Brain Research, 136 (1977) 475--486 © Elsevier/North-Holland Biomedical Press
475
REGIONAL ACETYLCHOLINE TURNOVER RATES IN THE BRAINS OF THREE INBRED STRAINS OF MICE: CORRELATION WITH SOME INTERSTRAIN BEHAVIOURAL DIFFERENCES
T. DURKIN, G. AYAD, A. EBEL and P. MANDEL Centre de Neurochimie du CNRS, and lnstitut de Chimie Biologique, Facultd de Mddecine, 11 Rue Humann, 67085 Strasbourg Cedex (France)
(Accepted March 3rd, 1977)
SUMMARY
The hypothesis that the genetically determined behavioural differences which exist between the inbred mouse strains Balb/c, DBA/2 and C57B1/6 may be related to differences in acetylcholine metabolism in certain regions of the brain has been tested. In vivo ACh turnover rates have been measured in three regions (hippocampus, caudate nucleus and frontal-parietal cortex) of the brains of each strain by following the rate of formation of labelled ACh, in these regions, after a pulse intravenous injection of a tracer dose of all-labelled choline. Focussed microwave procedures were used for the rapid fixation of brain tissue and Ch and ACh radioactivities were determined following their electrophoretic separation. Steady-state concentrations of Ch and ACh were measured by a sensitive radio-enzymatic method. Significant interstrain differences in ACh turnover rates are reported for each of the brain regions studied with the order of metabolic activity being Balb/c > DBA/2 > C57 B1/6 in each case. These results are interpreted as being in agreement with previous reports on correlations between learning ability or locomotor activity and regional activities of choline acetyltransferase in the brains of these inbred strains. The correlations between the in vivo ACh turnover rates and (1) interstrain differences in behavioural measures and (2) regional choline acetyltransferase activities are discussed.
INTRODUCTION
A very useful approach in the search for relationships between neurotransmitter biochemistry and the expression of behaviours has been the use of inbred strains of rodents which show stable and inherited behavioural differences2,2a, ca. Many reports have described differences in the activities of key enzymes in transmitter biosynthesis4,5,7,21,al and in brain neurotransmitter levels and turnoved,S, is,
476 a9,32 in several inbred strains of mice. The in vivo turnover rate studies have been conducted mainly on the noradrenergic, dopaminergic and serotonergic systems and have added significantly to the evidence available for an eventual understanding of the neurochemical bases for the observed behavioural variations. In regard to the cholinergic system previous reports 5,21,31 have shown that differences exist in the activities of choline acetyltransferase (CAT) and acetylcholinesterase (ACHE), both in whole brain and in brain regions between several of these inbred strains of mice, notably Balb/c, DBA/2 and C57B1/6. These enzyme activities have been correlated with interstrain differences in behavioural measures of locomotor activity and learning ability. However, the direct measurement of in vivo acetylcholine (ACh) turnover rates has, until recently, been hampered by interference in the measured steady-state levels of ACh and choline (Ch) by the extraordinarily rapid postmortem changes in the levels of the metabolites 6 and the lack of well adapted assay methods for the simultaneous measurement of Ch and ACh radioactivities and steady-state concentrations. Both these difficulties have been largely overcome by the development of assay methodology 12 and focussed microwave procedures11, z9 which fix brain tissue by a rapid heat inactivation of enzymes and greatly reduce postmortem changes in ACh and Ch concentrations. We have used both these developments, i.e. a focussed microwave procedure and a sensitive radio-enzymatic assay, for the measurement of the steady-state levels of Ch and ACh in order to determine the ACh turnover rates in three regions (hippocampus, caudate nucleus and frontal-parietal cortex) of the brain of the Balb/c, DBA/2 and C57B1/6 strains and to compare these to previous reports on the correlation of regional cholinergic activity and the behavioural differences. METHODS
Animals Male mice of the Balb/c ORL, D B A / 2 0 R L , and C 5 7 B 1 / 6 0 R L strains were obtained at the age of 6-7 weeks from the Centre d'Elevage du C.N.R.S., Orl6ansLa Source, France, and housed in controlled light and temperature conditions before the experiment. Standard rodent diet and water were fed ad libitum. All mice were aged between 8 and 12 weeks at the time of the experiments. In each experiment animals of the same age were compared. All experiments were conducted at the same time of day (6-8 h light) to avoid possible interference by circadian rhythms 13.
Injection procedure [methyl-all]Choline chloride (10.1 Ci/mmole) was obtained from the Radiochemical Centre, Amersham, U.K. and the ethanolic solution evaporated to dryness under a stream of dry nitrogen and redissolved in sterile physiological saline immediately before use. Mice were placed into a clear Perspex holder, similar to that described by Guidotti et al. 11 and injected via a tail vein with 10 nmoles [methyl-3H]choline chloride (101 #Ci )in a volume of 100 #1. The injection was given as a 2 see pulse and special efforts were taken in order to standardize this procedure.
477
Mode of sacrifice The mice were killed at various times after the injection by exposure to a beam of microwave radiation focussed onto the head for a period of 3.5 sec. A Litton microwave oven (model MM 70/50) as modified by Medical Engineering Consultants, Lexington, Mass., U.S.A., was used. The source generated 2 kW (effective) radiation at a frequency of 2.45 GHz.
Dissection procedure The brain was excised, cleared of dura and surface blood vessels and placed onto a glass plate cooled by liquid nitrogen vapours. The frontal-parietal cortex, hippocampus and caudate nucleus from both right and left halves of the brain were quickly dissected, the halves pooled and frozen in liquid nitrogen. The dissection of the brain regions is greatly facilitated following microwave fixation with easy separation of the regions along their natural mechanical boundaries. The samples were not stored but treated immediately.
Sample preparation The tissue samples were weighed frozen (sample weights were reproducible within and between strains) and homogenized at 0-2 °C in 5 vols of 1 N formic acid-acetone (15:85 v/v), after the extraction procedure of Toru and Aprison 3°, at 2500 rev./min in glass-Teflon Potter-Elvehjem homogenizers. The homogenates were transferred to sealed centrifuge tubes and left to stand on ice for 30 rain with occasional mixing. After centrifugation at 20,000 x g for 5 rain aliquots of the supernatant were taken to determine the amounts of labelled Ch and ACh and their steadystate concentrations.
Determination of labelled Ch and A Ch Duplicate aliquots (30/d) of the supernatant were transferred to plastic Eppendorf tubes, frozen in liquid nitrogen and freeze-dried (45 rain). The residue was dissolved in 50/A of 25 mM sodium phosphate-citrate buffer pH 6.5 by vigorous mixing for 30 see. Labelled Ch and ACh were then quantitatively (85 ~) extracted into 50/tl of a solution of sodium tetraphenylboron (Sigma Chemicals, St Louis, Mo. U.S.A.) in di-isobutylketone (15 mg/ml) by mixing vigorously for 30 sec1°. After a brief centrifugation at 20,000 × g to separate the phases 40 #1 of the organic phase were transferred to fresh plastic Eppendorf tubes containing 40 #1 of 0.4 N HC1. The labelled Ch and ACh were transferred to the acid phase by vigorous mixing for 30 sec. After a brief centrifugation at 20,000 x g the organic phase was aspirated (vacuum pump) and the acid phase freeze-dried (1 h). The residue was dissolved in 30 #1 of a carrier mixture (1:1 v/v) of ACh chloride and Ch chloride (10 mM in water) and 20/A was applied to a Beckman paper strip for low-voltage electrophoresis. Electrophoresis was conducted in a Beckman electrophoresis cell, Durrum type, model R, with the buffer system described by Potter and Murphy24:25 ml concentrated formic acid, 75 ml glacial acetic acid, 900 ml glass distilled water, at 500 V (approx. 12 mA per cell) for 1 h. The paper strips were then
478 dried in a stream of warm air and developed by brief exposure to iodine vapours in order to localize and mark the bands. Good separation of Ch from ACh and from other Ch containing compounds was achieved under these conditions. Migration characteristics were: ACh band centre 7.6 cm, band width 4 ram; Ch band centre 8.8 cm, band width 5 mm. This gives a separation of 7.5 ram. No overlapping was found when control experiments were performed with labelled Ch and ACh standards. After the complete disappearance of the colouration the bands were cut out and placed into scintillation vials and 10 ml of the following fluorophor added (I litre of a solution of Omnifluor (New England Nuclear) 4 g/1 in toluene, 1 litre water- free dioxane, 600 ml methanol and 208 g naphthalene). Radioactivity was counted in an |ntertechnique liquid scintillation spectrometer.
