Brain Research, 92 (1975) 157-164

157

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Norepinephrine uptake by cerebral synaptosomes of mouse: strain differences

BEATRIZ MOISSET*, EDITH D. HENDLEY** AND BRUCE L. WELCH*** Friends Medical Science Research Center, Baltimore, Md. (U.S.A.)

(Accepted March 24th, 1975)

Uptake of norepinephrine (NE) by the presynaptic membrane of noradrenergic neurons is a major mechanism of inactivation of this neurotransmitter 6, Drugs that inhibit this reuptake cause a prolongation of the action of N E at the receptor, and presumably this is the mechanism by which they cause an elevation of mood. This implies that the rate of uptake of N E plays an important role in the modulation of emotional behavior. I f genetic differences in the uptake mechanism of N E were found, they would contribute to an understanding of the genetic differences in emotional behavior found in man and animals. The genetic determination of emotional behavior has been studied in a number of inbred strains of mice by the open field method v,9,1°. In accordance with Broadhurst 1, a more emotional animal is one that reacts by urinating and defecating and by an inhibition of its locomotor activity when subjected to the moderately stressful novel situation of the oFen field, while a non-emotional animal is one that shows higher exploratory activity and low urination and defecation under these conditions. BALB/cJ and C57BL/10J inbred strains of mice differ widely in their open field behavior, with BALB/cJ being highly emotional and C57BL/10J nonemotional ~,4. In addition to this, BALB/cJ and C57BL/10J mice also differ in their behavioral response to D-amphetaminO 2 as measured in the open field. C57BL/10J mice showed an increase in locomotor activity in the first 4 days of testing, while BALB/cJ did not. Since amphetamine is considered to affect behavior by its actions upon release and uptake of NE, it would be possible that this strain difference in response to D-amphetamine is due to a genetic difference in N E reuptake by cerebral synaptic membranes. This paper reports strain differences in affinity for uptake of N E by crude synaptosomal preparations of 3 brain regions of male BALB/cJ and C57BL/I 0J mice, * Present address: The Jackson Laboratory, Bar Harbor, Maine 04609, U.S.A. ** Present address: Department of Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vt. 05401, U.S.A. *** Present address: Department of Psychiatry and Behavioral Science, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205, U.S.A.

158 and apparent strain differences in maximal velocity of uptake which were actually attributable to strain differences in fighting behavior. The subjects were male mice of the inbred strains BALB/cJ and C57BL/10J. They were received from the Jackson Laboratory at weaning age (3 weeks) and housed in opaque plastic cages (15 cm × 27 cm x 13 cm) with food and water ad libitum, under controlled conditions of temperature and lighting. They were kept either in groups of 10 or isolated (one per cage) from the time of arrival. Their age at the time of testing ranged between 3 and 5 months. In each experiment, one BALB/cJ and one C57BL/10J mouse, similarly housed, were tested simultaneously. All mice were sacrificed between 10 a.m. and 1 p.m. and special care was taken to treat the tissues of each pair of mice in an identical manner (time, temperature of incubation, centrifugation), to ensure a legitimate paired comparison. The animals were killed by decapitation, the brains dissected quickly and kept in cold isotonic sucrose. The 3 tissues used in these experiments were: cerebral cortex, diencephalon-mesencephalon and pons-medulla. The dissection procedure was a modification of that described by Glowinski and Iversen 3. A transverse section was made at the level of the optic chiasma, and the anterior part discarded. The telencephalon was separated from the rest of the encephalon using the lateral ventricles and the corpus callosum as boundaries. The hippocampus and striatum (including the putamen, caudate nucleus and globus pallidus) were discarded, leaving the cerebral cortex. The cerebellum was also discarded and a second transverse cut separated the diencephalon-mesencephalon from the pons-medulla. The uptake of NE was studied in vitro by the method described by Hendley et aLL The tissue samples were gently homogenized in 0.25 M sucrose and crude synaptosomal fractions rich in synaptosomes were prepared. Aliquots of this fraction equivalent to 5 mg of original tissue (or to 2.5 mg in a few experiments) were incubated for 5 rain at 37 °C in duplicate in the presence of 5 different concentrations of DL[7-3H]norepinephrine (New England Nuclear) in Krebs-Ringer phosphate medium, pH 7.4, to which a monoamine oxidase inhibitor, pargyline (0.01 mM) was added. The uptake at 0 °C was subtracted as a blank. In some experiments the Ki for D-amphetamine was simultaneously determined by incubating samples with a constant concentration of 0.1/~M D-amphetamine sulfate (Smith, Kline and French Labs.) The samples were centrifuged and the pellets, after being rinsed with 0.9 ~ NaC1, were extracted with a liquid scintillation fluid (Instagel, Packard). Tritium was determined in a Packard Tricarb model 3320 refrigerated liquid scintillation spectrophotometer at 35 ~ efficiency. The kinetic constants apparent Km and Vmax were calculated from LineweaverBurk plots 8 derived by least squares linear regression of the reciprocal of the velocity plotted against the reciprocal of the substrate concentration. The results were analyzed by paired t-test comparisons.

