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Diurnal rhythms in neurotransmitter receptor binding and choline acetyltransferase activity: Different patterns in two rat lines of Wistar origin
Brain Research, 370 (1986) 54-60
54
Elsevier BRE 11582
Diurnal Rhythms in Neurotransmitter Receptor Binding and Choline Acetyltransferase Activity: Different Patterns in Two Rat Lines of Wistar Origin SUSANNE JENNI-EIERMANN, H.P. von HAHN and C.G. HONEGGER
Institut fiir ExperirnentelleAlternsforschung, Felix Platter-Spital and Abteilung Neurobiologie, Departement Forschung, Kantonsspital, Basel (Switzerland) (Accepted August 13th, 1985)
Key words: rat line - - diurnal cycle - - ligand receptor binding - - choline acetyltransferase activity - - line-dependent cycle
Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (CHAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cyclesin the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings - - such major differences in two closely related rat lines - - is obvious in any attempt to compare receptor-binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain-dependency of cyclic functions, we are not aware that line-dependency has previouslybeen described. INTRODUCTION In the course of a study on diurnal changes in neurotransmitter receptor binding in rat brain during ageing6, we observed that in 2 commercially bred rat lines, both of which were of Wistar origin, the 3month-old animals that were to be used as the basis for the ageing study gave different diurnal patterns of receptor binding. This difference was found for a n u m b e r of ligands and in various brain regions. We therefore used only one of these rat lines for studying the ageing process, It appeared to be of importance to describe our findings with 'Wistar rats ~, as they are usually described in the literature, in order to put emphasis on the necessity of giving precise information on the origin of the animal line used in central nervous system (CNS) research. The few reports on diurnal variations in receptor binding in rat brain have been limited to a single animal line, and thus attempts to replicate these studies in different laboratories may lead to considerable difficulties if the diurnal cycles vary as much as they did in the two lines we compare here
(that were, of course, carefully controlled for all other methodological artefacts, including time of year). Furthermore, comparison of pharmacological studies involving receptor binding must take into account that experiments made at only one time of day may lead to erroneous conclusions not only due to disregarding temporal organization but also if the rat lines used in different studies have receptor cycles with different phase positions, such as those we demonstrate here. MATERIALS AND METHODS
Rat lines. Two rat lines, obtained from local commercial breeders, and both of Wistar origin, were used. The K f m : W I S T line (Mad6rin A G , Frillinsdorf) has been established since 1980, the Frillinsdorf Albino (Frill.) line (lnstitut for Biologisch-Medizinische Forschung, Fiillinsdorf) since 1955. Both lines are internationally registered. Male rats were purchased aged 3 months, and kept 4 to a cage for 3 weeks on a 12:12 h l i g h t - d a r k cycle (dark: 18.00-06.00 h) before the experiments were started.
Correspondence: H.P. von Hahn, Institut fiJr Experimentelle Alternsforschung, Felix Platter-Spiral, CH-4055 Basel, Switzerland. 0006-8993/86/$03.50© 1986Elsevier Science Publishers B.V. (Biomedical Division)
55
Experimental procedure. All preparative and analytical procedures were carried out by the same person (S.J.-E.) and at the same time of year for both rat lines. Four animals were killed by guillotine every 4 h sequentially during the same 24-h cycle. The brains were immediately removed, dissected on ice, and hippocampus, cerebellum, striatum and brainstem were frozen a t - 7 0 °C. Membrane preparations. Each individual brain region (there was no pooling of tissue from several animals) was homogenized twice with 20 vol. Trizma 7.7 buffer in a Polytron homogenizer and sedimented for 10 min at 48,000 g. The second pellet was diluted and resuspended in 100-200 parts buffer, depending on the receptor to be determined, and preincubated for 15 min at 37 °C. Receptor binding experiments. Standard methods were used. References to the original descriptions are given in Table I, together with the list of the labeled ligands and their displacing agents. Choline acetyltransferase (CHAT) activity. ChAT activity was determined by the method of Fonnum5 on 5/~1 of the first homogenate, using 0.2 mM labeled acetyl-coenzyme A as substrate. Protein. This was determined by the method of Lowry et al.8. Statistical analysis. Differences according to time of day were tested by one-way A N O V A . The Gtest 11was applied to test for significance in the differences in amplitude and time-locking of phase patterns in the 2 rat lines. Differences in the 24-h means