Determination of the steady-state concentrations of Ch and A Ch Steady-state concentrations of Ch and ACh were determined essentially as described previously2L In this experiment 5 aliquots (10#!) of the formic acidacetone supernatant were taken from each sample and freeze-dried. Two tubes were used for the determination of Ch, two tubes treated by alkaline hydrolysis and used for the determination of ACh and the extra tube was used to control for the presence of radioactivity associated with the 3H-labelled Ch and ACh in the sample. All samples plus an external standard curve of Ch (0-100 pmoles) were treated by liquid cation exchange into sodium tetraphenylboron-di-isobutylketone for quantitative extraction of quaternary ammonium compounds. The extracted endogenous Ch plus the Ch derived by hydrolysis of ACh was then re-acetylated by incubation at 37 °C for 1 h with [l-14C]acetyl CoA and a partially purified preparation of rat brain choline acetyltransferase. The resulting [l-14C]labelled ACh was extracted by liquid cation exchange and an aliquot counted in a liquid scintillation spectrometer. The control tubes were treated identically except water replaced [l-14C]acetyl CoA during the incubation. The radioactivity associated with the all-labelled ACh and Ch was counted with the samples in the 14C channel of the liquid-scintillation counter. This quantity was almost negligible (5 % av.) in comparison to that associated with the 14C-labelled ACh and was subtracted from the sample disint./min before calculation of ACh and Ch concentrations. RESULTS Preliminary studies using Balb/c whole brain indicated that the maximum labeling of ACh occurred 60-90 sec following injection. The range of time points chosen for this study was selected on this evidence.
Radioactivity-time curves for Ch and A Ch The radioactivity associated with Ch and ACh in the three strains at different times after injection is shown in Tables I (hippocampus), II (caudate nucleus) and III (frontal-parietal cortex). The radioactivity associated with choline in the hippocampus of the Balb/c strain is higher at all time points than that for the DBA/2 or
479 TABLE I ACh and Ch radioactivity in the hippocampus An interstrain comparison of the radioactivity associated with Ch and A C h in the hippocampus at different times (0-120 sec) following the administration of 101 izCi (10 nmoles) [methyl-3H]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g x 10 -5 dr S.D. See text for details and analysis. Sec
Balb/c
20 30 50 60 70 80 90 120
DBA/2
C57BI/6
Ch
A Ch
Ch
A Ch
Ch
A Ch
11.08 dr 0.56 11.75+0.42 7.94 dr 0.27 6.10 dr 0.36 5.24 dr 0.27 3.96 dr 0.38 4.25 dr 1.03 4.39 dr 0.23
2.73 dr 0.21 4.07+0.19 5.62 dr 0.38 6.25 dr 0.42 6.17 dr 1.30 7.26 dr 0.41 7.01 4- 0.27 6.17 dr 0.11
12.02 i 0.56 10.035_1.02 4.92 dr 1.02 3.81 -4- 0.60 3.90 dr 0.40 3.80 dr 0.07 3.54 dr 0.36 3.42 ± 0.19
2.90 dr 0.40 3.91dr0.88 4.17 dr 0.33 4.31 dr 0.68 5.12 dr 0.46 5.59 dr 0.22 4.89 dr 0.69 4.22 dr 0.24
10.23 ± 0.80 8.805-0.99 5.60 dr 0.68 4.40 dr 0.14 4.42 dr 0.27 4.08 dr 0.14 4.01 dr 0.61 2.85 dr 0.88
1.42 ± 0.64 2.305-0.80 3.55 dr 0.24 4.15 dr 0.31 4.46 dr 0.56 5.44 -4- 0.24 4.80 dr 0.76 4.40 :~ 0.68
C57B1/6 strains which are not significantly different from each other. The same trend was also observed but to a lesser extent in the caudate nucleus and no significant difference was observed between strains in the frontal-parietal cortex. The rate of formation of labelled ACh in the hippocampus
and caudate nucleus
is s i g n i f i c a n t l y h i g h e r i n t h e B a l b / c s t r a i n t h a n i n C57B1/6 ( P < 0.001, a t all t i m e p o i n t s , S t u d e n t s t-test). I n t h e f r o n t a l - p a r i e t a l c o r t e x , h o w e v e r , t h i s r a t e is h i g h e r i n B a l b / c t h a n i n C57B1/6 o n l y u p t o 60 sec ( P < 0.001 a t e a c h t i m e p o i n t t o 6 0 sec,
TABLE II A Ch and Ch radioactivity in the caudate nucleus An interstrain comparison of the radioactivity associated with Ch and ACh in the caudate nucleus at different times (0-120 sec) following the administration of 101/,Ci (10 nmoles) [methyl-all]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g × 10-5 dr S.D. See text for details and analysis. Sec
Balb/c Ch
20 30 50 60 70 80 90 120
7.98 7.37 5.44 4.77 4.39 4.16 3.99 4.12
DBA/2 ACh
dr 0.56 dr 0.47 dr 0.52 dr 0.36 ± 0.57 dr 0.57 dr 0.76 dr 0.68
4.62 6.93 8.33 8.58 8.77 8.18 7.31 4.75
Ch dr 0.62 dr 0.40 dr 0.42 dr 0.51 ± 0.87 dr 0.44 dr 0.75 dr 0.25
9.07 7.10 5.30 4.54 4.29 3.74 3.06 2.75
C57Bl/6 ACh
dr 0.26 dr 0.68 dr 0.88 dr 1.56 dr 0.59 dr 0.08 d_ 0.06 dr 0.42
4.15 4.34 4.84 6.10 6.63 7.53 5.70 5.22
Ch dr 0.12 dr 0.22 4- 1.05 ± 1.10 dr 0.90 dr 0.83 dr 0.03 dr 0.81
8.04 6.93 5.18 4.38 3.98 3.62 3.71 2.73
ACh dr 0.17 dr 0.39 dr 0.95 dr 0.62 dr 0.11 dr 0.59 ± 0.35 dr 0.27
2.46 3.87 4.97 6.33 5.85 6.50 7.30 5.16
± 0.56 ± 0.83 q- 0.18 4- 0.73 dr 1.04 dr 0.39 d= 0.49 dr 0.29
480 TABLE II1 ACh and Ch radioactivity in the frontal-parietal cortex An interstrain comparison of the radioactivity associated with Ch and ACh in the frontal-parietal cortex at different times (0-120 sec) following the administration of 101 t~Ci (10 nmoles) [methyl-all]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g × 10 ~ 5- S.D. See text for details and analysis. Sec
Balb/c Ch
20 30 50 60 70 80 90 120
13.01 12.65 8.33 6.67 5.94 4.81 4.92 4.43
5-0.88 5- 1.01 5- 0.76 5- 0.68 i 0.55 5- 0.35 5- 0.17 j- 0.57
DBA/2 ACh
Ch
3.73 5-0.56 5.81 5- 0.72 7.23 5- 1.37 7.32 ± 0.69 6.58 5- 0.72 7.71 i 0.68 6.83 5- 0.99 7.00 5~ 1.29
14.11 9.45 6.63 4.95 4.67 4.72 5.06 4.34
C57Bf/6 ACh
5-0.92 4.35 ±0.08 5- 0.65 4.87 5- 0.44 5-0.53 5.43 ± 0.55 ± 0.47 5.63 5- 0.42 5- 0.62 6.47 :£ 0.61 5- 0.41 6.71 £ 0.57 i 1.02 7.25 5- 0.80 ± 0.65 5.21 ~. 0.81
Ch 11.57 11.13 7.67 6.78 7.31 5.76 5.58 4.01
ACh ±0.56 5- 0.99 ~- 0.74 ± 0.53 5- 1.55 ± 0.62 i 0.63 ~ 0.72
1.59 5-0.64 3.07 £ 0.57 3.83 ± 1.03 5.37 ~: 0.72 5.41 i 0.87 5.83 ~:: 0:32 5.94 ± 1.14 6.08 5- 0.46
S t u d e n t s t-test). T h e rate o f f o r m a t i o n o f l a b e l l e d A C h in the D B A / 2 strain is intermediate. T h e m a x i m a o f t h e A C h c u r v e s o c c u r r e d w i t h i n the r a n g e 7 0 - 1 1 0 sec for all r e g i o n s a n d n o p a r t i c u l a r p a t t e r n was o b s e r v e d . Steady-state concentrations o f Ch and A C h T h e s t e a d y - s t a t e c o n c e n t r a t i o n s o f C h a n d A C h in t h e t h r e e r e g i o n s o f t h e i n b r e d strains a r e s h o w n in T a b l e IV. It c a n be seen t h a t : (1) t h e c o n c e n t r a t i o n s o f c h o l i n e are l o w f o l l o w i n g f o c u s s e d m i c r o w a v e radiationg,11; (2) m e a s u r e d c o n c e n t r a t i o n s o f c h o l i n e v a r y b e t w e e n t h e strains w i t h D B A / 2 s h o w i n g l o w e s t values, B a l b / c i n t e r m e d i a t e a n d C57B1/6 h i g h e s t v a l u e s in all t h r e e regions. (3) C57B1/6 s h o w TABLE IV lnterstrain comparison of the steady-state concentrations of Ch and ACh in the three brain regions Values are expressed as nmoles/g i S.D. Numbers in parentheses, n. Statistical evaluation was conducted by Students t-test. Strain
