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A new model for the detection of antidepressant drugs: Olfactory bulbectomy in the rat compared with existing models
A New Model for the Detection of Antidepressant Drugs: Olfactory Bulbectomy in the Rat Compared with Existing Models
K. D. CAIRNCROSS, B. Cox,
CHRISTINE FORSTER, AND ALLISON F. WREN
A new model for the detection of antidepressant drugs has been described and compared with two other tests. This new model depends on the ability of the antidepressant drugs to selectively modify some behavioral and biochemical changes which occur following bilateral ablation of the olfactory bulbs in rats. The bulbectomy syndrome has been characterized as a learning deficit on a stepdown passive-avoidance test; hyper-reactivity; and as an increase in circulating II-hydroxycorticosteroids (II-OHCS). The antidepressants, amitrip~line (10 mg/kg), mianserin (IO mg/kg), and viloxazine (IO mg/kg) administered i.p. daily for 7 days corrected the learning deficit, reduced the hyper-reactivity, and the elevated plasma (II-OHCS) in a reproducible manner. Thus this test indicates the potential antidepressant activity of mianserin, a drug which, although used in the clinic, was not detected by conventional screening tests. The present study not only confirms the apparent lack of activity of mianserin in two other tests (reserpine reversal and potentiation of noradrenaline) but also shows that, in the relatively low doses used in the bulbectomy model, the established antidepressants may also fail to give positive responses in these other tests.
KeyWords:
Olfactory bulbectomy;
Antidepressants;
Screening methods.
INTRODUCTION The problem of using animal models in predicting drugs with antidepressant potential is well known (Zetler, 1963; Barnett, Taber and Roth, 1969; Goodlet and Sugrue, 1974; van Riezen, Behagel and Chafik, 1975). Models currently in use for the detection of drugs with antidepressant activity include reserpine reversal (Garattini et al., 1962; Carattini and Jori, 1966; Spencer, 1966) and inhibition of the neuronal uptake for noradrenaline, which may be measured either directly or indirectly through the potentiation of responses to noradrenaline. All the above tests presuppose an action via neuronal catecholamine systems and are reported to give positive results with the established tricyclic antidepressants (~lowinski and Axelrod, 1964; Ross and Renyi, 1967). However, a new tetracyclic antidepressant (mianserin) has recently been introduced which apparently failed to Received February 3,1978; accepted March 15, 1978. From the Department of Pharmacology, Materia Medica and Therapeutics, Manchester Manchester, U. K. A.F.W. is an SRC (CASE award) Scholar. Address reprint requests to: K. D. Cairncross, School of Biological Sciences, Macquarie North Ryde, N.S.W. 2113, Australia.
University,
University,
131 @Elsevier North-Holland,
Inc., 1978, Journal of Pharmacological
Methods
1,131-143
(1978)
0160.5402/7W@X11-0131/$01.75
132
K. D. Cairncross et al.
give positive results in the established tests (van Riezen, 1972). The significance of this finding was twofold. Firstly, it indicated that the established models for the detection of drugs with antidepressant potential were indeed unreliable. Secondly, as these tests imply an interaction with central noradrenergic systems, it cast doubt on the accepted theories for the mode of action of the existing antidepressants. In the present study a new model, the olfactory bulbectomized rat, is compared with two other tests (reserpine reversal and potentiation of noradrenaline) in an attempt to determine if it provides a more reliable model for the prediction of antidepressant potential. METHODS Male Sprague-Dawley rats weighing 200-250 g were used throughout the experiments, which were carried out at the same time each day at an ambient temperature of 20 Zk 1°C. Olfactory Bulbectomy A rat was anesthetized with an i.p. injection of Equithesin (3.3 ml/kg) and its head immobilized in a David Kopf stereotaxic frame. After shaving the head a longitudinal incision was made through the skin and periosteum which extended at least 1 cm anterior and 1 cm posterior to the bregma. The skin and periosteum were retracted and any superficial bleeding points sealed off with bone wax (Ethicon Ltd). Using calipers, two points were marked on the skull surface which were 5 mm anterior to the bregma and 2 mm lateral to the midline (Fig. 1, top). Two holes, 2 mm in diameter, were then drilled through the skull at the marked points, care