63
Behavioural Brain Research, 39 (1990) 63-71 Elsevier BBR01064
Rats with anxious or non-anxious type of exploratory behaviour differ in their brain CCK-8 and benzodiazepine receptor characteristics J. H a r r o 1'2, R . - A . K i i v e t 2, A. L a n g 3 a n d E. V a s a r 1 ~Psychopharmacology Laboratory, 2Department of Pharmacology, and 3Hormonal Regulation Laboratory, Tartu University, Tartu, Estonia (U.S.S.R.) (Received 18 January 1990) (Revised version received 12 February 1990) (Accepted 19 February 1990)
Key words: Behavioral difference; Elevated plus-maze; Anxiety; Cholecystokinin octapeptide receptor; Benzodiazepine receptor; Benzodiazepine inverse agonist; Cholecystokinin octapeptide agonist; Rat
Rats with high and low exploratory activity in an elevated plus-maze model of anxiety were separated into subgroups termed 'non-anxious' and 'anxious' respectively according to the number of sectors the animals crossed and the total amount of time they spent in the open part of the plus-maze. The binding parameters ofbenzodiazepine and eholecystokininoctapeptide (CCK-8) receptors in frontal cortex and hippocampus of selected animals were studied and compared to an animal group representing the total mean scores and to home-cage controls. It was established that anxious rats had a significantly lower number of benzodiazepine receptors in frontal cortex as compared to non-anxious animals and in hippocampus as compared to home-cage controls. There was also a decreased number of CCK-8 receptors in hippocampus of anxious rats as compared to the non-anxious and control groups. Non-anxious animals had a significantly lower number of CCK-8 receptors in frontal cortex than anxious and control rats. Acute treatment of rats with anxiogenic benzodiazepine inverse agonist FG 7142 (10 and 20 mg/kg) did not influence benzodiazepine binding in brain regions under investigation but caused upregulation of CCK-8 receptor binding in frontal cortex. On the other hand, CCK-8 analogues caerulein and pentagastrin, administered in doses which inhibit exploratory activity in plus-maze (100 or 500 ng/kg respectively), decreased the number of benzodiazepine binding sites in rat frontal cortex if injected intraperitoneally but did not affect CCK-8 binding. The present findings indicate that benzodiazepine and CCK-8 receptor binding characteristics in brain undergo rapid and behaviourally specific changes during stressful events. INTRODUCTION
An enormous amount of evidence indicates that the GABA/benzodiazepine/barbiturate receptor complex plays a key role in the development of anxiety states. The qualitative nature of behavioural effects of several anxiolytic and anxiogenic drugs can be predicted by their certain biochemical effects on the subunits of this receptor c o m p l e x 29. Stressful manipulations have been shown to alter the radioligand binding character-
istics to GABA 2,13, benzodiazepine 13,2°,21 or Cl--channel-associated 27 binding sites. On the other hand, acute treatment with GABA antagonists which exert anxiogenic effects at subconvulsive doses also affects binding characteristics of GABA receptor complex ~,12. Cholecystokinin octapeptide (CCK-8), a neuropeptide that is localized in high concentrations in several brain regions ~, seems to be a comediator of GABA in cerebral cortex and hippocampus t4. It is well documented that there is a
Correspondence: J. Harro, Tartu University, Department of Pharmacology, 18 l~likooli Street, 202 400 Tartu, Estonia, U.S.S.R. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 functional antagonism between CCK-8 and benzodiazepine anxiolytics in the brain and in peripheral o r g a n s 15"16'3°. Benzodiazepines at low doses antagonize selectively the CCK-8-induced activation of rat hippocampal pyramidal neurones 3. Recently we have shown that CCK-8 receptor agonists (caerulein, pentagastrin) at very low doses have an anxiogenic-like effect on rodents in the elevated plus-maze test. Proglumide, an antagonist of CCK-8, completely attenuated these effects of CCK-8 agonists 8'9. Proglumide was also able to block the effects of the anxiogenic fl-carboline derivative D M C M and pentylenetetrazole, the compounds that are believed to exert their action through depression of GABAergic transmission 8. Therefore, it seems possible that the interaction between GABA- and CCK-8-ergic mechanisms takes place in the development of anxiety states. Recently a novel test of anxiety, an elevated plus-maze, was introduced and behaviourally, physiologically and pharmacologically validated 23. In experiments on mice substantial differences were detected between the individuals in the exploratory activity in this apparatus. There was a significantly lower number of benzodiazepine and GABA receptors in cerebral cortex of animals displaying more 'anxious' behaviour in the elevated plus-maze 25. Therefore we decided to study more completely the binding characteristics of benzodiazepine and CCK-8 receptors in the brains of rats, selected according to their exploratory activity in elevated plus-maze. Earlier studies have revealed that depending on the methods used to induce stress reactions in animals, quite inconsistent and even opposite results can be produced in radioreceptor binding in isolated brain regions. The main idea of this study was that if exposition to the plus-maze situation is a stressful event for rats as has been suggested 23, the rats who display more 'anxious' behaviour (i.e. less exploratory activity) would be more adequate subjects for evaluating the influence of distress on receptor binding characteristics in the brain. To verify further this initial hypothesis the effect of pretreatment of rats with anxiogenic drugs at doses in which they decrease exploratory activity in the plus-maze test on radioligand binding was determined.
MATERIALS AND METHODS
Animals and housing conditions Experiments were performed on adult male albino rats of no particular strain (Rappolovo Farm, Leningrad) weighing 225-250 g at the time of experiment. Rats were, upon arrival in our laboratory, randomly allocated to groups of 12 animals and were familiarized for at least 3 weeks to the new environmental conditions (a 12-h light/12-h dark cycle, lights on between 09.00 and 21.00 h; home cages 80 x 50 × 20 cm). Food and tap water were available ad libitum.
Procedure of behavioural experiments Apparatus. We have used an elevated plusmaze initially described by Pellow et al. 23 which consists of two open arms (45 × 10 cm) and two enclosed arms (45 x 10 x 40 cm) with an open roof. The maze was elevated to the height of 25 cm. To determine the exploratory activity of rats in the open part of the maze either open arm was divided into 3 equal sectors in our modification of apparatus 8. Assessment of anxiety. As the exploratory activity in an elevated plus-maze is thought to be a measure of state anxiety, the following parameters were registered by an observer: (1)the latency period of the first entry into the open part of the maze, (2)the number of sectors crossed in the open part, and (3) total time spent in the open part of the plus-maze. We have used a slight modification in one of the registration measures (the number of sectors crossed in the open part of the maze versus the percentage of open/total arm entries) because in the animal population used by us the total number of arm entries was quite low. Selectionprocedure. Behavioural tests were performed between 10.00 and 13.00 h. Each rat was placed at the centre of the plus-maze facing one of the closed arms of it. After a 4-min test period the animal was removed from the apparatus, rapidly carried to a separate room and killed by decapitation. When the behavioural testing was over the data were immediately calculated and the following groups were formed: (1)rats with high exploratory activity, (2) rats with low exploratory activity, and (3)rats with exploratory activity which could best represent the total measure, i.e.
65 intermediate exploratory activity. Two measures formed the basis of selection: (a) the number of sectors crossed, and (b) the total time spent in the open part of plus-maze. The control group was formed from rats that were decapitated without testing in the plus-maze. They were from the same population and the formation of this group was randomized.
Drug treatment Animals were taken from their home-cages and treated in a randomized order with vehicle, FG 7142 (N-methyl-/~-carboline-3-carboxamide) 10 mg/kg, FG 7142 20 mg/kg, or vehicle, caerulein 100 ng/kg, pentagastrin 500 ng/kg. These doses were used according to previous evidence from this and other laboratories 6,8,9 that they are anxiogenic in rats. All drugs were given intraperitoneally 30 min (FG 7142) or 15 min (CCK-8 agonists) prior to the decapitation. Each treatment group consisted of 5 rats. FG 7142 was dissolved in distilled water with the help of a drop of Tween80. Caerulein and pentagastrin were dissolved in saline.
