DL-/ :~H]norepinephrine uptake by cortical synaptosomes o t mice Kinetic constants were calculated as described in the legend to Table I, and the same statistical analysis was employed. Tukey's W - 1.111.
Strain
N
Vm~.~ (nmoles/g/5 min)
S.E.M.
CXBJ BALB/eBy CXBG CXBI CXBK CXBH CXBE C57BL/6By BXCFt CXBFj CXBD
~ 8 10 9 8 9 8 8 8 11 10
2.91 2.63 2.42 2.15 2.10 2.06 2.00 1.97 1.90 1.84 1.63
0.367 0.285 0.130 0.276 0.265 0.170 0.308 0.217 0.227 0.167 0.177
* ~ * * * * * * * *
* * * * * * * *
117
0 0 0 0 0
00 00 000 0 O0 0000 0000 O00 0 O00 00000
©00 0 O00 0 00000 O 0 0 0 0 0 0 0 O 0 0 0 0 0 O0 O00000 O0
0 0 0 0 0
ooooooooooooooo
ooo
o
o
,:o ,:s 2:0 £5 3:0 3:s g.o ~5 5:0 s'.s 6:0 6:5 ~.'o K m x 10 - 7 M
Fig. 1. K,,~ for NE uptake in C57BL/6By, BALB/cBy, F1 hybrids and R1 strains.
TABLE Ill
Open-fieM ambulation Number of squares traversed in the open field during a 3 rain test. See Table 1 for the statistical analysis. Tukey's W = 34.26.
Strain
N
Ambulation score
± S.E.M.
CXBD CXBK CXBE C57BL/6By CXBF1 BXCFI CXBI CXBJ CXBH BALB/cBy CXBG
12 12 12 12 12 12 12 12 12 12 12
108.0 107.9 93.6 93.2 84.4 76.5 55.1 48.2 43.9 29.2 20.3
7.34 6.27 6.98 8.95 8.47 9.91 9.36 4.41 6.64 6.44 3.88
* * * * *
TABLE IV
Pooled data on kinetic constants o f N E uptake by cortical synaptosomes in inbred strains o f mice with high and low open field ambulation (see text.) N
High ambulation strains (C57BL/6By, BXCF1, CXBF1, CXBD, CXBE and CXBK) 53 Low ambulation strains (BALB/cBy, CXBG, CXBH, CXBI, and CXBJ) 44
Apparent Km
Vmax
× 10 7 M
_+_S.E.M.
nmoles/g/5 min ± S.E.M.
2.84
0.138
1.89
0.089
3.72 P < 0.005
0.165
2.42 P < 0.005
0.116
118
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113
@ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
FACTORS C O N T R I B U T I N G TO T H E M O D U L A T I O N OF N O R E P I N E P H RINE U P T A K E BY SYNAPTOSOMES F R O M MOUSE BRAIN CORTEX
BEATRIZ MOISSET The Jackson Laboratory, Bar Harbor, Maine 04609 (U.S.A.)
(Accepted May 28th, 1976)
SUMMARY The mode of inheritance of the synaptosomal mechanism for uptake of norepinephrine (NE) was studied in two inbred strains of mice, BALB/cBy and C57BL/6By, along with the reciprocal F1 hybrids and 7 recombinant inbred strains, CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ and CXBK. All these strains were also tested in the open field as a measure of response to mild stress, since stress had been shown to affect the kinetic constants of synaptosomal uptake. The two parental strains show a significant difference in Km for NE uptake similar to that previously reported between BALB/cJ and C57BI_/10J, and no significant difference in Vma:,. The F1 hybrids resemble C57BL/6By, and the recombinant inbred strains show no significant differences from either parent with only minor exceptions. This makes further genetic analysis impossible with the data available at this time. A high positive correlation exists between Km and Vma,, (r = 0.89). The affinity for NE uptake and the number of uptake sites available seem to be modulated in a coordinated fashion. When the data on Km for all strains tested are pooled, a bimodal distribution is apparent. There are two populations with means of 2.25 and 4.03 × 10-7 M, respectively. Analysis of open field ambulation enables the strains to be divided into a high (C57BL/6By, BXCF1, CXBF1, CXBD, CXBE, and CXBK) and a low group (BALB/ cBy, CXBG, CXBH, CXBI and CXBJ). There is a significant negative interstrain correlation (r = 0.87) between open field ambulation and Km for NE uptake. If we take open field ambulation as an index of reactivity to stress (high ambulation-low reactivity and vice versa), then we can regroup the data on NE uptake into two categories: 78 ~ of the mice from the highly reactive strains presents high Km for NE uptake, while only 35 ~ of the non-reactive mice show high Kin. The bimodal distribution is apparent in both cases and the means of both high Km groups are
114 identical; the same is true of the means for both low K,,~ groups of strains of mice. It appears that Km for NE uptake does not vary along a continuum but it presents two discrete values. This would be suggestive of the existence of two distinct conformational states for the presumably proteinic uptake site. Stress presumably causes a switch from the high affinity confol mation to the low affinity conformation. Thus a higher percentage of individuals fi'om strains highly reactive to stress shows low affinity for NE uptake.