being taken not to penetrate the dura. After drilling, the area was cleaned with sterile saline and dried with sterile gauze swabs. The dura was then perforated and cut away using the point of a sterile 27 SWG hypodermic needle. If the rat was destined to become a sham-operated control, then at this point the holes were packed with Sterispon absorbable gelatin sponge (Allen and Hanburys Ltd). If the rat was to be bulbectomized, then the olfactory bulbs were severed using a small curved knife inserted through the holes (Fig 1, bottom). At this point care is necessary to avoid damage to the frontal cortex. The excised bulbs were then aspirated through a sterile Pasteur pipette using a water suction pump. After aspiration it was usual to see some bleeding and the excess blood was removed by means of swabs before continuing. The holes were then packed with absorbable gelatin. From this point the bulbectomized and sham-operated rats were treated identically. The retractors were removed from the skin and the wound closed with Ethicon sutures. Penicillin dusting powder was available but as wound infection did not frequently occur, it was not used. The duration of the operation was usually less than 15 min and recovery from the anesthetic commenced within 30 min. Postoperatively the rats were housed 5 per cage. Bulbectomized and sham-operated rats were not mixed together. Both groups were then left for 7 days to recover from the operation. On Day 8 and all following days, the rats were subjected to an identical standardized handling procedure involving their removal from the home cage in a constant order so that they become acclimatized to being removed from
Prediction of Antidepressant
Activity
the cage and to the experimenter. On Day 15 drug pretreatment began, arranged to coincide with the handling procedure. Drugs were given as one i.p. injection daily for a minimum of 7 days and then the rats were subjected to three standard tests. Two behavioral and one biochemical test was performed. The reactivity and step-down passive-avoidance tests took place between 9 AM and 1 PM for each individual rat. Rats were killed and blood collected between 2 and 4 PM. Bulbectomy
Syndrome
Tests
1. Reactivity A rat was removed from its home cage and placed on a demarcated area of bench. Reactivity was assessed on the basis of the rat’s response to two novel
I .
curved knife
FIGURE 1. Top: Exposed dorsal surface of rat skull showing position of burr holes overlying the olfactory bulbs. Bottom: Rat brain in saggital section showing correct placement of curved knife.
133
134
K. D. Cairncross et al. TABLE 1
Scoring System for Reactivity Test RESPONSE
SCORES 0
No response
1
Slight
jump
and moves
2
Slight
jump
and freezes
3
Rat jumps
and freezes
for 2-15 set
4
Rat jumps
and freezes
for > 15
“Maximum
away for O-2 set
score for the two novel stimuli
stimuli: (1) a puff of air blown whilst it was facing away from and in front of the rat’s nose. that of King (1958), based on (Table 1).
was 8.
sharply on to the rat’s back from a distance of IO cm the observer; and (2) a loud clap delivered close to An arbitrary scoring system was devised, similar to the freezing response of the rat to each stimulus
2. Passive Avoidance A perspex box with a 55-cm2 stainless steel grid floor, the bars of which were 1.5 cm apart and connected to a stimulator (SRI Model 6051) was used. A scrambled shock of 1.5 MA (7Ov, 6 ms, 6 Hz) was delivered to the grid. The rat’s paws were dampened with a wet cloth to improve electrical conductivity and placed on a central wooden platform (19 cm2). The latency time for the rat to step off the platform with all four paws was measured. The procedure was repeated with an intertrial interval of 1 min until the rat remained on the platform for at least 1 min. The number of trials needed by each rat to reach this learning criterion was recorded. 3. II-Hydroxycotficosteroid Assay Immediately after the completion of behavioral testing the rat was returned to its home cage. One hour afterwards it was removed again, ensuring that the established handling procedure was used. It was taken into an adjacent laboratory, sacrificed, and exsanguinated within 2 min. This prevented the parameter of novelty from affecting the concentration of the circulating II-hydroxycorticosteroids (II-OHCS). Blood was collected in heparinized tubes and centrifuged to obtain plasma. The plasma was then either assayed immediately to determine the ll-hydroxycorticosterone concentration (Mattingly, 1962), or stored at -20°C for assay at a later date.