Radioligand binding experiments Brain dissection procedure. Brain regions were dissected according to Heffner et al. lo. Briefly, rat brain was placed into cutting block and sliced at right angles to the sagittal axis with the help of razor blades. The initial razor blade sliced through the sagittal plane of the brain at the level of the body of the anterior commissure according to the atlas of KOnig and Klippe117. Three razor blades were inserted anterior to that blade along the rostral extent of the brain at intervals of 1.5 mm. Two razor blades were inserted posterior to the initial blade at intervals of 2.0 mm, followed by two more razor blades at intervals of 1.5 mm. The brain was thus divided into 9 sections, numbered rostrally to caudally. Brain regions were dissected from the slices obtained. Frontal cortex includes the cortical tissue from section 1 as well as the cortical tissue superior to the rhinal sulcus from sections 2 and 3. The hippocampus was separated from the midbrain and overlying cerebral cortex from sections 7 and 8 based on its distinct morphological appearance. The pooled
tissue of 5 animals per group was used in the subsequent binding studies.
Preparation of the crude synaptosomalfraction. This was performed as described elsewhere25. Dissected cerebral tissues were homogenized in 30 vols of ice-cold Tris-HC1 (pH = 7.4) using a Potter-S glass-Teflon homogenizer (1000rpm, 10 passings). The membranes were washed twice in Tris-HC1 buffer by centrifugation (48 000 g for 20 min) and resuspension. [3H]Flunitrazepam binding assay. In vitro binding studies to label benzodiazepine receptors were carried out in the presence of 0.125-8 nM of [3H]flunitrazepam (spec. act. 85.0Ci/mmol, Amersham Radiochemicals) using a total incubation volume of 500/~1. Ten #M offlunitrazepam was used to determine non-specific binding. After 60 min incubation on ice the reaction was stopped by rapid filtration over Whatman GF/B filters. The filters were washed with 4 x 3 ml of ice-cold Tris-HC1 buffer and dropped into scintillation vials. [3H]CCK-8 binding assay. The final pellets were resuspended in HEPES buffer (HEPES 10raM, NaC1 130mM, MgC1z 5 m M , KC1 5 mM, EDTA 1 mM, pH 6.5 adjusted with 1 N NaOH) containing bovine serum albumin (0.5 mg/ml). The CCK-8 receptor labelling was carried out in the presence of 0.05-2 nM labelled ligand ([propionyl-3H]CCK-8-sulfated, 60.5 Ci/ mmol, Amersham Radiochemicals) in polypropylene tubes. To determine non-specific binding 100 nM of caerulein was added. After 120min incubation at room temperature the reaction was terminated by centrifugation in a Beckman Microfuge 12 (11 000 rpm for 3 min). The supernatants were aspirated, and the pellets were washed with 2 × 0.5 ml of ice-cold HEPES buffer. The tips of the tubes were cut and dropped into scintillation vials.
Data analysis Mann-Whitney U-test was used for analysis of behavioural data. The specific binding of radioligands was defined as the difference between the degree of binding in the absence and in the presence of unlabelled ligand.
66 Proteins were determined according to the procedure of Lowry et all9, using bovine serum albumin as a standard. Saturation curves were initially analyzed according to the method of Scatchard 26 and then ftted using the non-linear least squares regression analysis. The apparent maximal number of binding sites (Bmax) is expressed as fmol/mg protein and constant K d as nmol/1.
RESULTS
mentors. Rats separated into the 'high activity' subgroup had a shorter latency period, they crossed more sectors and spent more time in the open part of the plus-maze. They seemed not to have any conflict between exploration drive and fear of open areas. The opposite was true in the case of rats separated into the 'low activity' subgroup. These rats did not explore open areas hardly at all. The 'intermediate representative' subgroup was formed from rats whose scores in measures (2) and (3) were the most correspondent to the respective values of the total group. We were not able to match the total group and the intermediate representative subgroup with regard to the latency period because the disposition of individual values did not correspond perfectly to normal distribution which was the case in another measures.
Description of rats with high, low and intermediate exploratory activity
Comparison of benzodiazepine receptor characteristics in selected subgroups of rats
As indicated in Table I, it was possible to separate subgroups of rats with high and low exploratory activity in an elevated plus-maze from a total group. The measures of exploratory activity of individual animals corresponded to normal distribution in the total group and the number of rats in subgroups reflects the discretion of experi-
In vitro experiments demonstrated that the number of [3H]flunitrazepam binding sites in frontal cortex was significantly lower in the low activity subgroup as compared to the high activity subgroup (Table II). The number of these binding sites in frontal cortex of the high activity subgroup was also higher if compared with home-cage con-
Drugs and chemicals F G 7142, flunitrazepam and caerulein were the generous gifts from Ferrosan, Hoffmann-La Roche and Farmitalia-Carlo Erba respectively. Tris and HEPES were purchased from Sigma. All other chemicals were from Reachim (U.S.S.R.).
TABLE I
Selection experiment according to the exploratory activity of rats in an elevated plus-maze All values are means + S.E.M. from three separate selection experiments. Statistical comparisons are done using M a n n - W h i t n e y U-tests.
Group
Number of rats
Latency of first open part entry (s)
Total group
31
47 + 12
8.5 + 1.1
High activity subgroup
5
9 + 1"
18.2 + 1.5'
Intermediate representative subgroup
5
Low activity subgroup
5
17 +_ 4 173 + 24*'**
* Significantly different from Total group, P < 0.01. ** Significantly different from High activity subgroup, P < 0.01.
Number of crossed sectors in open part
8.5 + 0.5 0.9 + 0.6*'**
Total time spent in open part (s) 43 _+ 6 94 _+ 19"
37 + 4 8 + 3*'**
67 TABLE II
Scatchard analysis of saturation data of [ 3H]flunitrazepam binding in rats selected according to their exploratory activity in an elevated plus-maze All values are means + S.E.M. from three separate selection experiments. Statistical comparisons are done using Student's t-test.
Test group
Frontal cortex B max
Home-cage controls High activity Intermediate activity Low activity
1367 1693 1487 1230
Hippocampus Kd
± 115 + 81" + 116 _+ 120"*
1.23 1.29 1.31 1.20
B max + + + +
0.21 0.05 0.16 0.13
1015 830 810 670
Kd + + + +
121 85 133 70*
2.01 1.65 1.60 1.03
+ 0.21 + 0.26 + 0.20 _+ 0.08*
* Significantly different from Home-cage controls, P < 0.05. ** Significantly different from High activity group, P < 0.05.
trois. Data obtained from the control group and intermediate activity subgroup were quite similar. There were no significant differences between any groups in binding affinity of [ 3H]flunitrazepam. On the other hand, both high activity and intermediate activity subgroups had slightly less benzodiazepine binding sites in hippocampus as compared to control animals, but these differences did not reach significance. However, the hippocampi of rats belonging to the low activity subgroup contained less benzodiazepine binding sites as compared to home-cage controls. The only detectable difference in the affinity of [ 3H]flunitrazepam binding in hippocampus was also between control group and low activity subgroup (Table II).
Comparison of CCK-8 receptor characteristics in selected subgroup of rats There was a significantly lower number of [ 3H]CCK-8 binding sites in frontal cortex of animals from the high activity subgroup as compared to the control rats and low activity subgroup (Table III). The number of CCK-8 binding sites in the frontal cortex of the intermediate activity subgroup was also decreased if compared with control rats, although not significantly. However, it also did not differ significantly from the score of the high activity subgroup. Scatchard analyses of binding in hippocampal membranes revealed that the low activity subgroup had a significantly lower density of CCK-8 binding sites than the control rats and high activity subgroup in this brain
TABLE III
Scatchard analysis of saturation data of FH]CCK-8 binding in rats selected according to their exploratory activity in an elevated plus-maze All values are means _+ S.E.M. from three separate selection experiments. Statistical comparisons are done using Student's t-test.