INTRODUCTION The neurotransmitter norepinephrine (NE} is taken up by the synaptlc membrane of noradrenergic nerve endings by an active transport mechanism which is saturable, requires sodium and potassium and is temperature dependentL The maximal rate of uptake of NE at infinitely high concentrations of substrate (Vm~xl can be regarded as an estimate of the total number of available uptake sites at a given time. The substrate concentration at which 1/2 of the maximal velocity is attained, apparent Michaelis constant (Kin) is an estimate of the affinity of the noradrenergic uptake site for the substrate. These two kinetic constants o f NE uptake can be determined in vitro in tissue slices 10 or in crude synaptosomal preparations 5 from brain tissue. It has been shown that fight stress and electroconvutsive shock cause a decrease in affinity (higher Kin) and an increase in the number ot availaNe uptake sites (higher Vm~x) of N E uptake by the synaptosomal preparation from brain cortex in mice ~,6.11 A strain of mice, BALB/cJ, is highly emotional as determined by the open field test (low ambulation and high defecation), while C57BL/10J is regarded as having low emotionality according to this test 8,4. These two strains differ in the quality of NE uptake by synaptosomal preparations from 3 brain regions (cortex, diencephalonmesencephalon and pons-medulla) with BALB/cJ presenting a higher Km than C57BL, 10J in all these tissues 9. In order to analyze the genetic determination of this strain difference in NE uptake as well as the interaction between N E uptake by cortical synaptosomes and emotionality or response to stress, 7 recombinant strains, along with the tw~ parental strains and the two reciprocal crosses, were studied. Recombinant inbred (RI) strains are bred by crossing two inbred strains and then by brother and sister mating from the F2 for at least 20 generations: at this point more than 99 ~ homozygosity is attained, and each recombinant strain carries some genes from each parental strain in different combinations. Such a battery of recombinant strains can be regarded as a replicable recombinant population, and thus it is very useful for a quick genetic analysis which often yields information on genetic linkage and models for genetic determination 1. All the strains were also tested in the open field for 3 min as a measure of their response to the mild stress of a novel environment.
115 METHODS The subjects were male mice of the inbred strains BALB/cBy, C57BL/6By, their reciprocal crosses CXBFa and BXCF1, and 7 recombinant strains: CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ and CXBK. The mice were kept 4 or 5 per cage, only mice from the same litter were caged together to avoid the occurrence of fighting with its concurrent stress that would affect NE uptake. They were given food and water ad lib., the temperature was kept constant at about 22 °C, and the day-night cycle was 12 12 h. They were killed at 60-90 days of age. Care was taken to randomize the subjects of different strains for the daily assays. The tissue used was brain cortex. The dissecting technique and in vitro synaptosomal uptake method have been described elsewhere 5,9. The tissue samples were homogenized in 10 vol. of 0.25 M sucrose, centrifuged at 1000 × g for 10 rain and the supernatant fraction rich in synaptosomes was decanted. Aliquots of this fraction equivalent to 10 mg of original tissue were incubated for 5 rain at 37 °C in duplicate in the presence of 5 different concentrations (0.03, 0.04, 0.07, 0.l and 0.2 #M) of DL-[7-3H]norepinephrine (spec. act., 6.9 Ci/nmole, New England Nuclear) in Krebs-Ringer phosphate medium, pH 7.4 with ethylenediamine tetraacetic acid (EDTA, 0.05 mg/ml), ascorbic acid (0.2 mg/ml), glucose (2 mg/ml) and an MAO inhibitor, nialamide (15 #M). The concentration of tritiated NE was kept constant in all the media and carrier DL-norepinephrine hydrochloride (Calbiochem) dissolved in 1 m M hydrochloric acid containing ethylenediaminetetraacetic acid (0.05 mg/ml) was added to vary the concentration of NE in the media. The uptake of 0 °C was subtracted as a blank. The samples were centrifuged for 20 min at 40,000 × g and the pellets were rinsed with 0.9 ~ saline. They were extracted with a liquid scintillation fluid, Triton X-100-toluene (1:4, v/v) (with 5.5 g PPO and 125 mg POPOP per litter). Tritium was determined in an Isocap/300 liquid scintillation system at 60 °Jo efficiency. The double reciprocal plot of 1/net uptake velocity in #moles/g of original tissue/5 rain versus 1/tritiated NE in #moles gives lines which are determined by linear regression least squares analysis. From these lines individual values of K.m and Vmax were obtained. The open field apparatus is a box 74 c m x 74 cm of square surface with 23 cm high walls. On the inside, the floor and walls are covered with white plastic sheeting. The floor is divided into 25 equal squares by black lines. The subjects were male mice of the strains mentioned above, between 60 and 75 days of age. The mouse is placed in a corner square of the open field and allowed to explore it for 3 min. The number of squares crossed is the ambulation score. The results were analyzed by Student-Newman-Keuls multiple range test and Tukey's W estimate. RESULTS The kinetic constants Km and Vmax for synaptosomal uptake of NE in R | strains are shown in Tables I and II, ranked from highest to lowest. The asterisks
116 TABLE 1
Apparent Km for DL-~ 3H]norepinephrine uptake by cortical svnaptosornes o f mice Kinetic constants were determined for each mouse by incubating duplicate samples of cerebral cortex homogenates with 5 different concentrations of substrate. Km and Vmax were derived for each subject by c o m p u t e r analysis of Lineweaver-Burk plots of initial velocities of uptake. Subjects were male mice of the recombinant inbred strains, their progenitor strains and reciprocal Fa crosses. G r o u p s with non-overlapping asterisks (vertical comparison) are significantly different at least at the 0.05 level by S t u d e n t - N e w m a n - K e u l s multiple range test. N represents the n u m b e r of subjects. Tukey's W = 1.673
Strain
N
Km ( ~ 10 7 M)
~ S.E.M.