between 2 lines were tested for significance by Student's t-test. RESULTS
Occurrence of circadian receptor and C h A T activity cycles Fig. 1 A - H presents the results for the 8 ligand-receptor pairs, and Fig. 2 A - C for the 3 ChAT activity determinations made at 4-h intervals during one complete 24-h cycle. [3H]Quinuclidinyl benzilate ([3H]QNB) binding in hippocampus and striatum, [3H]spiperone ([3H]SPI) binding in striatum, [3H]serotonin (5-[3H]HT) binding in brainstem and ChAT activity in hippocampus showed significant diurnal variations in both lines, ~,-[3H]aminobutyric acid ([3H]GABA) only in Ffillinsdorf cerebellum and ChAT activity only in Kfm striatum. Neither of the two lines showed significant diurnal cyclic variation in binding of [3H]dihydroalprenolol ([3H]DHA) in hippocampus, [3H]QNB and [3H]naloxone ([3H]NAL) in brainstem, and ChAT activity in brainstem. Thus, both lines had significant cyclic variation in 6 of 11 parameters tested. 24-Hour means The 24-h means were calculated from the individual determinations in each cycle. The differences in overall means between the two rat lines were highly significant in 6 out of the 11 parameters (Fig. 3). In all regions except brainstem the Kfm rats had means
TABLE I
Receptor types and labeledligands used in the binding experiments QNB, quinuclidinyl benzilate; DHA, dihydroalprenolol; SPI, spiperone; GABA, 7-aminobutyric acid; 5-HT, 5-hydroxytryptamine; LSD, lysergicacid diethylamide; NAL, naloxone.
Receptor type (brain region) Muscarinic-cholinergic antagonist (hippocampus, striatum, brainstem)
Labeled ligand
Concentration Displacing (nM) agent
Concentration Referenceto method (ltM)
[3H]QNB
1.0
atropine
a-Adrenergic antagonist (hippocampus) [3H]DHA
2.0
propanolol
1
Bylund and Snyderl
Dopaminergic antagonist (striatum)
[3H]SPI
1.0
haloperidol
1
Creese et al.2
GABAergic agonist (cerebellum)
[3H]GABA
Serotonergic agonist (brainstem)
5-[3H]HT
2.0
LSD
Opiate antagonist (brainstem)
[3H]NAL
5.0
levorphanol
25.0
muscimol
10
10
Wastek and Yamamura j2
Enna and Snyder4
1
Peroutka and Snyderl°
10
Pasternak and Snyder9
56
with respect to the light-dark cycle
100-250% above those of the Fiillinsdorf rats.
Independently of possible changes in the absolute levels of ligand binding or ChAT activity, as given by the 24-h means, a difference in the cycle phase (times of maximum and minimum) between the two rat lines may lead to erroneous conclusions if ligand binding is determined at different times of the day. It is therefore essential to know precisely the time-pattern of the respective cycle phases when testing for ligand binding to brain membrane preparations. To determine possible differences in phase position between the two rat lines, each individual time-point of a ligand-receptor or ChAT cycle was compared to the
Differences in cycle amplitudes The amplitude of a cycle is calculated as [maximum-minimum]/mean x 100, giving % of the 24-h mean. Fig. 4 shows the distribution of the amplitudes between the two lines. The Kfm rats have significantly higher cycle amplitudes than the Fiillinsdorf rats: the G-test for amplitudes under and over 50% (Kfm: 4 < 50%, 7 > 50%; Fiiltinsdorf: 10 < 50%, 1 > 50%) gave P < 0.01.
Differences in the phase position of receptor rhythms
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Fig. 1. Diurnal variations of 8 ligand receptor pairs in 4 regions of Kfm and Frillinsdorf (Frill) rat brains. Four individual brains at each time-point. The 07 h point is given both at the beginning and at the end of the 24-h period. Means + S.E.M. ~,, Kfm rats; ~ , Frillinsdoff rats. For each cycle, the amplitude (% of 24-h mean) and the significance of cyclic variation (P value for ANOVA) are as follows: A: [3H]QNB in hippocampus: Kfm: 85%, P < 0.001; Frill: 25%, P = 0.029. B: [3H]DHA in hippocampus: Kfm 20%, n.s. ; Frill: 17%, n.s. C: [3H]QNB in striatum: Kfm: 81%, P < 0.001; Frill: 53%, P = 0.049. D: [3H]SPI in striatum: Kfm: 72%, P = 0.045; Frill: 39%, P = 0.006. E: [3H]GABA in cerebellum: Kfm: 36%, n.s.; Frill: 37%, P = 0.003. F: [3H]QNB in brainstem: Kfm: 72%, n.s.; Frill: 25%, n.s. G: 5-[3H]HT in brainstem: Kfm: 65%, P = 0.023; Frill: 44%, P = 0.005. H: [3H]NAL in brainstem: Kfm: 46%, n.s.; Frill: 29%, n.s.