Balb/c DBA/2 C57B1/6
Hippocampus
Caudate n u c l e u s
Frontal-parietal cortex
Ch
ACh
Ch
ACh
Ch
ACh
14.6 ~ 3.0* (20) 10.5 ± 2.6* (17) 17.0±4.6 (16)
16.0 5- 3.8 (18) 14.6 £ 3.3* (17) 18.05-4.0 (17)
23.5 5- 4.7* (18) 15.8 ± 2.5** (17) 30.0±4.1 (10)
42.5 5- 7.0** (17) 45.3 ± 6.8** (14) 59.75-9.1 (11)
15.0 5- 3.7** (13) tl.9 5- 3.8** (14) 23.75-4.4 (12)
11.0 3- 2.3** (18) 11.5 ± 2.2** (15) 16.0±2.6 (10)
Different from C57BI/6: * P < 0.01; ** P < 0.001.
481 higher concentrations of ACh in all three regions than either DBA/2 or Balb/c which are not significantly different. The concentrations of Ch and ACh reported here are in the same range as those reported by Haubrich et al. 15 in a study using similar techniques for the cortex and striatum of rat brain. The steady-state concentrations of Ch and ACh are unaltered by the injection of the tracer dose (10 nmoles) of [methyl-3H]choline27.
Specific activity-time curves Jbr Ch and ACh The curves showing the specific activities of Ch and ACh as a function of time after injection are shown in Figs. 1 (hippocampus), 2 (caudate nucleus) and 3 (frontalparietal cortex). The curves have been generated from the means of the radioactivitytime curves for each strain (Tables I, II and III) and from the means of the steadystate concentrations of Ch and ACh for each strain (Table IV). For clarity of presentation only the curves for the Balb/c and C57B1/6 strains are shown; the curves for the DBA/2 strain being intermediate between these two extremes. It can be seen that the conditions for a precursor-product relationship in an open compartment model z8 are not completely fulfilled, i.e. the Ch and ACh specific activity curves do not cross at the maximum of the ACh specific activity but slightly before this is reached. This is more marked in the frontal-parietal cortex and hippocampus than in the caudate nucleus. Since Ch is undoubtedly the immediate precursor of ACh, a probable explanation of this is that a part of the free choline pool is used simultaneously in the synthesis of phosphorylated choline derivatives. In this respect,
15 m
10
\
e
\ 4 3
20
4O
t
eO
I
80
100
120
I
I
I
I
lk~s.
Fig. 1. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of lO nmoles (101 ~Ci) [methyl-aHlcholine in the hippocampus of the Balb/c and C57B1/6 strains. The curves have been generated from the data shown in Tables I and IV. Balb/c: Ch • 0, ACh © O ; C57BI/6: Ch [ ] - - - , , ACh [ ] - - - C ] .
482
a x
6
~s
4
.,
20
\
40
60
80
100
I
120
I
1
I
Se©s.
Fig. 2. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of 10 nmoles (101 #Ci) [methyl-3H]choline in the caudate nucleus of the Balb/c and C57B1[6 strains. The curves have been generated from the data shown in Tables II and IV. Balb/c: C h • - - - - - - • , ACh - ~ - - - - - - O ; C57B1/6: Ch I - - - I , ACh I ~ - - - [ ] .
D
x
E 1o
8
~7 m
6
"1 /
I 20
I
I
I
I
I
40
60
80
100
120
I
I
1
I
SlICI,
Fig, 3. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of 10 nmoles (101 ~tCi) [methyl-SH]choline in the frontal-parietal cortex of the Batb/c and C57B1/6 strains. The curves have been generated from the data shown in Tables III and IV. Balb/c: Ch • • , ACh 0 - - - - , ' 3 ; C57B1/6: Ch I - - - H I , ACh [ ] - - - [ ] .
483 TABLE V Interstrain comparison of ,4Ch turnover rates in the three brain regions
Values for ACh turnover rates were calculated using the differential method of Jenden et a1.17; see text for details. Slopes were obtained by linear regression analysis and are expressed as nmoles/g/h ± S.D. (n = 5 in all cases). Statistical evaluation was conducted by Students t-test. Strain
Hippocampus
Caudate n u c l e u s
Frontal-parietal cortex
Balb/c DBA/2 C57B1/6
389 ± 5*** 342 ~ 15" 289 ~ 37
2468 ± 165"** 1554 5- 37* 1345 -Z 161
675 & 77*** 477 ~ 15"* 354 ~: 45
Different from C57B1/6: * P < 0.05; ** P -< 0.01; *** P < 0.001. we observed that the only other labelled choline metabolite of importance (Balb/c whole brain) during the time period studied (0-120 sec) was phosphorylcholine (PCh). Radioactivity associated with PCh increased linearly within this period and, at 120 sec, was just greater than that associated with ACh. This observation is in agreement with that of Lundholm and Sparf from a similar experiment 2° using mouse whole brain. Calculation o f A Ch turnover rates
The calculation of A C h turnover rates is based on the differential method described by Jenden et al. 17, Briefly this method is based on the assumption that the rate of appearance of labelled ACh at any one time after injection of Ch (dy*/dt) is directly proportional to the difference in the mole ratios of Ch and ACh ( x * ] x - - y*/y, where x and y refer to Ch and ACh, respectively, and the asterisk denotes a labelled variant) at that time. A plot of the form V/S is thus produced where V = dy*/dt and S ---- (x*/x - - y*/y) and the slope being a measure of the turnover rate. The major assumption in this open compartment kinetic model is that the mean specific activity of the Ch pool is a reasonable estimate of the specific activity of Ch in the ACh synthesis pool. Calculated values for the regional turnover rates of ACh in the three strains are shown in Table V. Due to the fact that the specific activity-time curves for Ch and ACh do not cross at the m a x i m u m specific activity of A C h but slightly before this (in the cortex and hippocampus) the straight lines obtained by the differential method give a positive intercept (significantly different from zero) on the dy*/dt axis which is similar for all three strains. As was pointed out by Jenden et al. 17, the assumption mentioned above is commonly made in calculations of this type and is justified only in an approximate sense. Furthermore, as was mentioned in the previous section we have evidence, although indirect, that compartmentation of the choline pool in the synthesis of choline lipids probably reduces the validity of this assumption. However, since the shift of the crossover point from the maximum of the A C h specific activity curve is small the calculated turnover rates are probably only slightly over-estimated in absolute values and, in any case, retain their relative values.