Test group
Frontal cortex B,,,ax
Home-cage controls High activity Intermediate activity Low activity
36.6 19.8 29.5 36.5
+ + + +
Hippocarnpus Kd
3.4 2.8* 3.0 3.7**
0.52 0.52 0.43 0.49
* Significantly different from Home-cage controls, P < 0.05. ** Significantly different from High activity group, P < 0.05.
Bmax + 0.14 + 0.16 _+ 0.18 + 0.15
12.0 14.0 9.3 7.6
+ + + +
Kd
1.3 1.3 1.0 0.8"**
0.19 0.20 0.14 0.14
+ + + +
0.02 0.03 0.02 0.02
68 TABLE IV
Scatchard analysis of saturation data of [3H] CCK-8 and [3H ]flunitrazepam binding in ratfrontal cortex after systemic administration of FG 7142, caerulein, or pentagastrin Experiments were performed twice with very similar results. All values are from one representative binding study. Statistical comparisons are done using Student's t-test.
Test group
[ 3H / C CK-8 B,,,,~
Control F G 7142 10 mg/kg FG 7142 20 mg/kg Control Caerulein 100 ng/kg Pentagastrin 500 ng/kg
38.8 50.5 58.8 34.3 29.8 45.1
[ 3H ]flunitrazepam Kd
_+ 2.2 + 4.0* + 4.8** + 1.6 + 5.2 _+ 3.9
0.23 0.36 0.32 0.38 0.53 0.57
Bmax + 0.03 + 0.05 + 0.05 + 0.02 _+ 0.06 + 0.07
1461 1649 1683 1405 965 905
Kd + + + + + +
99 111 85 75 23* 40*
1.86 1.89 1.79 1.81 1.70 1.34
_+ 0.36 _+ 0.42 _+ 0.23 _+ 0.32 _+ 0.11 _+ 0.17
* Significantly different from respective Control, P < 0.05. ** Significantly different from respective Control, P < 0.01.
region. The intermediate activity subgroup had a score between those of the high and low activity subgroups, not significantly different from one or the other, and also not significantly different from controls (Table III). Binding affinity did not differ between the groups. Effect of anxiogenic drug treatment on rat brain benzodiazepine and CCK-8 receptor binding Acute administration of FG 7142 dose-dependently enhanced the number of [ 3H]CCK-8 binding sites in rat frontal cortex (Table IV). At the same time binding affinity was decreased but this effect just missed significance (P = 0.058). In
hippocampal membranes any effect of F G 7142 treatment on CCK-8 receptors was not observable. [ 3 H ]Flunitrazepam binding was not influenced by F G 7142 treatment in the brain regions under investigation. On the other hand, the treatment of rats with low doses of CCK-related peptides resulted in a decrease of [ 3 H ]flunitrazepam binding in frontal cortex (Table IV) without any change in hippocampus (Table V). CCK-8 receptors, neither in frontal cortex nor in hippocampus, were affected by that treatment as suggested by the results from Scatchard analysis (Tables IV and V).
TABLE V
Scatchard analysis of saturation data of [ 3H ] C CK-8 and [ 3H ]flunitrazepam binding in rat hippocampus after systemic administration of FG 7142, caerulein, or pentagastrin Experiments were performed twice with very similar results. All values are from one representative binding study.
Test group
[ 3H]CCK-8 Bmax
Control FG 7142 10 mg/kg FG 7142 20 mg/kg Control Caerulein 100 ng/kg Pentagastrin 500 ng/kg
13.3 15.6 14.3 12.1 14.3 13.1
+ + + + + +
[ 3H]flunitrazep am gd
3.4 4.7 2.1 2.2 2.5 1.9
0.18 0.22 0.13 0.17 0.21 0.17
+ 0.02 + 0.05 +_ 0.03 + 0.03 + 0.04 + 0.03
Bmax
Kd
884 931 863 973 840 830
1.18 1.47 1.38 1.25 1.87 1.35
_+ 87 + 94 + 99 +_ 43 + 16 _+ 25
+_. 0.19 +_ 0.18 +_ 0.27 _+ 0.26 + 0.10 + 0.17
69 DISCUSSION The behavioural, physiological and pharmacological validation of an elevated plus-maze as a measure of anxiety in the rat 23 gives rise to the idea that if individual animals display different exploratory activity in this apparatus, it would reflect differences in their anxiety rate. So we would like to regard the rats with high exploratory activity in the plus-maze as non-anxious and the rats with low exploratory activity as anxious animals. For selection procedure, we have used two measures, (a) the number of sectors crossed, and (b) the total time spent in the open part of plusmaze. The second one is essentially the same as one of the validated measures of Pellow et al. 23 and the first one correlates excellently with the second one (r = 0.98, Spearman rank correlation, P = 4.68 x 10-7). Rats confined to both the closed or open arms of the plus-maze had significantly elevated corticosterone concentrations compared with homecage controls 23. So the plus-maze test could be regarded as a stressful events for animals. There is evidence that 'stressed' rodents have increased 21"28 or decreased 13"18'2° benzodiazepine binding in forebrain. Lower benzodiazepine binding in hippocampus was attributed to 'anxious' rats selected according to the responsiveness in a conflict test 22. In the plus-maze selection experiment where anxious and non-anxious subgroups of mice were separated, it was shown that anxious mice had a lower number of benzodiazepine and G A B A receptors in cerebral cortex 25. Both binding measures did not differ between groups in cerebellar membranes. As there was not a control group from animals not exposed to the elevated plus-maze, it was impossible to say if these neurochemical differences had been present before the experimental procedure or arose during the behavioural test. We have now demonstrated that rats who seem to feel less anxious in a disturbing novel environment have a higher density of benzodiazepine binding sites in both frontal cortex and hippocampus as compared to anxious animals. However, if compared with home-cage controls it appears that in the case of high activity rats there is an upregulation of benzodiazepine
binding in frontal cortex whereas in the case of low activity rats downregulation of benzodiazepine binding sites in hippocampus occurs. So it should be emphasized that a stressful event may cause an increase as well as a decrease in the number of benzodiazepine binding sites depending on the adaptational level of the particular animal in the altered situation. The number of CCK-8 receptor sites in frontal cortex and hippocampus of anxious rats was different from the parameters of non-anxious animals. Namely, rats of the high activity group had considerably less [ 3H]CCK-8 sites in frontal cortex than control animals and rats of the low activity group. Rats with intermediate exploratory activity also had a number of CCK-8 receptor sites intermediate between the scores of the high and low activity groups. In the hippocampus, the number of CCK-8 binding sites was decreased after the plus-maze test in the low activity group, which differed from both the control and high activity group. Once more, the group with intermediate activity in the plus-maze had an intermediate score in CCK-8 binding sites between high and low activity groups. So it seems that downregulation of CCK-8 receptors in frontal cortex is required for the adaptation to novel environment, and downregulation of these sites in the hippocampus is connected with anxious behaviour. It has been recognized that benzodiazepines and CCK-8 act as functional antagonists. In this point of view it is interesting to note that in nonanxious rats benzodiazepine and CCK-8 receptors in frontal cortex are regulated in opposite directions during stressful events. To further verify the idea that balance between benzodiazepine and CCK-8 receptor function reflects the anxiety level of animals, we investigated the effect of anxiogenic drug administration on receptor characteristics. F G 7142, a benzodiazepine receptor inverse agonist, may induce severe anxiety in both animals 24 and man 5. Treatment of rats with this anxiogenic compound did not influence [3H]flunitrazepam binding in rat brain. This is consistent with results of Ito etal. ~1"12, who have shown that GABA antagonists bicuculline and picrotoxin, which also in-
70 duce anxiety at subconvulsive doses, do not affect benzodiazepine binding in brain after acute systemic administration. However, the number of CCK-8 receptor sites in frontal cortex following F G 7 1 4 2 treatment was dose-dependently increased, suggesting a predominance of CCK-8ergic processes over GABAergic ones in this brain region where G A B A and CCK-8 are believed to act as cotransmitters. On the other hand, it was recently found that CCK-related peptides may exert anxiogenic-like effects both in rodents 8'9 and in man 4. In a previous study we found that the systemic administration of caerulein and pentagastrin may downregulate benzodiazepine binding only in a certain (anxiogenic) dose range, larger, behaviourally sedative doses being inefficient 7. In this study we have reproduced the finding that cortical benzodiazepine receptor density is decreased following caerulein and pentagastrin administration at doses which depress exploratory activity in the plus-maze. However, it appears that CCK-8 binding was not affected. Thus it seems, that at least on the level of frontal cortex, in anxiety state there is CCK-8 receptor binding upregulation and/or benzodiazepine receptor binding downregulation, whereas in emotional well-being the opposite may be true. In summary, our results presented here support the idea that the characteristics o f b e n z o d i a z e p i n e and CCK-8 receptors are of importance in the genesis of anxiety. Secondly we would like to emphasize the importance of observing individual variability in animal populations when molecular mechanisms regulating emotionality are under investigation. If we do not know a great deal about how a manipulation affects the animal, not very much can be learned from subsequent biochemical analyses. ACKNOWLEDGEMENTS We are grateful to Ferrosan, H o f f m a n n - L a Roche and F a r m i t a l i a - C a r l o Erba for their generous supplying of F G 7142, flunitrazepam and caerulein respectively.
