Brain Research, 103 (1976) 617 621 © Elsevier Scientific Publishing Company, Amsterdam

617 Printed in The Netherlands

Brain glutamic acid decarboxylase and open field activity in ten inbred strains of mice

M. K. G A I T O N D E AND M. F. W. FESTING

(M.K.G.) Medical Research Council Laboratories, and Medical Research CouncilLaboratory Animal Centre, Woodmansterne Road, Carshalton, Surrey SM5 4EF (Great Britain) (Accepted November 6th, 1975)

During the past decade several studies have been reported which indicate a correlation between a behaviour pattern and a change, or changes, in a biochemical parameter in the brain. These studies may be classified broadly into two categories : (1) those in which a change in the behaviour was established by subjecting the animals of a given strain to a learning stress 4 by manipulating their environment s or by administering drugs 20, and (2) those in which a stable and specific behaviour pattern was 'isolated' or 'enriched' by selective breeding ~. Animals exposed to an externally imposed stimulus (category 1) often show behaviour change of limited duration, and, therefore, are not well suited for establishing neurochemical correlates of behaviour. To overcome limitations in experiments of this type 1~, Rick e t al. ~6 used animals of different strains, and exploited their stable inherited characteristics of behaviour in search of neurochemical changes in these strains. We have adopted this latter approach in the present investigation. Open-field ambulation was measured in 10 inbred strains of mice, details of which have been given previously 13 using a 36 sq.in, open field divided into 36 equal squares. Subjects were placed in a start box for 0.5 rain and the number of squares entered in the subsequent 2 minutes were recorded. Behavioural observations were carried out in two replicates each involving 4-8 mice per strain, and separated by a period of 3 months. The replicates were in close agreement and the results were pooled. All analyses were conducted on a square root transformation of the raw data. Home-cage wheel activity was measured using an 8.5 cm diameter exercise wheel suspended from the lid of a conventional plastic mouse cage. Revolutions were counted electronically over a 48 h period, using 5 male mice per strain, none of which had been used in any of the other studies. Animals had complete freedom of choice as to whether or not they ran in the exercise wheels. A separate group consisting of 4 animals of each strain which had not been subjected to these tests were used for measurement of glutamic acid decarboxylase ( G A D ) level in the brain using a randomized block experimental design. On each day, l animal of each of the 10 strains was decapitated, and the forebrain (cerebrum including the colliculi) was removed, weighed and placed in a heavy-wall test tube.

618 TABLE I M E A N V A L U E S OF

GAD,

O P E N - F I E L D A M B U L A T I O N A N D W H E E L A C T I V I T Y O F T E N I N B R E D S T R A I N S OI ~ MICE

Strain

GAD activity*

Open-fieM ambulation* *

Wheel activity* * *

B10.BR C57BL/10ScSn C57L ICFW DBA/I C3H/He A2G CBA-T6 C3H/He-mg NMRI

43.7 45.0 45.2 46.5 46.9 46.9 47.0 48.4 48.8 49.2

10.5 9.7 9.9 9.6 7.3 5.3 5.5 6.0 6.0 8.9

10.8 -10.6 8.2 5.2 5.6 1.4 5.8 6.0 5,2

* ffmole glutamate decarboxylated/gwet wt./h. ** Square-root transformation. *** 1000 revs./24 h.

The tissue was immediately suspended in an ice-cold medium (20 ml/g wet wt.) containing a mixture of 95 ml of freshly prepared 0.32 M sucrose, 5 ml of 1 0 ~ Triton X-100 and 1 ml (2.73 mg) of pyridoxal phosphate which had been gassed with N~ for 20-30 min: it was homogenized with 11 up- and 11 down-strokes of a teflon pestle at 830 rev./min. The homogenates were centrifuged at 3000 rev./min (2800 x g ) for 15 min at 5 °C. The opalescent supernatant solution was used for measurement of G A D activity in the brain. The substrate for the enzyme assay was prepared by dissolving 50 ffCi of DL-[1-14C]glutamic acid (Radiochemical Centre, Amersham, Bucks.) in a mixture of 150 ml of 60.4 × 10-3 M L-glutamic acid and 2.5 ml of pyridoxal phosphate (2.73 mg/ml). The G A D assay was carried out in 10 ml capacity conical flasks with a centre well which accommodated a small tube (16-20 mm long, 8-9 mm i.d.) containing 1 M hyamine in methanol (0.1 mI). The reaction was started by adding the enzyme extract (0.5 ml) to the substrate solution (0.6 ml). The flasks were immediately covered tightly with rubber closures, and shaken constantly for 30 min in a water bath at 36 °C. Each enzyme extract was incubated in triplicate as above, together with an enzyme blank consisting of subTABLE II CORRELATIONS BETWEEN TRAITS

GAD Open field

Open field

Wheel

-4).63*

~0.66" + 0.79" *

* Statistically significant at P < 0.05. Statistically significant at P < 0.01.