484 A number of other studies using focussed microwave procedures have estimated ACh turnover rates in mouse whole brain and in rat brain regions. Cheney et al. :~ using a pulse intravenous injection of phosphoryl [methyl-14C]-choline report a value for ACh turnover rate of 310 nmoles/g/h in mouse whole brain. Racagni et al. ~;. using an intravenous perfusion of phosphoryl [methyl-14C]choline, report values for ACh turnover rate of 1300 nmoles/g/h in the striatum and 200 nmoles/g/h in the cortex of the rat. Haubrich et al.l~, using a pulse intravenous injection of [methyl-3H]choline, reported values of 1447 nmoles/g/h for striatum and 367 nmoles/g,/h for cortex in rat brain. The values we report here for the three mouse strains are also in this range and, particularly for the C57B1/6 and DBA/2 strains, are very close to those reported for the same rat brain regions. DISCUSSION Previous studies on the correlation of behavioural patterns with the cholinergic system in inbred mouse strains have concentrated on the measurement of the activities of CAT and ACHE, the principal enzymes involved in ACh metabolism. Thus Tunnicliff et al. 31 in a study of 7 inbred strains reported that whole brain CAT levels were higher in Balb/c than in DBA/2 which were, in turn, higher than in C57B1/6 and that these activities showed a good inverse correlation with locomotor activity in these strains. In earlier reports from our laboratoryT, 21 it was observed that a good correlation existed between higher CAT activity in the frontal and temporal cortex and better learning ability in maze and avoidance testing procedures in the DBA/2 and C57B1/6 strains. Recently we have also reported a correlation between hippocampal CAT activity and the phenomenon underlying the improvement in performance ('reminiscence') observed in the Balb/c but not in the C57B1/6 strain when a 6-24 h interval is spaced between a first (partial acquisition) training session and the second trial I~. In all these cases it has been suggested that the differences in CAT activity may reflect underlying differences in ACh metabolism. Our findings of strain differences in regional ACh turnover rates provide some evidence for this postulate. There appears to be a good correlation between CAT activity measured in vitro or in vivo ACh turnover rate and the differences in avoidance, maze and wheel running behaviours observed between these strains. This may not, however, be absolute evidence of an obligatory link between in vivo CAT activity and ACh turnover rate. Although CAT has often been termed the rate-limiting enzyme in ACh synthesis it is clear that the availability of substrate, particularly Ch, is a major factor in the regulation of ACh synthesis in physiological conditions 14 and the possibility that the factors which determine this availability (for review see ref. 9) may also vary between strains cannot be excluded. Nevertheless, several observations can be made. The values we report for ACh turnover rates in the striatum show an inverse correlation with locomotor activity differences between the strains23, 81. It has already been proposed that the striatal cholinergic system exerts an inhibitory function on the level of locomotor activity and pharmacological inhibition of cholinergic transmission in the striatum is often
485 associated with m o t o r dysfunction o r elevated l o c o m o t o r activity (for review see ref. 25). T h e inverse c o r r e l a t i o n between strain differences in the A C h t u r n o v e r rate in the s t r i a t u m a n d l o c o m o t o r activity m a y be i n t e r p r e t e d in this w a y a n d thus lend s u p p o r t to this hypothesis. F u r t h e r m o r e , the higher A C h t u r n o v e r rate in the h i p p o c a m p u s o f the Balb/c as c o m p a r e d to the C57B1/6 strain agrees well with the previously o b s e r v e d higher h i p p o c a m p a l C A T activity in the Balb/c strain a n d p r o v i d e s further evidence for the h y p o t h e s i s t h a t the higher 'reminiscence' c a p a c i t y observed in this strain m a y be related to a m o r e active h i p p o c a m p a l cholinergic m e t a b o l i s m 16. Likewise the higher A C h t u r n o v e r rates in the f r o n t a l - p a r i e t a l cortex o f the Balb/c a n d D B A / 2 strains are in a g r e e m e n t with the p r e v i o u s l y observed c o r r e l a t i o n between better learning abilities o f these strains a n d the higher C A T activity in the frontal cortex o f the D B A / 2 as c o m p a r e d with the C57B1/6 strain 7. ACKNOWLEDGEMENTS A. Ebel is a Charg6e de R e c h e r c h e au C N R S . The a u t h o r s t h a n k Elisabeth G e r l i n g e r a n d D a n i e l G r u b e r for their excellent technical assistance.
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2 3 4 5 6 7 8 9 10 11 12 13 14
sterase activity and biogenic amine levels in brain for mouse strains differing in spontaneous activity and reactivity, Life Sci., 9 (1970) 21-27. Bovet, D., Bovet-Nitti, F. and Oliverio, A., Genetic aspects of learning and memory in mice, Science, 163 (1969) 139-149. Cheney, D. L., Costa, E., Hanin, I., Trabucchi, M. and Wang, C. T., Application of principles of steady-state kinetics to the in vivo estimation of ACh turnover rate in mouse brain, J. PharmacoL exp. Ther., 192 (1975) 288-296. Ciaranello, R. D., Barchas, R., Kessler, S. and Barchas, J. D., Catecholamines: Strain differences and biosynthetic enzyme activity in mice, Life Sci., II (1972) 565-572. Diez, J. A., Sze, P. Y. and Ginsburg, B. E., Genetic and developmental variation in mouse brain tryptophan hydroxylase activity, Brain Research, 109 (1976) 413-417. Dross, K. and Kewitz, M., Concentration and origin of choline in the rat brain, Naunyn-Schmiedebergs Arch, exp. Path. Pharmak., 274 (1972) 91-106. Ebel, A., Hermetet, J. C. and Mandel, P., Comparative study of acetylcholinesterase and choline acetyltransferase enzyme activity in brain of DBA and C57 mice, Nature New Biol., 242 (1973) 56-58. Eleftheriou, B. E., Regional brain norepinephrine turnover rates in four strains of mice, Neuroendocrinology, 7 (1971) 329-336. Freeman, J. J. and Jenden, D. J., The source of choline for acetylcholine synthesis in brain, Life ScL, 19 (1976) 949-962. Fonnum, F., Isolation of choline esters from aqueous solutions by extraction with sodium tetrapbenylboron in organic solvents, Biochem., J., 113 (1969) 291-298. Guidotti, A., Cheney, D. L., Trabucchi, M., Doteuchi, M., Wang, C. and Hawkins, R. A., Focussed microwave radiation: a technique to minimize postmortem changes of cyclic nucleotides, DOPA and choline and to preserve brain morphology, Neuropharmacology, 13 (1974) 1115-1122. Hanin, I., Choline and Acetylcholine: Handbook of Chemical Assay Methods, Raven Rress, New York, 1974. Hanin, I., Massarelli, R. and Costa, E., ACh concentrations in rat brain. Diurnal oscillation, Science, 197 (1970) 341-342. Haubrich, D. R., Wang, P. F., Clody, D. E. and Wedeking, P. W., Increase in rat brain acetylcholine induced by choline or deanol, Life Sci., 17 (1976) 975-980.