c o r r e s p o n d i n g 24-h m e a n , and classified ' + ' o r ' - ' if higher o r l o w e r t h a n t h e m e a n . T h e n , t h e n u m b e r o f coincidences in t h e signs b e t w e e n the t w o rat lines was c o u n t e d for e a c h o f the 11 cycle d e t e r m i n a t i o n s . T h e results o f this test are g i v e n in Fig. 5. T h e n u m ber of c o i n c i d e n c e s c o u l d r a n g e f r o m 0/6 (180 ° out of
phase) to 6/6 (identical p h a s e position). Six of o u r 11 d e t e r m i n a t i o n s i n d i c a t e d a large d i f f e r e n c e in p h a s e position. O n l y two of 11 d e t e r m i n a t i o n s had 5/6 coincidences, and n o n e 6 / 6 , i.e. a highly c o r r e l a t e d rhythm.
58 ditions (caging, nutrition, l i g h t - d a r k cycle, season, handling and experimentator) any differences must
DISCUSSION In discussing the data presented in this report we
be, we assume, the result of real differences in neuro-
are primarily concerned with the problem of different diurnal receptor ligand rhythms in the same brain
transmitter regulatory mechanisms. We find significant differences between the Kfm
regions of two rat lines of Wistar origin. Since both
and the Fiillinsdorf rats in the following points: (1) 24-h mean (Fig. 3) in 6 of 11 measurements, with
animal groups were kept under strictly identical con-
i strtatum
B
hippocarnpus
A
c 50
10
I
P
8 D
C
0
07
11
15
19
23
03
07
07 h
' brainstem
~. 15
11
15
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03
07 h
Cl
b
o Q.
ca E ~: 10 o
u
5
o E
0
07
11
15
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23
03
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h
Fig. 2. Diurnal variations of ChAT activity in 3 brain regions of Kfm and Frillinsdorf rats. Four individual brains at each time-point. Means _+S.E.M. ~1~,Kfm rats; ~ , Frillinsdorf (Frill) rats. For each cycle, the amplitude (% of 24-h mean) and the significanceof cyclic variation (P value for ANOVA) are as follows: A: striatum: Kfm: 83%, P = 0.002; Frill: 37%, n.s. B: hippocampus: Kfm: 83%, P = 0.036; Frill: 30%, P = 0.025. C: brainstem: Kfm: 27%, n.s.; Frill: 28%, n.s.
59 ii
receptor
binding 50
c
i
ChATactivity -
2000
i
o O.
= 30
E 0
E 1000 G
!'i
0 QNB DHA QNB SPI hippocamstriatum pus
GABA QNB 5HT NAL C&re-brain s t e m helium
hippo- s t r i campus atum
brainSt&m
Fig. 3.24-h means of 8 ligand receptor pairs, and of ChAT activity in 3 brain regions of Kfm and Fiillinsdorf rats. Means + S.E.M. Black columns: Kfm rats; white columns: Fiillinsdorf rats. ¢ indicates P < 0.001 for the difference between the two lines. Kfm values 2 0 0 - 2 5 0 % those of the Fiillinsdorf in 4 measurements. (2) Magnitude of cycle amplitudes: in 7 of 11 m e a s u r e m e n t s Kfm had amplitudes over 50%, Fiillinsdorf rats only in 1 of 11 (Fig. 4). Thus, the Kfm rats tended to show a more marked cyclic
variation than the Fiillinsdorf animals, whose cycles were more attenuated. (3) Cycle phases: in 6 of 1 1 measurements, m a r k e d difference in phase position was observed between the two lines (Fig. 5), with complete reversal of the acrophases in the case of
5
5
O
3
'o[D_ 1 11 21 31 41 51 61 71 81 --10 --20 --30 --40 --50 --60 --70 --80 --90 amplitudes in %
Fig. 4. Distribution of % amplitudes of 11 diurnal cycles (8 ligand receptor pairs, 3 ChAT activity) for Kfm rats (black columns) and Fiillinsdorf rats (white columns).