REFERENCES 1 Beinfeld, M.C., Cholecystokinin in the central nervous system: a minireview, Neuropeptides, 3 (1983) 421-427. 2 Biggio, G., The action of stress, ]~-carbolines, diazepam and Ro 15-1788 on GABA receptors in the rat brain. In G. Biggio and E. Costa (Eds.), Benzodiazepine recognition Site Ligands: Biochemistry and Pharmacology, Raven, New York, 1983, pp. 105-119. 3 Bradwejn, J. and De Montigny, C., Benzodiazepines antagonize cholecystokinin-induced activation of rat hippocampal neurones, Nature (Lond.), 312 (1984) 363-364. 4 De Montigny, C., Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers: preliminary findings, Arch. Gen. Psychiatry, 46 (1989) 511-517. 5 Dorow, R., Horowski, R., Paschelke, G., Amin, M. and Braestrup, C., Severe anxiety induced by FG 7142, a /3-carboline ligand for benzodiazepine receptors, Lancet, 9 (1983) 98-99. 6 File, S.E., Pellow, S. and Braestrup, C., Effects of the /3-carboline, FG 7142, in the social interaction test of anxiety and the holeboard: correlations between behaviour and plasma concentrations, Pharmacol. Biochem. Behav., 22 (1985) 941-944. 7 Harro, J., Kiivet, R.-A., Lang, A., P61d, M. and Vasar, E., The effect of systemic administration of CCK-8 agonists on CNS receptor binding parameters. In L. Allikmets (Ed.), Molecular Pharmacology of receptors III, Tartu University Press, Tartu, 1989, pp. 67-77. 8 Harro, J., P61d, M., Vasar, E. and Allikmets, L., The role of CCK-8-ergic mechanisms in the regulation of emotional behaviour in rodents, Zh. Vyss. Nerv. Deyat., 39 (1989) 877-883 (in Russian). 9 Harro, J., P61d, M. and Vasar, E., Anxiogenic-likeaction of caerulein, a CCK-8 receptor agonist, in the mouse: influence of acute and subchronic diazepam treatment, Naunyn-Schmiedeberg's Arch. PharmacoL, in press. 10 Heffner, T.G., Hartman, J.A. and Seiden, L. S., A rapid method for the regional dissection of the rat brain, Pharmacol. Biochem. Behav., 13 (1980)453-456. 11 Ito, Y., Lim, D. K., Hoskins, B. and Ho, I.K., BicucuUine up-regulation of GABAA receptors in rat brain, J. Neurochem., 51 (1988) 145-152. 12 Ito, Y., Lim, D.K., Nabeshima, T. and Ho, I.K., Effects ofpicrotoxin treatment on GABAAreceptor supramolecular complexes in rat brain, J. Neurochem., 52 (1989) 1064-1070. 13 Kiivet, R.-A., Harro, J., R~tgo,L. and P61d, M., Changes in GABA and benzodiazepine receptors after foot-shock in the rat: influence of diazepam. In L. Allikmets (Ed.), Molecular Pharmacology of receptors H, Tartu University Press, Tartu, 1988, p. 45-54. 14 Kosaka, T., Kosaka, K., Tateishi, K., Hamaoka, Y., Yanaihara, N., Wu, J.-Y. and Hama, K., GABAergic neurons containing CCK-8-1ike and/or VIP-like immu-
71
15
16
17
18
19
20
21
22
23
noreactivities in the rat hippocampus and dentate gyrus, J. Comp. Neurol., 239 (1985) 420-430. Kubota, K., Sugaya, K., Matsuda, I., Matsuoka, Y. and Itonaga, M., Caerulein antagonism by benzodiazepines in the food intake in mice, Jpn. J. Pharmacol., 39 (1985) 120-122. Kubota, K., Sun, F.-Y., Sugaya, K. and Sunagane, N., Reversal of antinociceptive effect of caerulein by benzodiazepine, J. Pharmacobio-Dyn., 9 (1986) 428-431. K6nig, J.F.R. and Klippel, R.A., The RatBrain:A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. Lippa, A.S., Kleifner, C.A., Yunger, L., Sano, C.M., Smith, W.V. and Beer, B., Relationship between benzodiazepine receptors and experimental anxiety in rats, Pharmacol. Biochem. Behav., 9 (1978) 853-856. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, RJ., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Medina, J.H., Novas, M.L., Wolfman, C.N.V., Levi de Stein, M. and De Robertis, E., Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress, Neuroscience, 9 (1983) 331-335. Miller, L.G., Thompson, M.L., Greenblatt, D.J., Deutsch, S.I., Shader, R.I. and Paul, S.M., Rapid increase in brain benzodiazepine receptor binding following defeat stress in mice, Brain Res., 414 (1987) 395-400. Patel, J.B., Stengel, J., Malik, J.B. and Enna, SJ., Neurochemical characteristics of rats distinguished as benzodiazepine responders and non-responders in a new conflict test, Life Sci., 34 (1984) 2647-2653. Pellow, S., Chopin, P., File, S.E. and Briley, M., Valid-
24
25
26 27
28
29
30
ation of open:closed arm entries in an elevated plusmaze as a measure of anxiety in the rat, J. Neurosci. Methods, 14 (1985) 149-167. Petersen, E.N. and Jensen, L.H., Proconflict effect of benzodiazepine receptor inverse agonists and other inhibitors of GABA function, Eur. J. Pharmacol., 103 (1984) 91-97. R~igo,L., Kiivet, R.-A., Harro, J. and P61d, M., Behavioral differences in an elevated plus-maze: correlation between anxiety and decreased number of GABA and benzodiazepine receptors in mouse cerebral cortex, NaunynSchmiedeberg's Arch. Pharmacol., 337 (1988) 675-678. Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N. IT. Acad. Sci., 54 (1949) 660-672. Schwartz, R.D., Wess, M.J., Labarca, R., Skolnick, P. and Paul, S.M., Acute stress enhances the activity of the GABA receptor-gated chloride ion channel in brain, Brain Res., 411 (1987) 151-155. Soubrie, P., Thiebot, M.H., Jobert, A., Montrastruc, S.L., Hery, F. and Hamon, M. Decreased convulsant potency of picrotoxin and pentylenetetrazol and enhanced [3H]flunitratepam cortical binding following stressful manipulations in rats, Brain Res., 189 (1980) 505-517. Stephens, D.N., Schneider, H.H., Kehr, W., Jensen, L.H., Petersen, E. and Honore, T., Modulation of anxiety by ~-carbolines and other benzodiazepine receptor ligands: relationship of pharmacological to biochemical measures of efficacy, Brain Res. Bull., 19 (1987) 309-318. Sugaya, K., Matsuda, I. and Kubota, K. Inhibition of hypothermic effect of cholecystokinin by benzodiazepines and a benzodiazepine antagonist, Ro 15-1788, in mice, Jpn. J. Pharmacol., 39 (1985) 277-280.