486 15 Haubrich, D. R., Wang, P. F., Herman, R. L. and Clody, D. E., Acetylcholine synthesis in rat brain: dissimilar effects of clozapine and chlorpromazine, Life Sci., 17 (1976) 739-748. 16 Jaffard, R., Ebel, A., Destrade, C., Durkin, T., Mandel, P. and Cardo, B., Effects of hippocampaI electrical stimulation on long-term memory and on cholinergic mechanisms in three inbred strains of mice, Brain Research, (1977) in press. 17 Jenden, D. J., Choi, L., Silverman, R. W., Steinborn, J. A., Roch, M. and Booth, R. A., Acetylcholine turnover estimation in brain by gas-chromatography/mass spectrometry, Lifo Sci., 14 (1974) 55 63. 18 Kellog, C., Serotonin metabolism in the brains of mice sensitive or resistant to audiogenic seizures, J. Neurobiol., 2 (1971) 209-219. 19 Kempf, E., Greilsamer, J., Mack, G. and Mandel, P., Correlation of behavioural differences in three strains of mice with differences in brain amines, Nature (Lond.), 247 (1974) 483-485. 20 Lundholm, B. and Sparf, B., The effect of atropine on the turnover of acetylcholine in the mouse brain, Europ. J. Pharmacol., 32 (1975) 287-292. 21 Mandel, P., Ayad, G., Hermetet, J. C. and Ebel, A., Correlation between choline acetyltransferase activity and learning ability in different mice strains and their offsprings, Brain Research, 72 (1974) 65-70. 22 Massarelli, R., Durkin, T., Niedergang, C. and Mandel, P., A simple radioenzymatic determination of Ch and ACh concentrations, Pharmacol. res. Commun., 8 (1976) 407--416. 23 Oliverio, A., Castellano, C. and Messeri, P., Genetic analysis of avoidance maze and wheel running behaviours in the mouse, J. comp. Physiol. Psychol., 79 (1972) 459-473. 24 Potter, L. T. and Murphy, W., Electrophoresis of acetylcholine, choline and related compounds, Biochem. Pharmacol., 16 (1967) 1386-1388. 25 Pradhan, S. N. and Dutta, S. N., Central cholinergic mechanism and behaviour, hlt. Rev. Neurobiol., 14 (1971) 173-231. 26 Racagni, C., Cheney, D. L., Trabucchi, M., Wang, C. and Costa, E., Measurement of ACh turnover rate in discrete areas of rat brain, Life Sci., 15 (1975) 1961-1975. 27 Sparf, B., On the turnover of acetylcholine in the brain, Acta physiol, stand., 397, Suppl. (1973) 1-47.
28 Sprott, R. L. and Staats, J., Behavioural studies using genetically defined mice, a bibliography, Behav. Genet., 5 (1975) 27-83. 29 Stavinoha, W. B., Weintraub, S. T. and Modak, A. T., The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine, J. Neurochem., 20 (1973) 361-371. 30 Toru, M. and Aprison, M. H., Brain acetylcholine studies: a new extraction procedure, J. Neurochem., 13 (1966) 1533-1544. 31 Tunnicliff, G., Wimer, C. C. and Wimer, R. E., Relationship between neurotransmitter metabolism and behaviour in seven inbred strains of mice, Brain Research, 61 (1973) 428-434. 32 Wimer, R. E., Norman, R. and Eleftheriou, B. E., Serotonin levels in hippocampus: striking variations associated with mouse strain and treatment, Brain Research, 63 (1973) 397-401. 33 Zilversmit, D. B., Entenman, C. and Fishier, M. C., On the calculation of 'turnover time' and 'turnover rate' from experiments involving the use of labeling agents, J. gen. PhysioL, 26 (1943) 325-331.
475
REGIONAL ACETYLCHOLINE TURNOVER RATES IN THE BRAINS OF THREE INBRED STRAINS OF MICE: CORRELATION WITH SOME INTERSTRAIN BEHAVIOURAL DIFFERENCES
T. DURKIN, G. AYAD, A. EBEL and P. MANDEL Centre de Neurochimie du CNRS, and lnstitut de Chimie Biologique, Facultd de Mddecine, 11 Rue Humann, 67085 Strasbourg Cedex (France)
(Accepted March 3rd, 1977)
SUMMARY
The hypothesis that the genetically determined behavioural differences which exist between the inbred mouse strains Balb/c, DBA/2 and C57B1/6 may be related to differences in acetylcholine metabolism in certain regions of the brain has been tested. In vivo ACh turnover rates have been measured in three regions (hippocampus, caudate nucleus and frontal-parietal cortex) of the brains of each strain by following the rate of formation of labelled ACh, in these regions, after a pulse intravenous injection of a tracer dose of all-labelled choline. Focussed microwave procedures were used for the rapid fixation of brain tissue and Ch and ACh radioactivities were determined following their electrophoretic separation. Steady-state concentrations of Ch and ACh were measured by a sensitive radio-enzymatic method. Significant interstrain differences in ACh turnover rates are reported for each of the brain regions studied with the order of metabolic activity being Balb/c > DBA/2 > C57 B1/6 in each case. These results are interpreted as being in agreement with previous reports on correlations between learning ability or locomotor activity and regional activities of choline acetyltransferase in the brains of these inbred strains. The correlations between the in vivo ACh turnover rates and (1) interstrain differences in behavioural measures and (2) regional choline acetyltransferase activities are discussed.
INTRODUCTION
A very useful approach in the search for relationships between neurotransmitter biochemistry and the expression of behaviours has been the use of inbred strains of rodents which show stable and inherited behavioural differences2,2a, ca. Many reports have described differences in the activities of key enzymes in transmitter biosynthesis4,5,7,21,al and in brain neurotransmitter levels and turnoved,S, is,
476 a9,32 in several inbred strains of mice. The in vivo turnover rate studies have been conducted mainly on the noradrenergic, dopaminergic and serotonergic systems and have added significantly to the evidence available for an eventual understanding of the neurochemical bases for the observed behavioural variations. In regard to the cholinergic system previous reports 5,21,31 have shown that differences exist in the activities of choline acetyltransferase (CAT) and acetylcholinesterase (ACHE), both in whole brain and in brain regions between several of these inbred strains of mice, notably Balb/c, DBA/2 and C57B1/6. These enzyme activities have been correlated with interstrain differences in behavioural measures of locomotor activity and learning ability. However, the direct measurement of in vivo acetylcholine (ACh) turnover rates has, until recently, been hampered by interference in the measured steady-state levels of ACh and choline (Ch) by the extraordinarily rapid postmortem changes in the levels of the metabolites 6 and the lack of well adapted assay methods for the simultaneous measurement of Ch and ACh radioactivities and steady-state concentrations. Both these difficulties have been largely overcome by the development of assay methodology 12 and focussed microwave procedures11, z9 which fix brain tissue by a rapid heat inactivation of enzymes and greatly reduce postmortem changes in ACh and Ch concentrations. We have used both these developments, i.e. a focussed microwave procedure and a sensitive radio-enzymatic assay, for the measurement of the steady-state levels of Ch and ACh in order to determine the ACh turnover rates in three regions (hippocampus, caudate nucleus and frontal-parietal cortex) of the brain of the Balb/c, DBA/2 and C57B1/6 strains and to compare these to previous reports on the correlation of regional cholinergic activity and the behavioural differences. METHODS
Animals Male mice of the Balb/c ORL, D B A / 2 0 R L , and C 5 7 B 1 / 6 0 R L strains were obtained at the age of 6-7 weeks from the Centre d'Elevage du C.N.R.S., Orl6ansLa Source, France, and housed in controlled light and temperature conditions before the experiment. Standard rodent diet and water were fed ad libitum. All mice were aged between 8 and 12 weeks at the time of the experiments. In each experiment animals of the same age were compared. All experiments were conducted at the same time of day (6-8 h light) to avoid possible interference by circadian rhythms 13.
Injection procedure [methyl-all]Choline chloride (10.1 Ci/mmole) was obtained from the Radiochemical Centre, Amersham, U.K. and the ethanolic solution evaporated to dryness under a stream of dry nitrogen and redissolved in sterile physiological saline immediately before use. Mice were placed into a clear Perspex holder, similar to that described by Guidotti et al. 11 and injected via a tail vein with 10 nmoles [methyl-3H]choline chloride (101 #Ci )in a volume of 100 #1. The injection was given as a 2 see pulse and special efforts were taken in order to standardize this procedure.