016 116
216
3/6
4/6
5/6 6/6
Fig. 5. Distribution of phase position of receptor binding and ChAT activity rhythms between the Kfm and Ffillinsdorf rat lines. For details of the calculation method, see Results. 0/6 = inverse phase position; 1/6 and 2/6 = marked difference in phase position; 3/6, 4/6, 5/6 = some difference in phase position, 6/6 = identical phase position.
60 ChAT activity in hippocampus. W h e t h e r the observed variations are true endogenous circadian rhythms is not the concern of this paper (see ref. 6 for discussion). W h a t we wish to emphasise is that: (1) diurnal variations in ligand receptor binding do occur, but (2) the amplitude and timing of these variations d e p e n d not only on brain region but on the rat line used. A l t h o u g h strain differences in such r e c e p t o r rhythms have been previously r e p o r t e d 14 and may be of functional significance, this variability between similar (but not identical) rat lines is not at all evident. Its importance is obvious in any attempt to compare receptor binding studies per se (Bmax varies according to time of day) between laboratories using the same rat strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of drugs. While there
REFERENCES 1 Bylund, D.B. and Snyder, S.H., Beta-adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol., 12 (1976) 568-580. 2 Creese, I., Schneider, R. and Snyder, S.H., 3H-Spiroperidol labels dopamine receptors in pituitary and brain, Eur. J. Pharmacol., 46 (1977) 377-381. 3 Daszuta, A., Faudon, M. and Ternaux, J.-P., Uptake of [3H]serotonin and [3H]noradrenaline in the Raphe nuclei and the locus coeruleus of C57BL/6 Rholco and BALB/c Cenclo mice at three times of the day, Neurosci. Lett., 29 (1982) 141-146. 4 Enna, S.J. and Snyder, S.H., Properties of gamma-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Research, 100 (1975) 81-97. 5 Fonnum, F., Radiochemical microassays for the determination of choline acetyltransferase and acetyl choline esterase activities, Biochem. J., 115 (1969) 465-472. 6 Jenni-Eiermann, S., von Hahn, H.P. and Honegger, C.G., Circadian variations of neurotransmitter binding in three age groups of rats, Gerontology (Basel), 31 (1985) 138-149. 7 Lemmer, B., Caspari-lrving, G. and Weimer, R., Straindependency in motor activity and in concentration and turnover of catecholamines in synchronized rats, Pharmacol. Biochem. Behav., 15 (1981) 173-178.
are some reports on tions such as m o t o r over 3,7.13,15, we are has previously been
strain-dependency of cyclic funcactivity and catecholamine turnnot aware that line-dependency described.
ACKNOWLEDGEMENTS We acknowledge gratefully support from the Fritz Hoffmann-La Roche F o n d s (Arbeitsgemeinschaft No. 157), the Emilia G u g g e n h e i m Stiftung der Naturforschenden Gesellschaft Basel, and the Stiftung fiir Experimentelle Alternsforschung, Felix PlatterSpital, Basel. S.J.-E. thanks Dr. A . Dravid, Sandoz Ltd., Basel, for permission to use l a b o r a t o r y space and for advice and assistance. Dr. A n n a Wirz-Justice, Psychiatrische Universit~itsklinik Basel, advised us in the p r e p a r a t i o n of the manuscript.
8 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 Pasternak, G.W. and Snyder, S.H., Identification of novel high affinity opiate receptor binding in rat brain, Nature (London), 253 (1975) 563-565. 10 Peroutka, S.J. and Snyder, S.H., Multiple serotonin receptors: differential binding of aH-5-hydroxytryptamine, 3Hlysergic acid diethylamide and 3H-spiroperidol, Mol. Pharmacol., 16 (1979) 687-699. 11 Sokal, R.R. and Rohlf, F.J., Biometry, 2nd edn., W.H. Freeman and Co., San Francisco, 1981. 12 Wastek, G.J. and Yamamura, H.I., Biochemical characterisation of the muscarinic-cholinergic receptor in human brain: alterations in Huntington's disease, Mol. Pharmacol., 14 (1978) 768-780. 13 Wax, T.M., Effects of age, strain and illumination intensity on activity and self-selection in light-dark schedules in mice, J. Comp. Physiol. Psychol., 91 (1977)51-62. 14 Wirz-Justice, A., Kr~iuchi, K., Campbell, I.C. and Feer, H., Adrenoceptor changes in spontaneous hypertensive rats: a circadian approach, Brain Research, 262 (1983) 233242. 15 Wollnik, F., Untersuchungen zur Genetik der Ultradian-und 24 h-Periodik bei den Ratteninzuchtstiimmen LEW/Ztm und A Cl/Ztm, Dissertation, Universitfit Hannover, 1984.