Reserpine
Pretreatment
1. Reserpine
Reversal
(Acute)
Rats were housed in groups of five. At time zero a rat received 2.5 mg/kg reserpine i.p. and 0.5 hr later it was injected with one of the following: mianserin, IO mg/kg; amitriptyline, IO mg/kg; viloxazine, 5 mg/kg; tranylcypromine, IO mg/kg; saline. All drugs were given by the i.p. route. At 1.5 hr after reserpine, the rat was placed on an LKB Animex activity meter (maximum sensitivity 40 PA) in the dark for 20 min in
Prediction of Antidepressant Activity a cage (floor area 1,000 cm2). The meter was connected to an external counter and the activity recorded every 5 min. The experiment was carried out between 11 AM and 4 PM. 2. Reserpine Prevention (Acute) In this variation the antidepressants were injected at time zero. One hour later the rat received an i.p. injection of reserpine, 2.5 mg/kg. After a further hour the locomotor activity was recorded for 20 min in a manner identical to that described above. 3. Reserpine Prevention (Chronic) Five groups of rats received either one of the four antidepressant drugs or saline injected daily i.p. for a total of 8 days. On Day 6 each rat received a saline injection 1 hr prior to the recording of locomotor activity. On Day 7 at 1 hr after either antidepressant or saline injection, each rat received an injection of reserpine 2.5 mg/kg i.p., and after a further hour locomotor activity was recorded as above. On Day 8, twenty-four hours after the reserpine treatment, a second period of locomotor activity recording was made. Isolated Rat Anococcygeus
Preparation
The isolated rat anococcygeus preparation (Gillespie, 1972) was set up in Krebs solution maintained at 37” ? 0.5”C aerated with 95% 02/5% CO, mixture, for the recording of isometric contractions. Log concentration effect curves were obtained to exogenously applied noradrenaline using a drug contact time of 60 sec. The log concentration effect curves were then repeated on some tissues in the absence of, and on other tissues in the presence of, concentrations of either amitriptyline (lO+M, lO-‘jM, IO-‘M) or mianserin (10P6M). The preincubation period with the modifying drugs present in the Krebs solution was 30 min. RESULTS Olfactory
Bulbectomy
The effect in rats of sham operation or olfactory bulbectomy on passive-avoidance behavior, reactivity, and the plasma concentrations of II-OHCS is shown in Fig. 2. The bulbectomized rat demonstrated a significant deficit in the acquisition of a passive-avoidance response and a significant increase in reactivity and plasma llOHCS levels when compared to the sham-operated rat. Chronic pretreatment with amitriptyline, mianserin, and viloxazine significantly reduced the reactivity, the elevated II-OHCS concentrations, and the number of trials required by bulbectomized rats to acquire the learning criterion. With the exception of the effect of mianserin on reactivity, these pretreatment schedules had no effect on the shamoperated controls. Tranylcypromine produced a different pattern of responses in the bulbectomized rat. The passive-avoidance deficit was exaggerated, there was no effect on reactivity but the plasma II-OHCS levels were significantly reduced. Tranylcypromine also modified the response of the sham-operated rats as the number of trials to acquire the step-down response were significantly increased, as was the reactivity and plasma II-OHCS concentrations.
135
136
K. D. Cairncross et al. TABLE 2
Effects of Antidepressant
Drugs on Reserpine (2.5 mg/kg)
MEAN CUMULATIVE LOCOMOTORACTIVIN SCORE RESERPINE” REVERSAL
RESERPINE~ PREVENTION
DRUG
N
SALINE
Saline Amitriptyline
7 4
688 1111
28 133
29 102
(IO mg/kg) Mianserin
5
1415”
237
104
(IO mg/kg) Viloxazine
4
1055
48
13
(5 mg/kg) Tranylcypromine
4
985
1861’
1841c
(10 mg/kg) a Antidepressant drugs given 4 hr after reserpine injection. b Antidepressant drugs given 1 hr prior to reserpine injection. c Indicates significant difference from corresponding control, P < 0.05 by Mann Whitney U-test.