477
Mode of sacrifice The mice were killed at various times after the injection by exposure to a beam of microwave radiation focussed onto the head for a period of 3.5 sec. A Litton microwave oven (model MM 70/50) as modified by Medical Engineering Consultants, Lexington, Mass., U.S.A., was used. The source generated 2 kW (effective) radiation at a frequency of 2.45 GHz.
Dissection procedure The brain was excised, cleared of dura and surface blood vessels and placed onto a glass plate cooled by liquid nitrogen vapours. The frontal-parietal cortex, hippocampus and caudate nucleus from both right and left halves of the brain were quickly dissected, the halves pooled and frozen in liquid nitrogen. The dissection of the brain regions is greatly facilitated following microwave fixation with easy separation of the regions along their natural mechanical boundaries. The samples were not stored but treated immediately.
Sample preparation The tissue samples were weighed frozen (sample weights were reproducible within and between strains) and homogenized at 0-2 °C in 5 vols of 1 N formic acid-acetone (15:85 v/v), after the extraction procedure of Toru and Aprison 3°, at 2500 rev./min in glass-Teflon Potter-Elvehjem homogenizers. The homogenates were transferred to sealed centrifuge tubes and left to stand on ice for 30 rain with occasional mixing. After centrifugation at 20,000 x g for 5 rain aliquots of the supernatant were taken to determine the amounts of labelled Ch and ACh and their steadystate concentrations.
Determination of labelled Ch and A Ch Duplicate aliquots (30/d) of the supernatant were transferred to plastic Eppendorf tubes, frozen in liquid nitrogen and freeze-dried (45 rain). The residue was dissolved in 50/A of 25 mM sodium phosphate-citrate buffer pH 6.5 by vigorous mixing for 30 see. Labelled Ch and ACh were then quantitatively (85 ~) extracted into 50/tl of a solution of sodium tetraphenylboron (Sigma Chemicals, St Louis, Mo. U.S.A.) in di-isobutylketone (15 mg/ml) by mixing vigorously for 30 sec1°. After a brief centrifugation at 20,000 × g to separate the phases 40 #1 of the organic phase were transferred to fresh plastic Eppendorf tubes containing 40 #1 of 0.4 N HC1. The labelled Ch and ACh were transferred to the acid phase by vigorous mixing for 30 sec. After a brief centrifugation at 20,000 x g the organic phase was aspirated (vacuum pump) and the acid phase freeze-dried (1 h). The residue was dissolved in 30 #1 of a carrier mixture (1:1 v/v) of ACh chloride and Ch chloride (10 mM in water) and 20/A was applied to a Beckman paper strip for low-voltage electrophoresis. Electrophoresis was conducted in a Beckman electrophoresis cell, Durrum type, model R, with the buffer system described by Potter and Murphy24:25 ml concentrated formic acid, 75 ml glacial acetic acid, 900 ml glass distilled water, at 500 V (approx. 12 mA per cell) for 1 h. The paper strips were then
478 dried in a stream of warm air and developed by brief exposure to iodine vapours in order to localize and mark the bands. Good separation of Ch from ACh and from other Ch containing compounds was achieved under these conditions. Migration characteristics were: ACh band centre 7.6 cm, band width 4 ram; Ch band centre 8.8 cm, band width 5 mm. This gives a separation of 7.5 ram. No overlapping was found when control experiments were performed with labelled Ch and ACh standards. After the complete disappearance of the colouration the bands were cut out and placed into scintillation vials and 10 ml of the following fluorophor added (I litre of a solution of Omnifluor (New England Nuclear) 4 g/1 in toluene, 1 litre water- free dioxane, 600 ml methanol and 208 g naphthalene). Radioactivity was counted in an |ntertechnique liquid scintillation spectrometer.
Determination of the steady-state concentrations of Ch and A Ch Steady-state concentrations of Ch and ACh were determined essentially as described previously2L In this experiment 5 aliquots (10#!) of the formic acidacetone supernatant were taken from each sample and freeze-dried. Two tubes were used for the determination of Ch, two tubes treated by alkaline hydrolysis and used for the determination of ACh and the extra tube was used to control for the presence of radioactivity associated with the 3H-labelled Ch and ACh in the sample. All samples plus an external standard curve of Ch (0-100 pmoles) were treated by liquid cation exchange into sodium tetraphenylboron-di-isobutylketone for quantitative extraction of quaternary ammonium compounds. The extracted endogenous Ch plus the Ch derived by hydrolysis of ACh was then re-acetylated by incubation at 37 °C for 1 h with [l-14C]acetyl CoA and a partially purified preparation of rat brain choline acetyltransferase. The resulting [l-14C]labelled ACh was extracted by liquid cation exchange and an aliquot counted in a liquid scintillation spectrometer. The control tubes were treated identically except water replaced [l-14C]acetyl CoA during the incubation. The radioactivity associated with the all-labelled ACh and Ch was counted with the samples in the 14C channel of the liquid-scintillation counter. This quantity was almost negligible (5 % av.) in comparison to that associated with the 14C-labelled ACh and was subtracted from the sample disint./min before calculation of ACh and Ch concentrations. RESULTS Preliminary studies using Balb/c whole brain indicated that the maximum labeling of ACh occurred 60-90 sec following injection. The range of time points chosen for this study was selected on this evidence.
Radioactivity-time curves for Ch and A Ch The radioactivity associated with Ch and ACh in the three strains at different times after injection is shown in Tables I (hippocampus), II (caudate nucleus) and III (frontal-parietal cortex). The radioactivity associated with choline in the hippocampus of the Balb/c strain is higher at all time points than that for the DBA/2 or
479 TABLE I ACh and Ch radioactivity in the hippocampus An interstrain comparison of the radioactivity associated with Ch and A C h in the hippocampus at different times (0-120 sec) following the administration of 101 izCi (10 nmoles) [methyl-3H]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g x 10 -5 dr S.D. See text for details and analysis. Sec
Balb/c
20 30 50 60 70 80 90 120
DBA/2
C57BI/6
Ch
A Ch
Ch
A Ch
Ch
A Ch
11.08 dr 0.56 11.75+0.42 7.94 dr 0.27 6.10 dr 0.36 5.24 dr 0.27 3.96 dr 0.38 4.25 dr 1.03 4.39 dr 0.23
2.73 dr 0.21 4.07+0.19 5.62 dr 0.38 6.25 dr 0.42 6.17 dr 1.30 7.26 dr 0.41 7.01 4- 0.27 6.17 dr 0.11
12.02 i 0.56 10.035_1.02 4.92 dr 1.02 3.81 -4- 0.60 3.90 dr 0.40 3.80 dr 0.07 3.54 dr 0.36 3.42 ± 0.19
2.90 dr 0.40 3.91dr0.88 4.17 dr 0.33 4.31 dr 0.68 5.12 dr 0.46 5.59 dr 0.22 4.89 dr 0.69 4.22 dr 0.24
10.23 ± 0.80 8.805-0.99 5.60 dr 0.68 4.40 dr 0.14 4.42 dr 0.27 4.08 dr 0.14 4.01 dr 0.61 2.85 dr 0.88