159 BALB/cJ--6" C57BL/IOJ--

CORTEX

Grouped mice

//

BALB/cJ - - -

t/

6'

C57BL/IOJ - -

5"

CORTEX Isoloted mice

5'

//

s/

/

~E

~E

~.~

~

2'

/"

!

-I0

-5

2 //II

0

1

1

t

5

I0

15

I / [3H-NOREPINEPHRINE]

I

20

-I0

(/Ji)

/

i'o

-5

I / [SH-NOREPINEPHRINE]

C~M)

Fig. '1. Lineweaver-Burk analysis of strain differences in [3H]NE uptake kinetics. Net uptake at 37 °C minus uptake at 0 °C was determined in 5 min in homogenates of cerebral cortex of male mice that had been living in groups of 10 since weaning age. BALB/cJ, broken line; C57BL/10J, solid line. Plotted values are averages derived from least squares regressions calculated for individual mice. Fig. 2. Lineweaver-Burk analysis of strain differences in [3H]NE uptake kinetics in homogenates from cerebral cortex from male BALB/cJ and C57BL/10J mice that had been kept in isolation since weaning age. Same code and experimental conditions as in Fig. 1.

Cerebral cortex ( A ) Grouped animals. B A L B / c J mice h a d 81 ~ higher a p p a r e n t Km for u p t a k e o f N E t h a n C57BL/10J mice, indicative o f a lower affinity for uptake, a n d 46 ~ higher Vmax (Fig. l, Table I), suggesting a larger n u m b e r o f active u p t a k e sites. N o significant difference in the K/ for i n h i b i t i o n o f u p t a k e b y D - a m p h e t a m i n e was f o u n d between the strains ( K / f o r D - a m p h e t a m i n e was 0.498 a n d 0.445 × 10 -7 M f o r B A L B / cJ a n d C57BL/10J respectively). The mice in this e x p e r i m e n t h a d been k e p t in groups o f 10 per cage. O r d i n a r i l y B A L B / c J mice are very aggressive a n d fight v i g o r o u s l y u n d e r these circumstances. T h e subjects o f this strain presented severe w o u n d s in their backs a n d tails as a result o f these encounters, while C57BL/10J mice d i d n o t present a n y traces o f fighting.

TABLE I UPTAKE OF DL-[aH]NOREPINEPHRINEINTO SYNAPTOSOME-RICHHOMOGENATESOF MOUSE CEREBRAL CORTEX. GROUPEDBALB/cJ AND C57BL/10J MICE Means J- S.E.M. Number of experiments between brackets. Paired t-test, two-tailed.

BALB/cJ C57BL/10J

Apparent Km ( × 10 7M)

Ki for D-amphetamine ( × lO-TM)

Vma, (nmole/g/5 rain)

2.04 -k 0.241 (11) 1.129 ± 0.167 (11) P < 0.005

0.498 ± 0.037 (7) 0.445 ± 0.024 (7) n.s.

0.843 ± 0.137 (11) 0.577 -k 0.056 (11) P < 0.05

160 TABLE II UPTAKE OF DL-[aH]NOREPINEPHRINE INTO SYNAPTOSOME-RICH HOMOGENATES OF MOUSE CEREBRAL CORTEX. ISOLATED B A L B / c J AND C57BL/10J MICE M e a n s ± S.E.M. N u m b e r of subjects between brackets. Paired t-test, two-tailed.

Apparent Km ( × 10-7M) BALB/cJ C57BL/10J

1.98 ± 0.157 1.42 -4- 0.200 P < 0.025

Vmaz (nmole/g/5 mitt) (10 (10)

0.962 :k 0.098 0.898 ± 0.139 n.s.