54
Elsevier BRE 11582
Diurnal Rhythms in Neurotransmitter Receptor Binding and Choline Acetyltransferase Activity: Different Patterns in Two Rat Lines of Wistar Origin SUSANNE JENNI-EIERMANN, H.P. von HAHN and C.G. HONEGGER
Institut fiir ExperirnentelleAlternsforschung, Felix Platter-Spital and Abteilung Neurobiologie, Departement Forschung, Kantonsspital, Basel (Switzerland) (Accepted August 13th, 1985)
Key words: rat line - - diurnal cycle - - ligand receptor binding - - choline acetyltransferase activity - - line-dependent cycle
Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (CHAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cyclesin the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings - - such major differences in two closely related rat lines - - is obvious in any attempt to compare receptor-binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain-dependency of cyclic functions, we are not aware that line-dependency has previouslybeen described. INTRODUCTION In the course of a study on diurnal changes in neurotransmitter receptor binding in rat brain during ageing6, we observed that in 2 commercially bred rat lines, both of which were of Wistar origin, the 3month-old animals that were to be used as the basis for the ageing study gave different diurnal patterns of receptor binding. This difference was found for a n u m b e r of ligands and in various brain regions. We therefore used only one of these rat lines for studying the ageing process, It appeared to be of importance to describe our findings with 'Wistar rats ~, as they are usually described in the literature, in order to put emphasis on the necessity of giving precise information on the origin of the animal line used in central nervous system (CNS) research. The few reports on diurnal variations in receptor binding in rat brain have been limited to a single animal line, and thus attempts to replicate these studies in different laboratories may lead to considerable difficulties if the diurnal cycles vary as much as they did in the two lines we compare here
(that were, of course, carefully controlled for all other methodological artefacts, including time of year). Furthermore, comparison of pharmacological studies involving receptor binding must take into account that experiments made at only one time of day may lead to erroneous conclusions not only due to disregarding temporal organization but also if the rat lines used in different studies have receptor cycles with different phase positions, such as those we demonstrate here. MATERIALS AND METHODS
Rat lines. Two rat lines, obtained from local commercial breeders, and both of Wistar origin, were used. The K f m : W I S T line (Mad6rin A G , Frillinsdorf) has been established since 1980, the Frillinsdorf Albino (Frill.) line (lnstitut for Biologisch-Medizinische Forschung, Fiillinsdorf) since 1955. Both lines are internationally registered. Male rats were purchased aged 3 months, and kept 4 to a cage for 3 weeks on a 12:12 h l i g h t - d a r k cycle (dark: 18.00-06.00 h) before the experiments were started.
Correspondence: H.P. von Hahn, Institut fiJr Experimentelle Alternsforschung, Felix Platter-Spiral, CH-4055 Basel, Switzerland. 0006-8993/86/$03.50© 1986Elsevier Science Publishers B.V. (Biomedical Division)
55
Experimental procedure. All preparative and analytical procedures were carried out by the same person (S.J.-E.) and at the same time of year for both rat lines. Four animals were killed by guillotine every 4 h sequentially during the same 24-h cycle. The brains were immediately removed, dissected on ice, and hippocampus, cerebellum, striatum and brainstem were frozen a t - 7 0 °C. Membrane preparations. Each individual brain region (there was no pooling of tissue from several animals) was homogenized twice with 20 vol. Trizma 7.7 buffer in a Polytron homogenizer and sedimented for 10 min at 48,000 g. The second pellet was diluted and resuspended in 100-200 parts buffer, depending on the receptor to be determined, and preincubated for 15 min at 37 °C. Receptor binding experiments. Standard methods were used. References to the original descriptions are given in Table I, together with the list of the labeled ligands and their displacing agents. Choline acetyltransferase (CHAT) activity. ChAT activity was determined by the method of Fonnum5 on 5/~1 of the first homogenate, using 0.2 mM labeled acetyl-coenzyme A as substrate. Protein. This was determined by the method of Lowry et al.8. Statistical analysis. Differences according to time of day were tested by one-way A N O V A . The Gtest 11was applied to test for significance in the differences in amplitude and time-locking of phase patterns in the 2 rat lines. Differences in the 24-h means