1.42 ± 0.64 2.305-0.80 3.55 dr 0.24 4.15 dr 0.31 4.46 dr 0.56 5.44 -4- 0.24 4.80 dr 0.76 4.40 :~ 0.68
C57B1/6 strains which are not significantly different from each other. The same trend was also observed but to a lesser extent in the caudate nucleus and no significant difference was observed between strains in the frontal-parietal cortex. The rate of formation of labelled ACh in the hippocampus
and caudate nucleus
is s i g n i f i c a n t l y h i g h e r i n t h e B a l b / c s t r a i n t h a n i n C57B1/6 ( P < 0.001, a t all t i m e p o i n t s , S t u d e n t s t-test). I n t h e f r o n t a l - p a r i e t a l c o r t e x , h o w e v e r , t h i s r a t e is h i g h e r i n B a l b / c t h a n i n C57B1/6 o n l y u p t o 60 sec ( P < 0.001 a t e a c h t i m e p o i n t t o 6 0 sec,
TABLE II A Ch and Ch radioactivity in the caudate nucleus An interstrain comparison of the radioactivity associated with Ch and ACh in the caudate nucleus at different times (0-120 sec) following the administration of 101/,Ci (10 nmoles) [methyl-all]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g × 10-5 dr S.D. See text for details and analysis. Sec
Balb/c Ch
20 30 50 60 70 80 90 120
7.98 7.37 5.44 4.77 4.39 4.16 3.99 4.12
DBA/2 ACh
dr 0.56 dr 0.47 dr 0.52 dr 0.36 ± 0.57 dr 0.57 dr 0.76 dr 0.68
4.62 6.93 8.33 8.58 8.77 8.18 7.31 4.75
Ch dr 0.62 dr 0.40 dr 0.42 dr 0.51 ± 0.87 dr 0.44 dr 0.75 dr 0.25
9.07 7.10 5.30 4.54 4.29 3.74 3.06 2.75
C57Bl/6 ACh
dr 0.26 dr 0.68 dr 0.88 dr 1.56 dr 0.59 dr 0.08 d_ 0.06 dr 0.42
4.15 4.34 4.84 6.10 6.63 7.53 5.70 5.22
Ch dr 0.12 dr 0.22 4- 1.05 ± 1.10 dr 0.90 dr 0.83 dr 0.03 dr 0.81
8.04 6.93 5.18 4.38 3.98 3.62 3.71 2.73
ACh dr 0.17 dr 0.39 dr 0.95 dr 0.62 dr 0.11 dr 0.59 ± 0.35 dr 0.27
2.46 3.87 4.97 6.33 5.85 6.50 7.30 5.16
± 0.56 ± 0.83 q- 0.18 4- 0.73 dr 1.04 dr 0.39 d= 0.49 dr 0.29
480 TABLE II1 ACh and Ch radioactivity in the frontal-parietal cortex An interstrain comparison of the radioactivity associated with Ch and ACh in the frontal-parietal cortex at different times (0-120 sec) following the administration of 101 t~Ci (10 nmoles) [methyl-all]choline as an intravenous pulse injection. Mice were killed by 3.5 sec exposure to a beam of (2 kW) focussed microwave radiation. Values are the means of, at least, three determinations in duplicate and are expressed as disint./min/g × 10 ~ 5- S.D. See text for details and analysis. Sec
Balb/c Ch
20 30 50 60 70 80 90 120
13.01 12.65 8.33 6.67 5.94 4.81 4.92 4.43
5-0.88 5- 1.01 5- 0.76 5- 0.68 i 0.55 5- 0.35 5- 0.17 j- 0.57
DBA/2 ACh
Ch
3.73 5-0.56 5.81 5- 0.72 7.23 5- 1.37 7.32 ± 0.69 6.58 5- 0.72 7.71 i 0.68 6.83 5- 0.99 7.00 5~ 1.29
14.11 9.45 6.63 4.95 4.67 4.72 5.06 4.34
C57Bf/6 ACh
5-0.92 4.35 ±0.08 5- 0.65 4.87 5- 0.44 5-0.53 5.43 ± 0.55 ± 0.47 5.63 5- 0.42 5- 0.62 6.47 :£ 0.61 5- 0.41 6.71 £ 0.57 i 1.02 7.25 5- 0.80 ± 0.65 5.21 ~. 0.81
Ch 11.57 11.13 7.67 6.78 7.31 5.76 5.58 4.01
ACh ±0.56 5- 0.99 ~- 0.74 ± 0.53 5- 1.55 ± 0.62 i 0.63 ~ 0.72
1.59 5-0.64 3.07 £ 0.57 3.83 ± 1.03 5.37 ~: 0.72 5.41 i 0.87 5.83 ~:: 0:32 5.94 ± 1.14 6.08 5- 0.46
S t u d e n t s t-test). T h e rate o f f o r m a t i o n o f l a b e l l e d A C h in the D B A / 2 strain is intermediate. T h e m a x i m a o f t h e A C h c u r v e s o c c u r r e d w i t h i n the r a n g e 7 0 - 1 1 0 sec for all r e g i o n s a n d n o p a r t i c u l a r p a t t e r n was o b s e r v e d . Steady-state concentrations o f Ch and A C h T h e s t e a d y - s t a t e c o n c e n t r a t i o n s o f C h a n d A C h in t h e t h r e e r e g i o n s o f t h e i n b r e d strains a r e s h o w n in T a b l e IV. It c a n be seen t h a t : (1) t h e c o n c e n t r a t i o n s o f c h o l i n e are l o w f o l l o w i n g f o c u s s e d m i c r o w a v e radiationg,11; (2) m e a s u r e d c o n c e n t r a t i o n s o f c h o l i n e v a r y b e t w e e n t h e strains w i t h D B A / 2 s h o w i n g l o w e s t values, B a l b / c i n t e r m e d i a t e a n d C57B1/6 h i g h e s t v a l u e s in all t h r e e regions. (3) C57B1/6 s h o w TABLE IV lnterstrain comparison of the steady-state concentrations of Ch and ACh in the three brain regions Values are expressed as nmoles/g i S.D. Numbers in parentheses, n. Statistical evaluation was conducted by Students t-test. Strain
Balb/c DBA/2 C57B1/6
Hippocampus
Caudate n u c l e u s
Frontal-parietal cortex
Ch
ACh
Ch
ACh
Ch
ACh
14.6 ~ 3.0* (20) 10.5 ± 2.6* (17) 17.0±4.6 (16)
16.0 5- 3.8 (18) 14.6 £ 3.3* (17) 18.05-4.0 (17)
23.5 5- 4.7* (18) 15.8 ± 2.5** (17) 30.0±4.1 (10)
42.5 5- 7.0** (17) 45.3 ± 6.8** (14) 59.75-9.1 (11)
15.0 5- 3.7** (13) tl.9 5- 3.8** (14) 23.75-4.4 (12)
11.0 3- 2.3** (18) 11.5 ± 2.2** (15) 16.0±2.6 (10)
Different from C57BI/6: * P < 0.01; ** P < 0.001.
481 higher concentrations of ACh in all three regions than either DBA/2 or Balb/c which are not significantly different. The concentrations of Ch and ACh reported here are in the same range as those reported by Haubrich et al. 15 in a study using similar techniques for the cortex and striatum of rat brain. The steady-state concentrations of Ch and ACh are unaltered by the injection of the tracer dose (10 nmoles) of [methyl-3H]choline27.
Specific activity-time curves Jbr Ch and ACh The curves showing the specific activities of Ch and ACh as a function of time after injection are shown in Figs. 1 (hippocampus), 2 (caudate nucleus) and 3 (frontalparietal cortex). The curves have been generated from the means of the radioactivitytime curves for each strain (Tables I, II and III) and from the means of the steadystate concentrations of Ch and ACh for each strain (Table IV). For clarity of presentation only the curves for the Balb/c and C57B1/6 strains are shown; the curves for the DBA/2 strain being intermediate between these two extremes. It can be seen that the conditions for a precursor-product relationship in an open compartment model z8 are not completely fulfilled, i.e. the Ch and ACh specific activity curves do not cross at the maximum of the ACh specific activity but slightly before this is reached. This is more marked in the frontal-parietal cortex and hippocampus than in the caudate nucleus. Since Ch is undoubtedly the immediate precursor of ACh, a probable explanation of this is that a part of the free choline pool is used simultaneously in the synthesis of phosphorylated choline derivatives. In this respect,
15 m
10
\
e
\ 4 3
20
4O
t
eO
I
80
100
120
I
I
I
I
lk~s.
Fig. 1. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of lO nmoles (101 ~Ci) [methyl-aHlcholine in the hippocampus of the Balb/c and C57B1/6 strains. The curves have been generated from the data shown in Tables I and IV. Balb/c: Ch • 0, ACh © O ; C57BI/6: Ch [ ] - - - , , ACh [ ] - - - C ] .
482
a x
6
~s
4
.,
20
\
40
60
80
100
I
120
I
1
I
Se©s.