Behavioural Brain Research, 39 (1990) 63-71 Elsevier BBR01064
Rats with anxious or non-anxious type of exploratory behaviour differ in their brain CCK-8 and benzodiazepine receptor characteristics J. H a r r o 1'2, R . - A . K i i v e t 2, A. L a n g 3 a n d E. V a s a r 1 ~Psychopharmacology Laboratory, 2Department of Pharmacology, and 3Hormonal Regulation Laboratory, Tartu University, Tartu, Estonia (U.S.S.R.) (Received 18 January 1990) (Revised version received 12 February 1990) (Accepted 19 February 1990)
Key words: Behavioral difference; Elevated plus-maze; Anxiety; Cholecystokinin octapeptide receptor; Benzodiazepine receptor; Benzodiazepine inverse agonist; Cholecystokinin octapeptide agonist; Rat
Rats with high and low exploratory activity in an elevated plus-maze model of anxiety were separated into subgroups termed 'non-anxious' and 'anxious' respectively according to the number of sectors the animals crossed and the total amount of time they spent in the open part of the plus-maze. The binding parameters ofbenzodiazepine and eholecystokininoctapeptide (CCK-8) receptors in frontal cortex and hippocampus of selected animals were studied and compared to an animal group representing the total mean scores and to home-cage controls. It was established that anxious rats had a significantly lower number of benzodiazepine receptors in frontal cortex as compared to non-anxious animals and in hippocampus as compared to home-cage controls. There was also a decreased number of CCK-8 receptors in hippocampus of anxious rats as compared to the non-anxious and control groups. Non-anxious animals had a significantly lower number of CCK-8 receptors in frontal cortex than anxious and control rats. Acute treatment of rats with anxiogenic benzodiazepine inverse agonist FG 7142 (10 and 20 mg/kg) did not influence benzodiazepine binding in brain regions under investigation but caused upregulation of CCK-8 receptor binding in frontal cortex. On the other hand, CCK-8 analogues caerulein and pentagastrin, administered in doses which inhibit exploratory activity in plus-maze (100 or 500 ng/kg respectively), decreased the number of benzodiazepine binding sites in rat frontal cortex if injected intraperitoneally but did not affect CCK-8 binding. The present findings indicate that benzodiazepine and CCK-8 receptor binding characteristics in brain undergo rapid and behaviourally specific changes during stressful events. INTRODUCTION
An enormous amount of evidence indicates that the GABA/benzodiazepine/barbiturate receptor complex plays a key role in the development of anxiety states. The qualitative nature of behavioural effects of several anxiolytic and anxiogenic drugs can be predicted by their certain biochemical effects on the subunits of this receptor c o m p l e x 29. Stressful manipulations have been shown to alter the radioligand binding character-
istics to GABA 2,13, benzodiazepine 13,2°,21 or Cl--channel-associated 27 binding sites. On the other hand, acute treatment with GABA antagonists which exert anxiogenic effects at subconvulsive doses also affects binding characteristics of GABA receptor complex ~,12. Cholecystokinin octapeptide (CCK-8), a neuropeptide that is localized in high concentrations in several brain regions ~, seems to be a comediator of GABA in cerebral cortex and hippocampus t4. It is well documented that there is a
Correspondence: J. Harro, Tartu University, Department of Pharmacology, 18 l~likooli Street, 202 400 Tartu, Estonia, U.S.S.R. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 functional antagonism between CCK-8 and benzodiazepine anxiolytics in the brain and in peripheral o r g a n s 15"16'3°. Benzodiazepines at low doses antagonize selectively the CCK-8-induced activation of rat hippocampal pyramidal neurones 3. Recently we have shown that CCK-8 receptor agonists (caerulein, pentagastrin) at very low doses have an anxiogenic-like effect on rodents in the elevated plus-maze test. Proglumide, an antagonist of CCK-8, completely attenuated these effects of CCK-8 agonists 8'9. Proglumide was also able to block the effects of the anxiogenic fl-carboline derivative D M C M and pentylenetetrazole, the compounds that are believed to exert their action through depression of GABAergic transmission 8. Therefore, it seems possible that the interaction between GABA- and CCK-8-ergic mechanisms takes place in the development of anxiety states. Recently a novel test of anxiety, an elevated plus-maze, was introduced and behaviourally, physiologically and pharmacologically validated 23. In experiments on mice substantial differences were detected between the individuals in the exploratory activity in this apparatus. There was a significantly lower number of benzodiazepine and GABA receptors in cerebral cortex of animals displaying more 'anxious' behaviour in the elevated plus-maze 25. Therefore we decided to study more completely the binding characteristics of benzodiazepine and CCK-8 receptors in the brains of rats, selected according to their exploratory activity in elevated plus-maze. Earlier studies have revealed that depending on the methods used to induce stress reactions in animals, quite inconsistent and even opposite results can be produced in radioreceptor binding in isolated brain regions. The main idea of this study was that if exposition to the plus-maze situation is a stressful event for rats as has been suggested 23, the rats who display more 'anxious' behaviour (i.e. less exploratory activity) would be more adequate subjects for evaluating the influence of distress on receptor binding characteristics in the brain. To verify further this initial hypothesis the effect of pretreatment of rats with anxiogenic drugs at doses in which they decrease exploratory activity in the plus-maze test on radioligand binding was determined.
MATERIALS AND METHODS
Animals and housing conditions Experiments were performed on adult male albino rats of no particular strain (Rappolovo Farm, Leningrad) weighing 225-250 g at the time of experiment. Rats were, upon arrival in our laboratory, randomly allocated to groups of 12 animals and were familiarized for at least 3 weeks to the new environmental conditions (a 12-h light/12-h dark cycle, lights on between 09.00 and 21.00 h; home cages 80 x 50 × 20 cm). Food and tap water were available ad libitum.
Procedure of behavioural experiments Apparatus. We have used an elevated plusmaze initially described by Pellow et al. 23 which consists of two open arms (45 × 10 cm) and two enclosed arms (45 x 10 x 40 cm) with an open roof. The maze was elevated to the height of 25 cm. To determine the exploratory activity of rats in the open part of the maze either open arm was divided into 3 equal sectors in our modification of apparatus 8. Assessment of anxiety. As the exploratory activity in an elevated plus-maze is thought to be a measure of state anxiety, the following parameters were registered by an observer: (1)the latency period of the first entry into the open part of the maze, (2)the number of sectors crossed in the open part, and (3) total time spent in the open part of the plus-maze. We have used a slight modification in one of the registration measures (the number of sectors crossed in the open part of the maze versus the percentage of open/total arm entries) because in the animal population used by us the total number of arm entries was quite low. Selectionprocedure. Behavioural tests were performed between 10.00 and 13.00 h. Each rat was placed at the centre of the plus-maze facing one of the closed arms of it. After a 4-min test period the animal was removed from the apparatus, rapidly carried to a separate room and killed by decapitation. When the behavioural testing was over the data were immediately calculated and the following groups were formed: (1)rats with high exploratory activity, (2) rats with low exploratory activity, and (3)rats with exploratory activity which could best represent the total measure, i.e.
65 intermediate exploratory activity. Two measures formed the basis of selection: (a) the number of sectors crossed, and (b) the total time spent in the open part of plus-maze. The control group was formed from rats that were decapitated without testing in the plus-maze. They were from the same population and the formation of this group was randomized.
Drug treatment Animals were taken from their home-cages and treated in a randomized order with vehicle, FG 7142 (N-methyl-/~-carboline-3-carboxamide) 10 mg/kg, FG 7142 20 mg/kg, or vehicle, caerulein 100 ng/kg, pentagastrin 500 ng/kg. These doses were used according to previous evidence from this and other laboratories 6,8,9 that they are anxiogenic in rats. All drugs were given intraperitoneally 30 min (FG 7142) or 15 min (CCK-8 agonists) prior to the decapitation. Each treatment group consisted of 5 rats. FG 7142 was dissolved in distilled water with the help of a drop of Tween80. Caerulein and pentagastrin were dissolved in saline.