between 2 lines were tested for significance by Student's t-test. RESULTS
Occurrence of circadian receptor and C h A T activity cycles Fig. 1 A - H presents the results for the 8 ligand-receptor pairs, and Fig. 2 A - C for the 3 ChAT activity determinations made at 4-h intervals during one complete 24-h cycle. [3H]Quinuclidinyl benzilate ([3H]QNB) binding in hippocampus and striatum, [3H]spiperone ([3H]SPI) binding in striatum, [3H]serotonin (5-[3H]HT) binding in brainstem and ChAT activity in hippocampus showed significant diurnal variations in both lines, ~,-[3H]aminobutyric acid ([3H]GABA) only in Ffillinsdorf cerebellum and ChAT activity only in Kfm striatum. Neither of the two lines showed significant diurnal cyclic variation in binding of [3H]dihydroalprenolol ([3H]DHA) in hippocampus, [3H]QNB and [3H]naloxone ([3H]NAL) in brainstem, and ChAT activity in brainstem. Thus, both lines had significant cyclic variation in 6 of 11 parameters tested. 24-Hour means The 24-h means were calculated from the individual determinations in each cycle. The differences in overall means between the two rat lines were highly significant in 6 out of the 11 parameters (Fig. 3). In all regions except brainstem the Kfm rats had means
TABLE I
Receptor types and labeledligands used in the binding experiments QNB, quinuclidinyl benzilate; DHA, dihydroalprenolol; SPI, spiperone; GABA, 7-aminobutyric acid; 5-HT, 5-hydroxytryptamine; LSD, lysergicacid diethylamide; NAL, naloxone.
Receptor type (brain region) Muscarinic-cholinergic antagonist (hippocampus, striatum, brainstem)
Labeled ligand
Concentration Displacing (nM) agent
Concentration Referenceto method (ltM)
[3H]QNB
1.0
atropine
a-Adrenergic antagonist (hippocampus) [3H]DHA
2.0
propanolol
1
Bylund and Snyderl
Dopaminergic antagonist (striatum)
[3H]SPI
1.0
haloperidol
1
Creese et al.2
GABAergic agonist (cerebellum)
[3H]GABA
Serotonergic agonist (brainstem)
5-[3H]HT
2.0
LSD
Opiate antagonist (brainstem)
[3H]NAL
5.0
levorphanol
25.0
muscimol
10
10
Wastek and Yamamura j2
Enna and Snyder4
1
Peroutka and Snyderl°
10
Pasternak and Snyder9
56
with respect to the light-dark cycle
100-250% above those of the Fiillinsdorf rats.
Independently of possible changes in the absolute levels of ligand binding or ChAT activity, as given by the 24-h means, a difference in the cycle phase (times of maximum and minimum) between the two rat lines may lead to erroneous conclusions if ligand binding is determined at different times of the day. It is therefore essential to know precisely the time-pattern of the respective cycle phases when testing for ligand binding to brain membrane preparations. To determine possible differences in phase position between the two rat lines, each individual time-point of a ligand-receptor or ChAT cycle was compared to the
Differences in cycle amplitudes The amplitude of a cycle is calculated as [maximum-minimum]/mean x 100, giving % of the 24-h mean. Fig. 4 shows the distribution of the amplitudes between the two lines. The Kfm rats have significantly higher cycle amplitudes than the Fiillinsdorf rats: the G-test for amplitudes under and over 50% (Kfm: 4 < 50%, 7 > 50%; Fiiltinsdorf: 10 < 50%, 1 > 50%) gave P < 0.01.
Differences in the phase position of receptor rhythms
,
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200
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_
-
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, 07
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03
07 h
Fig. 1. Diurnal variations of 8 ligand receptor pairs in 4 regions of Kfm and Frillinsdorf (Frill) rat brains. Four individual brains at each time-point. The 07 h point is given both at the beginning and at the end of the 24-h period. Means + S.E.M. ~,, Kfm rats; ~ , Frillinsdoff rats. For each cycle, the amplitude (% of 24-h mean) and the significance of cyclic variation (P value for ANOVA) are as follows: A: [3H]QNB in hippocampus: Kfm: 85%, P < 0.001; Frill: 25%, P = 0.029. B: [3H]DHA in hippocampus: Kfm 20%, n.s. ; Frill: 17%, n.s. C: [3H]QNB in striatum: Kfm: 81%, P < 0.001; Frill: 53%, P = 0.049. D: [3H]SPI in striatum: Kfm: 72%, P = 0.045; Frill: 39%, P = 0.006. E: [3H]GABA in cerebellum: Kfm: 36%, n.s.; Frill: 37%, P = 0.003. F: [3H]QNB in brainstem: Kfm: 72%, n.s.; Frill: 25%, n.s. G: 5-[3H]HT in brainstem: Kfm: 65%, P = 0.023; Frill: 44%, P = 0.005. H: [3H]NAL in brainstem: Kfm: 46%, n.s.; Frill: 29%, n.s.