**

619 11 k

BIO.BR

4,-',1 ~ / u I~

I

C57L x x

"4~7BL~10

LCFW x

N.R,

~6

44

45

46

L7

L8

/.9

50

GAD revel Fig. I. The inverse correlation between brain glutamic acid decarboxylase level (ffmole glutamate decarboxylated/g wet wt./h) and the exploratory activity of 10 inbred strains of mice. The data fitted the equation y :- 42.805--0.747 x with a coefficient of correlation r - - - 0.64 (P < 0.05). Ambulation score is the square root of the number of squares entered in a 2 rain period.

strate (0.6 ml), 20'}; trichloroacetic acid (0.3 ml), and enzyme extract (0.5 ml). The enzyme activity was terminated by injecting a solution of 20};~ trichloroacetic acid (0.3 ml) through the rubber seal, directly into the reaction mixture, (except in the enzyme blank). The incubation was continued for a further period of 75 min to complete the absorption of 14CO2 evolved in hyamine solution in the small tube placed in the centre well. The tube was removed, and placed in a glass vial containing a mixture of 2 ml of methoxyethanol and 10 ml of toluene scintillation fluid 7. The contents of the vial were stirred well and assayed for t4C. Under these conditions, the presence of small amounts of methanol in hyamine had no inhibitory effect on the G A D level. The enzyme assay described is an adaptation of the methods described by several workers 17,2t,22,24. G A D levels, ambulation scores and wheel activity and the correlations between them in the 10 strains are given in Tables I and il and the relationship between GAD and open-field activity is shown in Fig. 1. G A D levels ranged from 43.7 units (ffmole glutamate decarboxylated/g wet wt./h) in BI0.BR to 49.2 units in NMR1, the analysis of variance indicating that the differences between strains were highly significant (P < 0.001). The intraclass correlation (heritability in the broad sense 14) between members of the same strain was 0.73, indicating a high degree of genetic determination, and closely related strains (e.g. C57BL/10 and BI0.BR) had similar G A D levels. Similarly, strain differences in exploratory activity were highly correlated with published results 19. The correlation o f - - 0 . 6 3 between G A D level and exploratory activity was significant (P < 0.05), even though the exploratory activity level in NMRI was substantially higher than predicted from the G A D level. Had N M R I not been included in the experiment the correlation would have been --0.83 (P < 0.01). However, the 4 estimates of G A D levels and the replicated behavioural observations in N M R I were in close agreement suggesting that the anomalous behaviour of NMRI is a real effect and is worth studying in more detail.

620 The finding of a higher G A D level in A2G than C57BL/10 mice would imply that GABA production was also higher in strain A2G, a result in apparent contradiction to the findings of Al-Ani et a l . k However, GABA production as measured by A1-Ani et al. represents the turnover of GABA, which is dependent on several enzymes, rather than the true G A D level in the brain, as in this study. Our results are in close agreement with those of Tunnicliff et al. ~3 who studied 5 transmitter metabolizing enzymes in the whole brain of 7 inbred strains of mice, and found a correlation o f - - 0 . 5 2 between one measure of open-field activity and G A D activity, though their correlation was not statistically significant. Our experiment was, however, more sensitive in that we used 3 more strains, and there were differences in the methods of G A D assay in that we prepared tissue homogenates in the presence of Triton X-100, and therefore our values represent total brain GAD activity. This is an important consideration, since 50-60 ~o of the G A D in the brain is found in the cytoplasm of the nerve ending particleslS, ~4,25 and the enzyme homogenates, whether prepared in sucrose, as in the case of the present work, or in water, as in the case of work reported by Tunnicliff et al., give approximately 33-60 ~,~,of the total G A D activity in the brain, unless the homogenates are pretreated with Triton to release the activity trapped in the nerve ending particles. Since G A D levels were measured in the whole forebrain, it is not possible to assess the contribution of G A D levels in the basal ganglia (see refs. 3, 6), which is considered to play a role in the facilitation of locomotor activity. The observed inverse correlation of G A D level with the exploratory activity, an innate trait, suggests the existence of a genetic factor which may be expressed at the functional level as a morphological structure, rather than the concentration of G A D p e r :s'e, in the brain. It may be assumed from the observed localization of G A D predominantly at the synapse 2,1°A~ that a finite number of G A D molecules (quanta) are present per GABA-dependent inhibitory synapse ~. Our finding of a lower G A D level would suggest a lower number of such synapses in mice with higher exploratory activity. In this connection it is of interest to note a recent reporO 2 of differences in the affinity for uptake of norepinephrine by crude synaptosomal preparations of two strains of mice. We thank Miss Gwyneth Evans, Mr. M. Robins and Mr. W. Peacock for their excellent technical assistance.

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