Radioligand binding experiments Brain dissection procedure. Brain regions were dissected according to Heffner et al. lo. Briefly, rat brain was placed into cutting block and sliced at right angles to the sagittal axis with the help of razor blades. The initial razor blade sliced through the sagittal plane of the brain at the level of the body of the anterior commissure according to the atlas of KOnig and Klippe117. Three razor blades were inserted anterior to that blade along the rostral extent of the brain at intervals of 1.5 mm. Two razor blades were inserted posterior to the initial blade at intervals of 2.0 mm, followed by two more razor blades at intervals of 1.5 mm. The brain was thus divided into 9 sections, numbered rostrally to caudally. Brain regions were dissected from the slices obtained. Frontal cortex includes the cortical tissue from section 1 as well as the cortical tissue superior to the rhinal sulcus from sections 2 and 3. The hippocampus was separated from the midbrain and overlying cerebral cortex from sections 7 and 8 based on its distinct morphological appearance. The pooled
tissue of 5 animals per group was used in the subsequent binding studies.
Preparation of the crude synaptosomalfraction. This was performed as described elsewhere25. Dissected cerebral tissues were homogenized in 30 vols of ice-cold Tris-HC1 (pH = 7.4) using a Potter-S glass-Teflon homogenizer (1000rpm, 10 passings). The membranes were washed twice in Tris-HC1 buffer by centrifugation (48 000 g for 20 min) and resuspension. [3H]Flunitrazepam binding assay. In vitro binding studies to label benzodiazepine receptors were carried out in the presence of 0.125-8 nM of [3H]flunitrazepam (spec. act. 85.0Ci/mmol, Amersham Radiochemicals) using a total incubation volume of 500/~1. Ten #M offlunitrazepam was used to determine non-specific binding. After 60 min incubation on ice the reaction was stopped by rapid filtration over Whatman GF/B filters. The filters were washed with 4 x 3 ml of ice-cold Tris-HC1 buffer and dropped into scintillation vials. [3H]CCK-8 binding assay. The final pellets were resuspended in HEPES buffer (HEPES 10raM, NaC1 130mM, MgC1z 5 m M , KC1 5 mM, EDTA 1 mM, pH 6.5 adjusted with 1 N NaOH) containing bovine serum albumin (0.5 mg/ml). The CCK-8 receptor labelling was carried out in the presence of 0.05-2 nM labelled ligand ([propionyl-3H]CCK-8-sulfated, 60.5 Ci/ mmol, Amersham Radiochemicals) in polypropylene tubes. To determine non-specific binding 100 nM of caerulein was added. After 120min incubation at room temperature the reaction was terminated by centrifugation in a Beckman Microfuge 12 (11 000 rpm for 3 min). The supernatants were aspirated, and the pellets were washed with 2 × 0.5 ml of ice-cold HEPES buffer. The tips of the tubes were cut and dropped into scintillation vials.
Data analysis Mann-Whitney U-test was used for analysis of behavioural data. The specific binding of radioligands was defined as the difference between the degree of binding in the absence and in the presence of unlabelled ligand.
66 Proteins were determined according to the procedure of Lowry et all9, using bovine serum albumin as a standard. Saturation curves were initially analyzed according to the method of Scatchard 26 and then ftted using the non-linear least squares regression analysis. The apparent maximal number of binding sites (Bmax) is expressed as fmol/mg protein and constant K d as nmol/1.
RESULTS
mentors. Rats separated into the 'high activity' subgroup had a shorter latency period, they crossed more sectors and spent more time in the open part of the plus-maze. They seemed not to have any conflict between exploration drive and fear of open areas. The opposite was true in the case of rats separated into the 'low activity' subgroup. These rats did not explore open areas hardly at all. The 'intermediate representative' subgroup was formed from rats whose scores in measures (2) and (3) were the most correspondent to the respective values of the total group. We were not able to match the total group and the intermediate representative subgroup with regard to the latency period because the disposition of individual values did not correspond perfectly to normal distribution which was the case in another measures.
Description of rats with high, low and intermediate exploratory activity
Comparison of benzodiazepine receptor characteristics in selected subgroups of rats
As indicated in Table I, it was possible to separate subgroups of rats with high and low exploratory activity in an elevated plus-maze from a total group. The measures of exploratory activity of individual animals corresponded to normal distribution in the total group and the number of rats in subgroups reflects the discretion of experi-
In vitro experiments demonstrated that the number of [3H]flunitrazepam binding sites in frontal cortex was significantly lower in the low activity subgroup as compared to the high activity subgroup (Table II). The number of these binding sites in frontal cortex of the high activity subgroup was also higher if compared with home-cage con-
Drugs and chemicals F G 7142, flunitrazepam and caerulein were the generous gifts from Ferrosan, Hoffmann-La Roche and Farmitalia-Carlo Erba respectively. Tris and HEPES were purchased from Sigma. All other chemicals were from Reachim (U.S.S.R.).
TABLE I
Selection experiment according to the exploratory activity of rats in an elevated plus-maze All values are means + S.E.M. from three separate selection experiments. Statistical comparisons are done using M a n n - W h i t n e y U-tests.
Group
Number of rats
Latency of first open part entry (s)
Total group
31
47 + 12
8.5 + 1.1
High activity subgroup
5
9 + 1"
18.2 + 1.5'
Intermediate representative subgroup
5
Low activity subgroup
5
17 +_ 4 173 + 24*'**
* Significantly different from Total group, P < 0.01. ** Significantly different from High activity subgroup, P < 0.01.
Number of crossed sectors in open part
8.5 + 0.5 0.9 + 0.6*'**
Total time spent in open part (s) 43 _+ 6 94 _+ 19"
37 + 4 8 + 3*'**
67 TABLE II
Scatchard analysis of saturation data of [ 3H]flunitrazepam binding in rats selected according to their exploratory activity in an elevated plus-maze All values are means + S.E.M. from three separate selection experiments. Statistical comparisons are done using Student's t-test.
Test group
Frontal cortex B max
Home-cage controls High activity Intermediate activity Low activity
1367 1693 1487 1230
Hippocampus Kd
± 115 + 81" + 116 _+ 120"*
1.23 1.29 1.31 1.20
B max + + + +
0.21 0.05 0.16 0.13
1015 830 810 670
Kd + + + +
121 85 133 70*
2.01 1.65 1.60 1.03
+ 0.21 + 0.26 + 0.20 _+ 0.08*
* Significantly different from Home-cage controls, P < 0.05. ** Significantly different from High activity group, P < 0.05.
trois. Data obtained from the control group and intermediate activity subgroup were quite similar. There were no significant differences between any groups in binding affinity of [ 3H]flunitrazepam. On the other hand, both high activity and intermediate activity subgroups had slightly less benzodiazepine binding sites in hippocampus as compared to control animals, but these differences did not reach significance. However, the hippocampi of rats belonging to the low activity subgroup contained less benzodiazepine binding sites as compared to home-cage controls. The only detectable difference in the affinity of [ 3H]flunitrazepam binding in hippocampus was also between control group and low activity subgroup (Table II).
Comparison of CCK-8 receptor characteristics in selected subgroup of rats There was a significantly lower number of [ 3H]CCK-8 binding sites in frontal cortex of animals from the high activity subgroup as compared to the control rats and low activity subgroup (Table III). The number of CCK-8 binding sites in the frontal cortex of the intermediate activity subgroup was also decreased if compared with control rats, although not significantly. However, it also did not differ significantly from the score of the high activity subgroup. Scatchard analyses of binding in hippocampal membranes revealed that the low activity subgroup had a significantly lower density of CCK-8 binding sites than the control rats and high activity subgroup in this brain
TABLE III
Scatchard analysis of saturation data of FH]CCK-8 binding in rats selected according to their exploratory activity in an elevated plus-maze All values are means _+ S.E.M. from three separate selection experiments. Statistical comparisons are done using Student's t-test.