(10) (10)

This behavioral difference had to be taken into consideration in the study of strain differences of N E uptake for the reasons described below. The effects of fight stress on NE uptake mechanism of mouse cerebral synaptosomes have been studied in our laboratory 5. This kind of stress induced a significant increase in Km and Vmax of N E uptake in synaptosomal preparations of cerebral cortex of Swiss mice. Chronic fight stress reduced the affinity for N E uptake at the synapse and increased the number of uptake sites, indicating that the NE uptake mechanism in the brain is not fixed but plastic, and capable of responding to environmental stimulations with modifications of its kinetic constants. The strain found to have higher Km and Vmax for uptake of NE is also the more aggressive one. In view of the experiments mentioned above, it would seem that we were possibly not dealing with a true genetic difference in uptake of NE, but rather that this difference might have occurred due to the higher level of social stress to which the members of one strain are subjected as compared with the other one. In order to elucidate the causes of the uptake differences between the 2 strains it became necessary to repeat the experiment with animals that had been prevented from fighting by keeping them in isolation. (B) Isolated animals. BALB/cJ had a 40 ~ higher Km for uptake of NE than C57BL/10J isolated mice, but no difference in Vma,: (Fig. 2, Table II). The persistence in difference in apparent Km between the 2 strains whether they had been kept in groups or isolation suggests that there might be a true genetic difference in affinity for uptake of NE in cerebral cortex. Although it is not possible to compare grouped with isolated mice of each strain, since the 2 series of experiments were performed at different times, it is interesting to note that the strain difference in Km was smaller when isolated animals were tested instead of grouped ones (40 ~ as compared with 81 ~). This is consistent with the findings with fight-stressed Swiss mice. The difference in the Vmax between the 2 strains when grouped animals were considered is probably due to adaptive changes in the uptake mechanism caused by fighting that occurs among BALB/cJ mice, since this difference was not present when isolated animals were compared.

161 DIENCEPHALON-MESENCEPHALON BALB/oJ ~ - -

A. Grouped Mice

C57BL/IOJ ~

7'

B. Isolated Mice

7' ,.-

~ . ~ 6, / /

/

~$4.

/

.. " " "

~'6

I

-5

I

0 5 io I/[~H-NOREPINEPHRINE] (uM)

I

I

Is

zo

2, ;S

I

-to

-5

f

3'

ZE ---~ /

-10

/

I

0 5 10 I/[3H-NOREPINEPHRINE] (aM)

I

I

15

20

Fig. 3. Lineweaver-Burk analysis of strain differences in [~H]NE uptake kinetics in homogenates from diencephalon-mesencephalon of male BALB/cJ and C57BL/10J mice. A: grouped mice, B: isolated mice. Same code and experimental conditions as in Fig. 1.

Dieneephalon-mesencephalon In this series of experiments, grouped BALB/cJ mice fought to the same extent as previously observed in these mice, whereas no fighting was observed among C57BL/10J. There was a difference in the apparent Km between the 2 strains but this difference was smaller than that found in brain cortex, and it was significant only when we pooled the results of the grouped plus the isolated animals (P < 0.025). The apparent Km in grouped animals was not significantly different from that of isolated mice of the same strain. There was a moderate, but significant, difference in Vmax between the 2 strains in grouped animals (34 ~ higher in BALB/cJ), but no difference in isolates (Fig. 3, Table III). TABLE III UPTAKE OF DL-[3H]NOREPINEPHRINE INTO SYNAPTOSOME-RICH HOMOGENATES OF DIENCEPHALONMESENCEPHALON. BALB/cJ AND C57BL/10J MICE Means ± S.E.M. Number of subjects between brackets. Paired t-test, two-tailed.

Grouped

Isolated

Apparent Km ( × IO-VM) BALB/cJ C57BL/10J

1.63 -Lz 0.234 1.08 ~- 0.056 n.s.

Vmax (nmole/g/5 min)

Apparent Km ( × 10 7M)

(10) 0.713 ± 0.071 (10) 1.304 4- 0.349 (10) 0.557 -E 0.039 (10) (10) 0.530 4- 0.011 (10) 1.09 4- 0.148 (10) 0.547 ~ 0.041 (10) P < 0.05 n.s. n.s.