BALB/cBy CXBG CXBJ CXBI CXBH CXBE CXBK BXCFI CXBF1 C57BL/6By CXBD
8 10 8 9 9 8 8 8 11 8 10
4.43 3.87 3.81 3.36 3.21 2.97 2.96 2.95 2.90 2.80 2.51
0.536 0.278 0.327 0.381 0.24l 0.531 0.348 0.304 0.286 0.358 0.284
* * * * * * * * * *
* * * * * * * * * *
indicate non-significant differences by Student-Newman-Keuls test. C57BL/6By and BALB/cBy show a significant difference in Km by Student t-test ~t ~ 2.55, P < 0.025), with BALB/cBy having a higher Km than C57BL/6By. This agrees with the previous findings in the closely related strains BALB/cJ and C57BL/10J. One-way analysis of variance of all strains shows no significant difference in Km (F---- 2.55, P ~ 0.05), nor in Vmax (F 2.45. P > 0.05). The only strains that show significant difference in Km by Student-Newman-Keuls test are BALB/cBy T A B L E I1
Maximal velocity for DL-/ :~H]norepinephrine uptake by cortical synaptosomes o t mice Kinetic constants were calculated as described in the legend to Table I, and the same statistical analysis was employed. Tukey's W - 1.111.
Strain
N
Vm~.~ (nmoles/g/5 min)
S.E.M.
CXBJ BALB/eBy CXBG CXBI CXBK CXBH CXBE C57BL/6By BXCFt CXBFj CXBD
~ 8 10 9 8 9 8 8 8 11 10
2.91 2.63 2.42 2.15 2.10 2.06 2.00 1.97 1.90 1.84 1.63
0.367 0.285 0.130 0.276 0.265 0.170 0.308 0.217 0.227 0.167 0.177
* ~ * * * * * * * *
* * * * * * * *
117
0 0 0 0 0
00 00 000 0 O0 0000 0000 O00 0 O00 00000
©00 0 O00 0 00000 O 0 0 0 0 0 0 0 O 0 0 0 0 0 O0 O00000 O0
0 0 0 0 0
ooooooooooooooo
ooo
o
o
,:o ,:s 2:0 £5 3:0 3:s g.o ~5 5:0 s'.s 6:0 6:5 ~.'o K m x 10 - 7 M
Fig. 1. K,,~ for NE uptake in C57BL/6By, BALB/cBy, F1 hybrids and R1 strains.
TABLE Ill
Open-fieM ambulation Number of squares traversed in the open field during a 3 rain test. See Table 1 for the statistical analysis. Tukey's W = 34.26.
Strain
N
Ambulation score
± S.E.M.
CXBD CXBK CXBE C57BL/6By CXBF1 BXCFI CXBI CXBJ CXBH BALB/cBy CXBG
12 12 12 12 12 12 12 12 12 12 12
108.0 107.9 93.6 93.2 84.4 76.5 55.1 48.2 43.9 29.2 20.3
7.34 6.27 6.98 8.95 8.47 9.91 9.36 4.41 6.64 6.44 3.88
* * * * *
TABLE IV
Pooled data on kinetic constants o f N E uptake by cortical synaptosomes in inbred strains o f mice with high and low open field ambulation (see text.) N
High ambulation strains (C57BL/6By, BXCF1, CXBF1, CXBD, CXBE and CXBK) 53 Low ambulation strains (BALB/cBy, CXBG, CXBH, CXBI, and CXBJ) 44
Apparent Km
Vmax
× 10 7 M
_+_S.E.M.
nmoles/g/5 min ± S.E.M.
2.84
0.138
1.89
0.089
3.72 P < 0.005
0.165
2.42 P < 0.005
0.116
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