Brain Research, 122 (1977) 59-69

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© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

GENETIC VARIATION IN BRAIN L-GLUTAMATE DECARBOXYLASE ACTIVITY FROM TWO INBRED STRAINS OF MICE

PAUL Y. SZE Department of Biobehavioral Sciences, University of Connecticut, Storrs, Conn. 06268 (U.S.A.)

(Accepted June 6th, 1976)

SUMMARY Two inbred strains of mice, C57BL/6Bg and DBA/1Bg, were compared for genetic variation in brain L-glutamate decarboxylase (GAD) activity. Although no large difference was found between the strains in whole brain GAD activity at adult age (27-45 days postnatally), regional examination revealed a difference in GAD activity in the cerebral cortex (15~o higher in DBA); subcellular examination revealed a difference in synaptosomal fraction (21 ~o higher in DBA). When GAD activity was measured in synaptosomal fractions prepared from dissected cerebral cortex, DBA was 34 ~o higher than C57BL. In addition, differences in GAD activity between the two strains could be observed even in the whole brain (10-15 ~o higher in DBA) during earlier development (15-23 days postnatally). These data indicate that at adult age, genetic difference in GAD activity between the two strains exists mainly in nerve terminals of the cerebral cortex. It is postulated that the difference may be due to a genetically mediated mechanism regulating GAD in the presynaptic terminals of GABA neurons of the cerebral cortex.

INTRODUCTION The effects of genetic differences on biochemical function of synaptic transmission have been investigated in inbred strains of mice. Such genetically determined neurochemical differences have been used to define molecular mechanisms regulating synaptic events4, or to provide clues for understanding neural basis of behavior (for review, see ref. 20). The latter approach appears particularly tempting, partly because in no other species have the genetic differences been better characterized than in inbred strains of mice for a diversity of complex behaviors (for review, see ref. 35). In two of the strains, C57BL and DBA, there are extensive data not only on dif-

60 ferences in behavioral capacities, but also on neurochemical differences in cholinergic 6, catecholaminergic17, 32, and serotonergic5,17 systems of the brain. In this study, the two well studied strains of mice, C57BL/6 and DBA/I, were further compared for phenotypic variation in brain L-glutamate decarboxylase (GAD) activity. GAD is the rate-limiting enzyme which determines endogenous levels of 7-aminobutyric acid (GABA) 29, and it is a well characterized protein42, 43 present in high concentrations in specific neurons including presynaptic terminals throughout the brain 7,23,~5,26,36. Therefore, the activity of GAD was measured as a phenotype characteristic of the type of neurons utilizing GABA as the transmitter. Two previous studies have reported the lack of difference in whole brain GAD activity between similar strains of C57BL and DBA mice2S,4L However, a combination of developmental, regional and subcellular approaches in this study revealed strain difference in GAD activity between C57BL/6 and DBA/I mice, localized mainly in nerve terminals of their cerebral cortex. METHODS

Animals The two inbred strains of Mus musculus domesticus, C57BL/6Bg and DBA/ IBg, were obtained from Dr. B. E. Ginsburg's colony presently at the University of Connecticut. Both strains were originally acquired from The Roscoe B. Jackson Memorial Laboratory (Bar Harbor, Maine) and have been separately inbred in Dr. Ginsburg's colony for at least 50 generations. The substrain suffix Bg is hereafter omitted. The mice were maintained under specific pathogen-free conditions in a 12 h light/dark cycle and at a temperature of 22 -q- 1 °C and a relative humidity of 52 :k 2 70- They were given Charles River mouse chow and water ad libitum. Pregnant females were housed singly and litters, with their birth monitored to an accuracy of =]=0.5 day, were not disturbed until the time of experimental use. Tissue preparation All mice were sacrificed between 9:00 and 11:00 a.m. by decapitation. Five gross anatomical regions (cerebral cortex, rhinencephalon-basal ganglia-diencephalon, mesencephalon, cerebellum, and medulla pons) were immediately dissected from whole brain at 4 °C. The brain tissue (whole brain or region) was weighed on an analytical balance and homogenized in 6 vol. of distilled water containing 0.2 (v/v) Triton X-100 for enzyme assay. Subcellular fractions were prepared according to the method of Gray and Whittaker 13. Brain homogenate (10 70 in 0.32 M sucrose) was centrifuged at 850 × g for 15 min. The sediment was washed once with 0.32 M sucrose to obtain the crude nuclear fraction (Px). The combined supernatant was centrifuged at 12,000 × g for 20 min. The supernatant ($2) was then recentrifuged at 105,000 × g for 60 min to obtain the cytosol fraction. The 12,000 × g pellet (P2) was resuspended in 0.32 M sucrose and layered on the discontinuous sucrose gradient as previously described la. After centrifugation at 27,000 × g for 70 min, the fraction at 0.8 M-1.2 M interphase