Apparent Km of groupedplus isolated mice ( x lO-TM) BALB/eJ C57BL/10J

1.466 ~- 0.134 1.087 ± 0.079 P < 0.025

Vmax (nmoIe/g/5 min)

(20) (20)

162 PONS-MEDULLA

BALB/cJ - - -

A. Grouped Mice

C57BL/IOJ ~

>"

i

z

: ~ 1 -15

-I0

"~"

I -5

,~ 4" o

E

I

0

/

5

I / [3H-NOREPINEPHRINE]

(P,M)

B. Isolated

Mice

>,

I

I

I0

15

20

! io

I ~5

I zo

~e4"

: -zo

"

~ -I~

~

~" ~ ' ~ -wb

" -~

o

I 5

I/[3H-NOREPmEPHRmE] O,M) Fig. 4. Lineweaver-Burk analysis of strain differences in lZH]NE uptake kinetics in homogenates from pons-medulla of male BALB/cJ and C57BL/10J mice. A: grouped mice. B: isolated mice. Same code and experimental conditions as in Fig. 1.

The difference in Kin, although smaller, was in the same direction as that observed in cortex; BALB/cJ synaptosomes had a lower affinity for uptake of NE than those of C57BL/10J mice. Although, once again, the experimental design does not permit comparison of grouped and isolated animals of the same strain, there is the same general trend toward a higher Km in grouped animals that fight (BALB/cJ) than in isolates of the same strain and no differences between grouped and isolated animals of the strain that does not fight (C57BL/10J). As in cerebral cortex the difference in Vmax between the 2 strains in diencephalon-mesencephalon seems to be caused by the chronic fighting of BALB/cJ mice, since there was no strain difference in Vmax in isolated animals.

Pons-medulla The grouped BALB/cJ mice used in this series of experiments showed no spontaneous fighting behavior, as occasionally happens with this strain. C57BL/10J did not fight either, as usual. Grouped BALB/cJ mice averaged higher apparent Km values (61 ~ higher) than grouped C57BL/10J mice; isolated animals showed a similar

163 TABLE IV UPTAKE OF DL-[3H]NOREPINEPHRINE INTO SYNAPTOSOME-RICH HOMOGENATES OF PONS--MEDULLA. BALB/cJ AND C57BL/10J M~CE Mean ± S.E.M. Number of subjects between brackets. Paired t-test, two-tailed.

Grouped

BALB/cJ C57BL/10J

Isolated

Apparent Km ( H 10 7M)

Vmaz (nmole/g/5 rain)

Apparent Km ( x lO-TM)

Vmaz (nmole/g/5 min)

1.153 ± 0.111 (7) 0.718 ± 0.128 (7) P < 0.01

0.591 ~z 0.051 (7) 0.577 zk 0.066 (7) n.s.

0.823 -4- 0.051 (8) 0.552 zk 0.048 (8) P < 0.005

0.533 i 0.033 (8) 0.538 ± 0.037 (8) n.s.

difference (49 ~). There was no difference in Vmax between the 2 strains whether they were grouped or isolated (Fig. 4, Table IV). Synaptosomes of pons-medulla from BALB/cJ mice had a lower affinity for uptake of NE than those of C57BL/10J. The difference in apparent Km was larger in this tissue than in the diencephalon-mesencephalon. The fact that no difference in Vmax was found in grouped animals of the 2 strains might be due to the different tissue, or it might be a reflection of the absence of fighting in this particular group of animals. The affinity of the norepinephrine uptake mechanism for D-amphetamine does not seem to be different in the 2 strains of mice, since no differences in K~ for Damphetamine were found in cerebral cortex. The strain difference in maximal velocity for uptake of N E seems to be a result, and not a cause, of the behavioral difference between the 2 strains. It is possible that some other 'genetic' differences reported in the literature, such as strain differences in endogenous levels, turnover and synthesis of brain amines, have a similar environmental origin. Psychosocial stress is known to accelerate the synthesis, release and breakdown of brain NE13,15. Thus, if the members of one strain are subjected to a high level of stress due to being grouped with other highly aggressive animals, while the mice of the other strain are not under such stress, this difference in social environment would be the real cause of the neurochemical differences between the 2 strains. When studying genetic differences in neurochemistry it is not enough to control the physical environment of the subjects but also their social environment, since there are genetic differences in social interactions, and these in turn modify the activity of various enzymes and transport systems involved in neurotransmission. The affinity of the synaptic membrane for the neurotransmitter N E is also modulated by stress 5A4. The difference in apparent Km for uptake of N E that we found in grouped animals of the 2 strains, could also be caused by this stress; however, it persisted in isolated animals which were not allowed to fight, which gives a little more certainty to the conclusion that there is a genetic difference in the affinity for uptake of NE by brain synaptosomes between BALB/cJ and C57BL/10J mice. Whether this difference in affinity is due to a genetically determined structural difference in uptake