Test group
Frontal cortex B,,,ax
Home-cage controls High activity Intermediate activity Low activity
36.6 19.8 29.5 36.5
+ + + +
Hippocarnpus Kd
3.4 2.8* 3.0 3.7**
0.52 0.52 0.43 0.49
* Significantly different from Home-cage controls, P < 0.05. ** Significantly different from High activity group, P < 0.05.
Bmax + 0.14 + 0.16 _+ 0.18 + 0.15
12.0 14.0 9.3 7.6
+ + + +
Kd
1.3 1.3 1.0 0.8"**
0.19 0.20 0.14 0.14
+ + + +
0.02 0.03 0.02 0.02
68 TABLE IV
Scatchard analysis of saturation data of [3H] CCK-8 and [3H ]flunitrazepam binding in ratfrontal cortex after systemic administration of FG 7142, caerulein, or pentagastrin Experiments were performed twice with very similar results. All values are from one representative binding study. Statistical comparisons are done using Student's t-test.
Test group
[ 3H / C CK-8 B,,,,~
Control F G 7142 10 mg/kg FG 7142 20 mg/kg Control Caerulein 100 ng/kg Pentagastrin 500 ng/kg
38.8 50.5 58.8 34.3 29.8 45.1
[ 3H ]flunitrazepam Kd
_+ 2.2 + 4.0* + 4.8** + 1.6 + 5.2 _+ 3.9
0.23 0.36 0.32 0.38 0.53 0.57
Bmax + 0.03 + 0.05 + 0.05 + 0.02 _+ 0.06 + 0.07
1461 1649 1683 1405 965 905
Kd + + + + + +
99 111 85 75 23* 40*
1.86 1.89 1.79 1.81 1.70 1.34
_+ 0.36 _+ 0.42 _+ 0.23 _+ 0.32 _+ 0.11 _+ 0.17
* Significantly different from respective Control, P < 0.05. ** Significantly different from respective Control, P < 0.01.
region. The intermediate activity subgroup had a score between those of the high and low activity subgroups, not significantly different from one or the other, and also not significantly different from controls (Table III). Binding affinity did not differ between the groups. Effect of anxiogenic drug treatment on rat brain benzodiazepine and CCK-8 receptor binding Acute administration of FG 7142 dose-dependently enhanced the number of [ 3H]CCK-8 binding sites in rat frontal cortex (Table IV). At the same time binding affinity was decreased but this effect just missed significance (P = 0.058). In
hippocampal membranes any effect of F G 7142 treatment on CCK-8 receptors was not observable. [ 3 H ]Flunitrazepam binding was not influenced by F G 7142 treatment in the brain regions under investigation. On the other hand, the treatment of rats with low doses of CCK-related peptides resulted in a decrease of [ 3 H ]flunitrazepam binding in frontal cortex (Table IV) without any change in hippocampus (Table V). CCK-8 receptors, neither in frontal cortex nor in hippocampus, were affected by that treatment as suggested by the results from Scatchard analysis (Tables IV and V).
TABLE V
Scatchard analysis of saturation data of [ 3H ] C CK-8 and [ 3H ]flunitrazepam binding in rat hippocampus after systemic administration of FG 7142, caerulein, or pentagastrin Experiments were performed twice with very similar results. All values are from one representative binding study.
Test group
[ 3H]CCK-8 Bmax
Control FG 7142 10 mg/kg FG 7142 20 mg/kg Control Caerulein 100 ng/kg Pentagastrin 500 ng/kg
13.3 15.6 14.3 12.1 14.3 13.1
+ + + + + +
[ 3H]flunitrazep am gd
3.4 4.7 2.1 2.2 2.5 1.9
0.18 0.22 0.13 0.17 0.21 0.17
+ 0.02 + 0.05 +_ 0.03 + 0.03 + 0.04 + 0.03
Bmax
Kd
884 931 863 973 840 830
1.18 1.47 1.38 1.25 1.87 1.35
_+ 87 + 94 + 99 +_ 43 + 16 _+ 25
+_. 0.19 +_ 0.18 +_ 0.27 _+ 0.26 + 0.10 + 0.17
69 DISCUSSION The behavioural, physiological and pharmacological validation of an elevated plus-maze as a measure of anxiety in the rat 23 gives rise to the idea that if individual animals display different exploratory activity in this apparatus, it would reflect differences in their anxiety rate. So we would like to regard the rats with high exploratory activity in the plus-maze as non-anxious and the rats with low exploratory activity as anxious animals. For selection procedure, we have used two measures, (a) the number of sectors crossed, and (b) the total time spent in the open part of plusmaze. The second one is essentially the same as one of the validated measures of Pellow et al. 23 and the first one correlates excellently with the second one (r = 0.98, Spearman rank correlation, P = 4.68 x 10-7). Rats confined to both the closed or open arms of the plus-maze had significantly elevated corticosterone concentrations compared with homecage controls 23. So the plus-maze test could be regarded as a stressful events for animals. There is evidence that 'stressed' rodents have increased 21"28 or decreased 13"18'2° benzodiazepine binding in forebrain. Lower benzodiazepine binding in hippocampus was attributed to 'anxious' rats selected according to the responsiveness in a conflict test 22. In the plus-maze selection experiment where anxious and non-anxious subgroups of mice were separated, it was shown that anxious mice had a lower number of benzodiazepine and G A B A receptors in cerebral cortex 25. Both binding measures did not differ between groups in cerebellar membranes. As there was not a control group from animals not exposed to the elevated plus-maze, it was impossible to say if these neurochemical differences had been present before the experimental procedure or arose during the behavioural test. We have now demonstrated that rats who seem to feel less anxious in a disturbing novel environment have a higher density of benzodiazepine binding sites in both frontal cortex and hippocampus as compared to anxious animals. However, if compared with home-cage controls it appears that in the case of high activity rats there is an upregulation of benzodiazepine
binding in frontal cortex whereas in the case of low activity rats downregulation of benzodiazepine binding sites in hippocampus occurs. So it should be emphasized that a stressful event may cause an increase as well as a decrease in the number of benzodiazepine binding sites depending on the adaptational level of the particular animal in the altered situation. The number of CCK-8 receptor sites in frontal cortex and hippocampus of anxious rats was different from the parameters of non-anxious animals. Namely, rats of the high activity group had considerably less [ 3H]CCK-8 sites in frontal cortex than control animals and rats of the low activity group. Rats with intermediate exploratory activity also had a number of CCK-8 receptor sites intermediate between the scores of the high and low activity groups. In the hippocampus, the number of CCK-8 binding sites was decreased after the plus-maze test in the low activity group, which differed from both the control and high activity group. Once more, the group with intermediate activity in the plus-maze had an intermediate score in CCK-8 binding sites between high and low activity groups. So it seems that downregulation of CCK-8 receptors in frontal cortex is required for the adaptation to novel environment, and downregulation of these sites in the hippocampus is connected with anxious behaviour. It has been recognized that benzodiazepines and CCK-8 act as functional antagonists. In this point of view it is interesting to note that in nonanxious rats benzodiazepine and CCK-8 receptors in frontal cortex are regulated in opposite directions during stressful events. To further verify the idea that balance between benzodiazepine and CCK-8 receptor function reflects the anxiety level of animals, we investigated the effect of anxiogenic drug administration on receptor characteristics. F G 7142, a benzodiazepine receptor inverse agonist, may induce severe anxiety in both animals 24 and man 5. Treatment of rats with this anxiogenic compound did not influence [3H]flunitrazepam binding in rat brain. This is consistent with results of Ito etal. ~1"12, who have shown that GABA antagonists bicuculline and picrotoxin, which also in-
70 duce anxiety at subconvulsive doses, do not affect benzodiazepine binding in brain after acute systemic administration. However, the number of CCK-8 receptor sites in frontal cortex following F G 7 1 4 2 treatment was dose-dependently increased, suggesting a predominance of CCK-8ergic processes over GABAergic ones in this brain region where G A B A and CCK-8 are believed to act as cotransmitters. On the other hand, it was recently found that CCK-related peptides may exert anxiogenic-like effects both in rodents 8'9 and in man 4. In a previous study we found that the systemic administration of caerulein and pentagastrin may downregulate benzodiazepine binding only in a certain (anxiogenic) dose range, larger, behaviourally sedative doses being inefficient 7. In this study we have reproduced the finding that cortical benzodiazepine receptor density is decreased following caerulein and pentagastrin administration at doses which depress exploratory activity in the plus-maze. However, it appears that CCK-8 binding was not affected. Thus it seems, that at least on the level of frontal cortex, in anxiety state there is CCK-8 receptor binding upregulation and/or benzodiazepine receptor binding downregulation, whereas in emotional well-being the opposite may be true. In summary, our results presented here support the idea that the characteristics o f b e n z o d i a z e p i n e and CCK-8 receptors are of importance in the genesis of anxiety. Secondly we would like to emphasize the importance of observing individual variability in animal populations when molecular mechanisms regulating emotionality are under investigation. If we do not know a great deal about how a manipulation affects the animal, not very much can be learned from subsequent biochemical analyses. ACKNOWLEDGEMENTS We are grateful to Ferrosan, H o f f m a n n - L a Roche and F a r m i t a l i a - C a r l o Erba for their generous supplying of F G 7142, flunitrazepam and caerulein respectively.