c o r r e s p o n d i n g 24-h m e a n , and classified ' + ' o r ' - ' if higher o r l o w e r t h a n t h e m e a n . T h e n , t h e n u m b e r o f coincidences in t h e signs b e t w e e n the t w o rat lines was c o u n t e d for e a c h o f the 11 cycle d e t e r m i n a t i o n s . T h e results o f this test are g i v e n in Fig. 5. T h e n u m ber of c o i n c i d e n c e s c o u l d r a n g e f r o m 0/6 (180 ° out of
phase) to 6/6 (identical p h a s e position). Six of o u r 11 d e t e r m i n a t i o n s i n d i c a t e d a large d i f f e r e n c e in p h a s e position. O n l y two of 11 d e t e r m i n a t i o n s had 5/6 coincidences, and n o n e 6 / 6 , i.e. a highly c o r r e l a t e d rhythm.
58 ditions (caging, nutrition, l i g h t - d a r k cycle, season, handling and experimentator) any differences must
DISCUSSION In discussing the data presented in this report we
be, we assume, the result of real differences in neuro-
are primarily concerned with the problem of different diurnal receptor ligand rhythms in the same brain
transmitter regulatory mechanisms. We find significant differences between the Kfm
regions of two rat lines of Wistar origin. Since both
and the Fiillinsdorf rats in the following points: (1) 24-h mean (Fig. 3) in 6 of 11 measurements, with
animal groups were kept under strictly identical con-
i strtatum
B
hippocarnpus
A
c 50
10
I
P
8 D
C
0
07
11
15
19
23
03
07
07 h
' brainstem
~. 15
11
15
19
23
03
07 h
Cl
b
o Q.
ca E ~: 10 o
u
5
o E
0
07
11
15
19
23
03
07
h
Fig. 2. Diurnal variations of ChAT activity in 3 brain regions of Kfm and Frillinsdorf rats. Four individual brains at each time-point. Means _+S.E.M. ~1~,Kfm rats; ~ , Frillinsdorf (Frill) rats. For each cycle, the amplitude (% of 24-h mean) and the significanceof cyclic variation (P value for ANOVA) are as follows: A: striatum: Kfm: 83%, P = 0.002; Frill: 37%, n.s. B: hippocampus: Kfm: 83%, P = 0.036; Frill: 30%, P = 0.025. C: brainstem: Kfm: 27%, n.s.; Frill: 28%, n.s.
59 ii
receptor
binding 50
c
i
ChATactivity -
2000
i
o O.
= 30
E 0
E 1000 G
!'i
0 QNB DHA QNB SPI hippocamstriatum pus
GABA QNB 5HT NAL C&re-brain s t e m helium
hippo- s t r i campus atum
brainSt&m
Fig. 3.24-h means of 8 ligand receptor pairs, and of ChAT activity in 3 brain regions of Kfm and Fiillinsdorf rats. Means + S.E.M. Black columns: Kfm rats; white columns: Fiillinsdorf rats. ¢ indicates P < 0.001 for the difference between the two lines. Kfm values 2 0 0 - 2 5 0 % those of the Fiillinsdorf in 4 measurements. (2) Magnitude of cycle amplitudes: in 7 of 11 m e a s u r e m e n t s Kfm had amplitudes over 50%, Fiillinsdorf rats only in 1 of 11 (Fig. 4). Thus, the Kfm rats tended to show a more marked cyclic
variation than the Fiillinsdorf animals, whose cycles were more attenuated. (3) Cycle phases: in 6 of 1 1 measurements, m a r k e d difference in phase position was observed between the two lines (Fig. 5), with complete reversal of the acrophases in the case of
5
5
O
3
'o[D_ 1 11 21 31 41 51 61 71 81 --10 --20 --30 --40 --50 --60 --70 --80 --90 amplitudes in %
Fig. 4. Distribution of % amplitudes of 11 diurnal cycles (8 ligand receptor pairs, 3 ChAT activity) for Kfm rats (black columns) and Fiillinsdorf rats (white columns).
016 116
216
3/6
4/6
5/6 6/6
Fig. 5. Distribution of phase position of receptor binding and ChAT activity rhythms between the Kfm and Ffillinsdorf rat lines. For details of the calculation method, see Results. 0/6 = inverse phase position; 1/6 and 2/6 = marked difference in phase position; 3/6, 4/6, 5/6 = some difference in phase position, 6/6 = identical phase position.