61 (P2B) was aspirated and the sucrose concentration was adjusted to 0.32 M. It was re-sedimented at 12,000 × g for 20 min to obtain the synaptosomal fraction. The 27,000 × g pellet (P2C) was used directly as the mitochondrial fraction without further washing. All particulate fractions were resuspended by homogenization in distilled water containing 0.2 ~ (v/v) Triton X-100 for enzyme assay.

Biochemical assays GAD activity was assayed according to a procedure and with apparatus described by Roberts and Simonsen 3°, measurement being made of the formation of 14CO2 from L-[1-14C]glutamic acid under nitrogen as the gas phase. The 14CO2 formed was absorbed in hyamine and the radioactivity was measured by liquid scintillation spectrometry. In a typical assay, the incubation medium (final volume 1.0 ml) contained 0.1 M potassium phosphate, pH 6.2, 25 mM L-[1-14C]glutamic acid (spec. act. 5 #Ci/mmole; Calbiochem Company), 0.2 mM pyridoxal 5'-phosphate, and 1 mM aminoethylisothiouronium bromide. After incubation at 37 °C for 20 min, the reaction was stopped by the addition of 0.1 ml of 8 N H2SO4. The flasks were allowed to remain at 37 °C with continuous shaking for another 60 min to insure a complete release of 14COz and absorption in the hyamine. DOPA decarboxylase (DOPA-D) activity was assayed by the method of Sims et al. 34 using the same measurement of 14CO2 as described for GAD. The incubation medium (1.0 ml) contained 0.1 M sodium phosphate, pH 6.9, 3.6 mM L-[carboxyl-14C]-3,4-dihydroxyphenylalanine (spec. act. 20 #Ci/mmole, New England Nuclear), 0.15 mM pyridoxal 5'-phosphate, and 10-5 M isoniazid. After incubation at 37 °C for 30 min, the reaction was stopped by the addition of 0.1 ml glacial acetic acid. Synaptosomal uptake of [aH]GABA was measured as described previously 37. The incubation medium (1.0 ml) contained 50 mM Tris. HC1, pH 7.3, 0.2 M sodium chloride, 1 mM aminooxyacetic acid, 1.0 # M [U-aH]GABA (spec. act. 1 Ci/mmole; New England Nuclear), and 2 mg of synaptosomal protein. After incubation at 30 °C for 5 mln, the synaptosomal suspension was pelleted at 10,000 × g (10 min) and the radioactivity associated with the pellet was measured. Protein was measured by the method of Lowry et al. 19. Linear function was determined by regression analysis 40 on a Wang 700 series programmable calculator. Significance of differences between groups was determined by the use of Student's two-tailed t-test. RESULTS

Strain comparison of brain GAD activity during postnatal development In preliminary experiments using 45-day-old mice, GAD activity in whole brain was found only slightly higher in DBA/1 than in C57BL/6. The difference, although small, was consistently obtained at 5 other ages when GAD activity in whole brain already reached adult levels: days 27, 28, 29, 30, and 31 (Fig. 1). The difference became considerably larger before 25 days of age when GAD activity was ontogenetically still rising. Brains of DBA/1 mice had persistently higher GAD

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Genetic variation in brain L-glutamate decarboxylase activity from two inbred strains of mice.

Brain Research, 122 (1977) 59-69 59 © Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands GENETIC VARIATION IN BRAIN L-GLU...
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