Fig. 2. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of 10 nmoles (101 #Ci) [methyl-3H]choline in the caudate nucleus of the Balb/c and C57B1[6 strains. The curves have been generated from the data shown in Tables II and IV. Balb/c: C h • - - - - - - • , ACh - ~ - - - - - - O ; C57B1/6: Ch I - - - I , ACh I ~ - - - [ ] .
D
x
E 1o
8
~7 m
6
"1 /
I 20
I
I
I
I
I
40
60
80
100
120
I
I
1
I
SlICI,
Fig, 3. The specific activities of Ch and ACh as a function of time after pulse intravenous injection of 10 nmoles (101 ~tCi) [methyl-SH]choline in the frontal-parietal cortex of the Batb/c and C57B1/6 strains. The curves have been generated from the data shown in Tables III and IV. Balb/c: Ch • • , ACh 0 - - - - , ' 3 ; C57B1/6: Ch I - - - H I , ACh [ ] - - - [ ] .
483 TABLE V Interstrain comparison of ,4Ch turnover rates in the three brain regions
Values for ACh turnover rates were calculated using the differential method of Jenden et a1.17; see text for details. Slopes were obtained by linear regression analysis and are expressed as nmoles/g/h ± S.D. (n = 5 in all cases). Statistical evaluation was conducted by Students t-test. Strain
Hippocampus
Caudate n u c l e u s
Frontal-parietal cortex
Balb/c DBA/2 C57B1/6
389 ± 5*** 342 ~ 15" 289 ~ 37
2468 ± 165"** 1554 5- 37* 1345 -Z 161
675 & 77*** 477 ~ 15"* 354 ~: 45
Different from C57B1/6: * P < 0.05; ** P -< 0.01; *** P < 0.001. we observed that the only other labelled choline metabolite of importance (Balb/c whole brain) during the time period studied (0-120 sec) was phosphorylcholine (PCh). Radioactivity associated with PCh increased linearly within this period and, at 120 sec, was just greater than that associated with ACh. This observation is in agreement with that of Lundholm and Sparf from a similar experiment 2° using mouse whole brain. Calculation o f A Ch turnover rates
The calculation of A C h turnover rates is based on the differential method described by Jenden et al. 17, Briefly this method is based on the assumption that the rate of appearance of labelled ACh at any one time after injection of Ch (dy*/dt) is directly proportional to the difference in the mole ratios of Ch and ACh ( x * ] x - - y*/y, where x and y refer to Ch and ACh, respectively, and the asterisk denotes a labelled variant) at that time. A plot of the form V/S is thus produced where V = dy*/dt and S ---- (x*/x - - y*/y) and the slope being a measure of the turnover rate. The major assumption in this open compartment kinetic model is that the mean specific activity of the Ch pool is a reasonable estimate of the specific activity of Ch in the ACh synthesis pool. Calculated values for the regional turnover rates of ACh in the three strains are shown in Table V. Due to the fact that the specific activity-time curves for Ch and ACh do not cross at the m a x i m u m specific activity of A C h but slightly before this (in the cortex and hippocampus) the straight lines obtained by the differential method give a positive intercept (significantly different from zero) on the dy*/dt axis which is similar for all three strains. As was pointed out by Jenden et al. 17, the assumption mentioned above is commonly made in calculations of this type and is justified only in an approximate sense. Furthermore, as was mentioned in the previous section we have evidence, although indirect, that compartmentation of the choline pool in the synthesis of choline lipids probably reduces the validity of this assumption. However, since the shift of the crossover point from the maximum of the A C h specific activity curve is small the calculated turnover rates are probably only slightly over-estimated in absolute values and, in any case, retain their relative values.
484 A number of other studies using focussed microwave procedures have estimated ACh turnover rates in mouse whole brain and in rat brain regions. Cheney et al. :~ using a pulse intravenous injection of phosphoryl [methyl-14C]-choline report a value for ACh turnover rate of 310 nmoles/g/h in mouse whole brain. Racagni et al. ~;. using an intravenous perfusion of phosphoryl [methyl-14C]choline, report values for ACh turnover rate of 1300 nmoles/g/h in the striatum and 200 nmoles/g/h in the cortex of the rat. Haubrich et al.l~, using a pulse intravenous injection of [methyl-3H]choline, reported values of 1447 nmoles/g/h for striatum and 367 nmoles/g,/h for cortex in rat brain. The values we report here for the three mouse strains are also in this range and, particularly for the C57B1/6 and DBA/2 strains, are very close to those reported for the same rat brain regions. DISCUSSION Previous studies on the correlation of behavioural patterns with the cholinergic system in inbred mouse strains have concentrated on the measurement of the activities of CAT and ACHE, the principal enzymes involved in ACh metabolism. Thus Tunnicliff et al. 31 in a study of 7 inbred strains reported that whole brain CAT levels were higher in Balb/c than in DBA/2 which were, in turn, higher than in C57B1/6 and that these activities showed a good inverse correlation with locomotor activity in these strains. In earlier reports from our laboratoryT, 21 it was observed that a good correlation existed between higher CAT activity in the frontal and temporal cortex and better learning ability in maze and avoidance testing procedures in the DBA/2 and C57B1/6 strains. Recently we have also reported a correlation between hippocampal CAT activity and the phenomenon underlying the improvement in performance ('reminiscence') observed in the Balb/c but not in the C57B1/6 strain when a 6-24 h interval is spaced between a first (partial acquisition) training session and the second trial I~. In all these cases it has been suggested that the differences in CAT activity may reflect underlying differences in ACh metabolism. Our findings of strain differences in regional ACh turnover rates provide some evidence for this postulate. There appears to be a good correlation between CAT activity measured in vitro or in vivo ACh turnover rate and the differences in avoidance, maze and wheel running behaviours observed between these strains. This may not, however, be absolute evidence of an obligatory link between in vivo CAT activity and ACh turnover rate. Although CAT has often been termed the rate-limiting enzyme in ACh synthesis it is clear that the availability of substrate, particularly Ch, is a major factor in the regulation of ACh synthesis in physiological conditions 14 and the possibility that the factors which determine this availability (for review see ref. 9) may also vary between strains cannot be excluded. Nevertheless, several observations can be made. The values we report for ACh turnover rates in the striatum show an inverse correlation with locomotor activity differences between the strains23, 81. It has already been proposed that the striatal cholinergic system exerts an inhibitory function on the level of locomotor activity and pharmacological inhibition of cholinergic transmission in the striatum is often
485 associated with m o t o r dysfunction o r elevated l o c o m o t o r activity (for review see ref. 25). T h e inverse c o r r e l a t i o n between strain differences in the A C h t u r n o v e r rate in the s t r i a t u m a n d l o c o m o t o r activity m a y be i n t e r p r e t e d in this w a y a n d thus lend s u p p o r t to this hypothesis. F u r t h e r m o r e , the higher A C h t u r n o v e r rate in the h i p p o c a m p u s o f the Balb/c as c o m p a r e d to the C57B1/6 strain agrees well with the previously o b s e r v e d higher h i p p o c a m p a l C A T activity in the Balb/c strain a n d p r o v i d e s further evidence for the h y p o t h e s i s t h a t the higher 'reminiscence' c a p a c i t y observed in this strain m a y be related to a m o r e active h i p p o c a m p a l cholinergic m e t a b o l i s m 16. Likewise the higher A C h t u r n o v e r rates in the f r o n t a l - p a r i e t a l cortex o f the Balb/c a n d D B A / 2 strains are in a g r e e m e n t with the p r e v i o u s l y observed c o r r e l a t i o n between better learning abilities o f these strains a n d the higher C A T activity in the frontal cortex o f the D B A / 2 as c o m p a r e d with the C57B1/6 strain 7. ACKNOWLEDGEMENTS A. Ebel is a Charg6e de R e c h e r c h e au C N R S . The a u t h o r s t h a n k Elisabeth G e r l i n g e r a n d D a n i e l G r u b e r for their excellent technical assistance.
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