164 site, or to a n adaptive response of the uptake m e c h a n i s m to a genetic difference i n rate of synthesis, or d e g r a d a t i o n or i n receptor sensitivity, remains to be seen. Some of the work reported here was included i n the dissertation submitted by Beatriz Moisset to the School of Sciences, U n i v e r s i d a d N a c i o n a l de C o r d o b a , Cordoba, A r g e n t i n a as partial fulfillment of the requirements for a Ph.D. degree 11. Drs. A. A. K u r l a n d a n d R. W. Von Korff provided facilities at the M a r y l a n d Psychiatric Research Center. This research was supported by N a t i o n a l Institutes of M e n t a l Health G r a n t MH-23478-01 to Beatriz Moisset a n d R01-DA-00053 to Bruce L. Welch.

1 BROADHURST,P. L., The inheritance of behavior, Sci. J., 65 (1965) 3943. 2 DEFRIES,J. C., AND HEGMANN,J. P., Genetic analysis of open field behavior. In C. LINDZEVAND D. D. THIESSEN(Eds.), Contributions to Behavior - - Genetic Analysis - - The Mouse as a Prototype, Appleton-Century-Crofts, New York, 1970, pp. 23-56. 3 GLOWaNSKt,J., AND IVERSEN,L. L., Regional studies of catecholamines in the rat brain. I. The disposition of [ZH]norepinephrine, [aH]dopamine and [3H]DOPA in various regions of the brain, J. Neuroehem., 13 (1966) 655-669. 4 HENDERSON,N. D., Prior treatment effects on open field behavior of mice - - a genetic analysis, Anim. Behav., 15 (1967) 364-376. 5 HENDLEY, E. D., MOISSET,B., AND WELCH, B. L., Catecholamine uptake in cerebral cortex: adaptive change induced by fighting, Science, 180 (1973) 1050-1052. 6 IVERSEN, L. L., The Uptake and Storage o f Noradrenaline in Sympathetic Nerves, Cambridge University Press, Cambridge, 1967. 7 LINDZEY,G., AND THIESSEN,D. D. (Eds.), Contributions to Behavior - - Genetic Analysis - - The Mouse as a Prototype, Appleton-Century-Crofts, New York, 1970. 8 LINEWEAVER,H., AND BURK, D., The determination of enzyme dissociation constants, J. Amer. Chem. Soc., 56 (1934) 658-666. 9 MANOSEVITZ,M., LINDZEY, C., AND THIESSEN,D. D. (Eds.), Behavioral Genetics: Method and Research, Appleton-Century-Crofts, New York, 1969. 10 MANOSEVITZ,M., AND MONTEMAYOR,R. J., Interaction of environmental enrichment and genotype, J. comp. physioL PsychoL, 79 (1972) 67-76. 11 MOISSET,B., Diferencias Neuroquimicas en Cepas Puras de Ratones de Diferente Emocionalidad, Ph.D. Dissertation, Universidad Nacional de Cordoba, 1974. 12 MOISSET,B., ANDWELCH,B. L., Effects of D-amphetamine upon open field behavior in two inbred strains of mice, Experientia (Basel), 29 (1973) 625-626. 13 WELCH,A. S., ANDWELCH,B. L., Isolation, reactivity and aggression: evidence for an involvement of brain catecholamines and serotonin. In B. E. ELEFTHERIOUANDJ. P. SCOTT(Eds), The Physiology o f Aggression and Defeat, Plenum Press, New York, 1971, pp. 91-142. 14 WELCH, B. L., HENDLEY, E. D., AND TUREK,I., Norepinephrine uptake into cerebral cortical synaptosomes after one fight or electroconvulsive shock, Science, 183 (1974) 220-221. 15 WELCH,B. L., AND WELCH, A. S., Control of brain catecholamines and serotonin during acute stress and after D-amphetamine by natural inhibition of monoamine oxidase: an hypothesis. In E. COSTAAND S. GARATTINI(Eds), Amphetamines and Related Compounds, Raven Press, New York, 1970, pp. 415~t45.