60 ChAT activity in hippocampus. W h e t h e r the observed variations are true endogenous circadian rhythms is not the concern of this paper (see ref. 6 for discussion). W h a t we wish to emphasise is that: (1) diurnal variations in ligand receptor binding do occur, but (2) the amplitude and timing of these variations d e p e n d not only on brain region but on the rat line used. A l t h o u g h strain differences in such r e c e p t o r rhythms have been previously r e p o r t e d 14 and may be of functional significance, this variability between similar (but not identical) rat lines is not at all evident. Its importance is obvious in any attempt to compare receptor binding studies per se (Bmax varies according to time of day) between laboratories using the same rat strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of drugs. While there
REFERENCES 1 Bylund, D.B. and Snyder, S.H., Beta-adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol., 12 (1976) 568-580. 2 Creese, I., Schneider, R. and Snyder, S.H., 3H-Spiroperidol labels dopamine receptors in pituitary and brain, Eur. J. Pharmacol., 46 (1977) 377-381. 3 Daszuta, A., Faudon, M. and Ternaux, J.-P., Uptake of [3H]serotonin and [3H]noradrenaline in the Raphe nuclei and the locus coeruleus of C57BL/6 Rholco and BALB/c Cenclo mice at three times of the day, Neurosci. Lett., 29 (1982) 141-146. 4 Enna, S.J. and Snyder, S.H., Properties of gamma-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Research, 100 (1975) 81-97. 5 Fonnum, F., Radiochemical microassays for the determination of choline acetyltransferase and acetyl choline esterase activities, Biochem. J., 115 (1969) 465-472. 6 Jenni-Eiermann, S., von Hahn, H.P. and Honegger, C.G., Circadian variations of neurotransmitter binding in three age groups of rats, Gerontology (Basel), 31 (1985) 138-149. 7 Lemmer, B., Caspari-lrving, G. and Weimer, R., Straindependency in motor activity and in concentration and turnover of catecholamines in synchronized rats, Pharmacol. Biochem. Behav., 15 (1981) 173-178.
are some reports on tions such as m o t o r over 3,7.13,15, we are has previously been
strain-dependency of cyclic funcactivity and catecholamine turnnot aware that line-dependency described.
ACKNOWLEDGEMENTS We acknowledge gratefully support from the Fritz Hoffmann-La Roche F o n d s (Arbeitsgemeinschaft No. 157), the Emilia G u g g e n h e i m Stiftung der Naturforschenden Gesellschaft Basel, and the Stiftung fiir Experimentelle Alternsforschung, Felix PlatterSpital, Basel. S.J.-E. thanks Dr. A . Dravid, Sandoz Ltd., Basel, for permission to use l a b o r a t o r y space and for advice and assistance. Dr. A n n a Wirz-Justice, Psychiatrische Universit~itsklinik Basel, advised us in the p r e p a r a t i o n of the manuscript.
8 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 Pasternak, G.W. and Snyder, S.H., Identification of novel high affinity opiate receptor binding in rat brain, Nature (London), 253 (1975) 563-565. 10 Peroutka, S.J. and Snyder, S.H., Multiple serotonin receptors: differential binding of aH-5-hydroxytryptamine, 3Hlysergic acid diethylamide and 3H-spiroperidol, Mol. Pharmacol., 16 (1979) 687-699. 11 Sokal, R.R. and Rohlf, F.J., Biometry, 2nd edn., W.H. Freeman and Co., San Francisco, 1981. 12 Wastek, G.J. and Yamamura, H.I., Biochemical characterisation of the muscarinic-cholinergic receptor in human brain: alterations in Huntington's disease, Mol. Pharmacol., 14 (1978) 768-780. 13 Wax, T.M., Effects of age, strain and illumination intensity on activity and self-selection in light-dark schedules in mice, J. Comp. Physiol. Psychol., 91 (1977)51-62. 14 Wirz-Justice, A., Kr~iuchi, K., Campbell, I.C. and Feer, H., Adrenoceptor changes in spontaneous hypertensive rats: a circadian approach, Brain Research, 262 (1983) 233242. 15 Wollnik, F., Untersuchungen zur Genetik der Ultradian-und 24 h-Periodik bei den Ratteninzuchtstiimmen LEW/Ztm und A Cl/Ztm, Dissertation, Universitfit Hannover, 1984.