REFERENCES 1 Beinfeld, M.C., Cholecystokinin in the central nervous system: a minireview, Neuropeptides, 3 (1983) 421-427. 2 Biggio, G., The action of stress, ]~-carbolines, diazepam and Ro 15-1788 on GABA receptors in the rat brain. In G. Biggio and E. Costa (Eds.), Benzodiazepine recognition Site Ligands: Biochemistry and Pharmacology, Raven, New York, 1983, pp. 105-119. 3 Bradwejn, J. and De Montigny, C., Benzodiazepines antagonize cholecystokinin-induced activation of rat hippocampal neurones, Nature (Lond.), 312 (1984) 363-364. 4 De Montigny, C., Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers: preliminary findings, Arch. Gen. Psychiatry, 46 (1989) 511-517. 5 Dorow, R., Horowski, R., Paschelke, G., Amin, M. and Braestrup, C., Severe anxiety induced by FG 7142, a /3-carboline ligand for benzodiazepine receptors, Lancet, 9 (1983) 98-99. 6 File, S.E., Pellow, S. and Braestrup, C., Effects of the /3-carboline, FG 7142, in the social interaction test of anxiety and the holeboard: correlations between behaviour and plasma concentrations, Pharmacol. Biochem. Behav., 22 (1985) 941-944. 7 Harro, J., Kiivet, R.-A., Lang, A., P61d, M. and Vasar, E., The effect of systemic administration of CCK-8 agonists on CNS receptor binding parameters. In L. Allikmets (Ed.), Molecular Pharmacology of receptors III, Tartu University Press, Tartu, 1989, pp. 67-77. 8 Harro, J., P61d, M., Vasar, E. and Allikmets, L., The role of CCK-8-ergic mechanisms in the regulation of emotional behaviour in rodents, Zh. Vyss. Nerv. Deyat., 39 (1989) 877-883 (in Russian). 9 Harro, J., P61d, M. and Vasar, E., Anxiogenic-likeaction of caerulein, a CCK-8 receptor agonist, in the mouse: influence of acute and subchronic diazepam treatment, Naunyn-Schmiedeberg's Arch. PharmacoL, in press. 10 Heffner, T.G., Hartman, J.A. and Seiden, L. S., A rapid method for the regional dissection of the rat brain, Pharmacol. Biochem. Behav., 13 (1980)453-456. 11 Ito, Y., Lim, D. K., Hoskins, B. and Ho, I.K., BicucuUine up-regulation of GABAA receptors in rat brain, J. Neurochem., 51 (1988) 145-152. 12 Ito, Y., Lim, D.K., Nabeshima, T. and Ho, I.K., Effects ofpicrotoxin treatment on GABAAreceptor supramolecular complexes in rat brain, J. Neurochem., 52 (1989) 1064-1070. 13 Kiivet, R.-A., Harro, J., R~tgo,L. and P61d, M., Changes in GABA and benzodiazepine receptors after foot-shock in the rat: influence of diazepam. In L. Allikmets (Ed.), Molecular Pharmacology of receptors H, Tartu University Press, Tartu, 1988, p. 45-54. 14 Kosaka, T., Kosaka, K., Tateishi, K., Hamaoka, Y., Yanaihara, N., Wu, J.-Y. and Hama, K., GABAergic neurons containing CCK-8-1ike and/or VIP-like immu-
71
15
16
17
18
19
20
21
22
23
noreactivities in the rat hippocampus and dentate gyrus, J. Comp. Neurol., 239 (1985) 420-430. Kubota, K., Sugaya, K., Matsuda, I., Matsuoka, Y. and Itonaga, M., Caerulein antagonism by benzodiazepines in the food intake in mice, Jpn. J. Pharmacol., 39 (1985) 120-122. Kubota, K., Sun, F.-Y., Sugaya, K. and Sunagane, N., Reversal of antinociceptive effect of caerulein by benzodiazepine, J. Pharmacobio-Dyn., 9 (1986) 428-431. K6nig, J.F.R. and Klippel, R.A., The RatBrain:A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. Lippa, A.S., Kleifner, C.A., Yunger, L., Sano, C.M., Smith, W.V. and Beer, B., Relationship between benzodiazepine receptors and experimental anxiety in rats, Pharmacol. Biochem. Behav., 9 (1978) 853-856. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, RJ., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Medina, J.H., Novas, M.L., Wolfman, C.N.V., Levi de Stein, M. and De Robertis, E., Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress, Neuroscience, 9 (1983) 331-335. Miller, L.G., Thompson, M.L., Greenblatt, D.J., Deutsch, S.I., Shader, R.I. and Paul, S.M., Rapid increase in brain benzodiazepine receptor binding following defeat stress in mice, Brain Res., 414 (1987) 395-400. Patel, J.B., Stengel, J., Malik, J.B. and Enna, SJ., Neurochemical characteristics of rats distinguished as benzodiazepine responders and non-responders in a new conflict test, Life Sci., 34 (1984) 2647-2653. Pellow, S., Chopin, P., File, S.E. and Briley, M., Valid-
24
25
26 27
28
29
30
ation of open:closed arm entries in an elevated plusmaze as a measure of anxiety in the rat, J. Neurosci. Methods, 14 (1985) 149-167. Petersen, E.N. and Jensen, L.H., Proconflict effect of benzodiazepine receptor inverse agonists and other inhibitors of GABA function, Eur. J. Pharmacol., 103 (1984) 91-97. R~igo,L., Kiivet, R.-A., Harro, J. and P61d, M., Behavioral differences in an elevated plus-maze: correlation between anxiety and decreased number of GABA and benzodiazepine receptors in mouse cerebral cortex, NaunynSchmiedeberg's Arch. Pharmacol., 337 (1988) 675-678. Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N. IT. Acad. Sci., 54 (1949) 660-672. Schwartz, R.D., Wess, M.J., Labarca, R., Skolnick, P. and Paul, S.M., Acute stress enhances the activity of the GABA receptor-gated chloride ion channel in brain, Brain Res., 411 (1987) 151-155. Soubrie, P., Thiebot, M.H., Jobert, A., Montrastruc, S.L., Hery, F. and Hamon, M. Decreased convulsant potency of picrotoxin and pentylenetetrazol and enhanced [3H]flunitratepam cortical binding following stressful manipulations in rats, Brain Res., 189 (1980) 505-517. Stephens, D.N., Schneider, H.H., Kehr, W., Jensen, L.H., Petersen, E. and Honore, T., Modulation of anxiety by ~-carbolines and other benzodiazepine receptor ligands: relationship of pharmacological to biochemical measures of efficacy, Brain Res. Bull., 19 (1987) 309-318. Sugaya, K., Matsuda, I. and Kubota, K. Inhibition of hypothermic effect of cholecystokinin by benzodiazepines and a benzodiazepine antagonist, Ro 15-1788, in mice, Jpn. J. Pharmacol., 39 (1985) 277-280.