At the completion of this study, each rat brain was examined to determine the success or failure of the bulbectomy operation. A successful bulbectomy was accepted if more than 70% of the olfactory bulb tissue had been removed with no evidence of cortical damage (Fig. 3, top). Results from animals in which either less than 70% of the bulbs had been removed (Fig. 3, middle) or macroscopic examination revealed damage to the frontal cortex (Fig. 3, bottom) were discarded. Reserpine
Pretreatment
Saline pretreated animals responded to acute antidepressant treatment by an apparent increase in locomotor activity, as shown by the group means being higher than in the control group (Table 2). However, because of the wide variability in the individual responses, the increase in locomotor activity rarely achieved the accepted level of statistical significance when tested by the nonparametric Mann Whitney U-test. In animals pretreated with reserpine (2.5 mg/kg) and receiving a saline injection 0.5 hr later, there was a significant loss of locomotor activity when compared to the appropriate saline control (Table 2). Of the antidepressants tested, only an acute injection of tranylcypromine significantly increased the locomotor activity when compared with that recorded after reserpine alone (Table 2). The results of injecting a single dose of the antidepressant drugs 1 hr prior to the reserpine treatment are also shown in Table 2. Reserpine administration following saline produced a significant decrease in locomotor activity. Again, of the antidepressants tested, only tranylcypromine prevented the reserpine-induced changes. The effects of chronic pretreatment with the antidepressants on reserpine-induced loss of locomotor activity is shown in Table 3. In this situation all three antidepressants caused a significant increase in the activity of the saline controls, measured 6 days after treatment began. Reserpine injection brought about a significant loss of locomotor activity both at 1 hr and 24 hr after injection. Although
Prediction of Antidepressant
Activity
137
6
1
41?
.E
z
% 2z” o-
7
x x
i3 IIYizl;c x
x
x
MI
lo
IIAN
10
VIL
5
‘CP
10
FIGURE 2. Effect of antidepressant drugs on passive avoidance (top), reactivity (middle), and plasma ll-OHCS concentrations of sham-operated (open columns) and olfactory bulbectomized (hatched columns) rats (bottom). Each column represents the mean 2 SE (except reactivity score where the Mann Whitney U-test was used for statistical comparisons) for groups of at least 10 rats. (O), significant difference between saline pretreated sham operated and bulbectomized rats. (x), significant change induced by drug pretreatment when compared with appropriate control. P < 0.05.
138
K. D. Cairncross et al.
Prediction of Antidepressant TABLE 3 Effects of Chronic 8-Day Pretreatment Reserpine (2.5 mg/kg)
with Antidepressant
Activity
Drugs on
MEAN CUMULATIVE LOCOMOTORACTIVITY SCORE DAY 7 (RESERPINE,1 DAY 8 (RESERPINE, 24 HR) HR)
DRUG
N
DAY 6 (SALINE)
Saline Amitriptyline
7 5
668 987”
28 964
30 69
(IO mg/kg) Mianserin
5
1460a
65
264
(IO n-g/kg) Viloxazine
5
1128”
624
1 54a
(5 mg/kg) a Indicates significant difference U-test.
from corresponding
control, P < 0.05, by Mann Whitney
the loss of activity in rats pretreated with the antidepressants appeared not to be as marked as in the controls, only one value (that for viloxazine at 24 hr after reserpine) achieved the accepted level of statistical significance. No results were obtained for tranylcypromine because the combination of chronic pretreatment with this drug and reserpinization caused death in the majority of the group of rats under test. Rat Isolated Anococcygeus
Preparation
The effects of amitriptyline and mianserin on the response of the rat anococcygeus preparation to noradrenaline is shown in Fig. 4. Neither amitriptyline nor mianserin potentiated the response to noradrenaline. Mianserin (10e6M) had no significant effect and with amitriptyline there was a depression of the maximum response, which was particularly evident at a concentration of 10m5M. DISCUSSION A general dissatisfaction with methods currently in use for the prediction of antidepressant activity (see Introduction) has led to a search for new models. It was decided that any new model should fulfill the following criteria. Firstly, it should identify antidepressant drugs and differentiate between them and other psychotropic drugs. Secondly, it should be capable of giving reproducible results with data which are quick and easy to obtain. Thirdly, it should be an in vivo test so that drugs which do not penetrate the central nervous system can be eliminated or that other central nervous system actions (e.g., stimulation or depression) can be simultaneously assessed. Fourthly, to mimic the clinical situation, it would be useful
FIGURE 3. Rat brains following bulbectomy operations. Top, successful bulbectomy; Middle, incomplete bulbectomy; Bottom, successful operation but slight damage to frontal cortex (arrow).
139
140 K. D. Cairncrosset al.
if the antidepressants were active when administered chronically. Finally, as there is still doubt about the underlying biochemical disorder in depressive illness, the test should not presuppose any particular mechanism of action. Of a number of animal models considered, the olfactory bulbectomized rat seemed worthy of further investigation. Some years ago Cairncross and King (1971) had shown that amitriptyline reversed the deficit in acquisition of a one-way avoidance test exhibited by bulbectomized rats. Consequently, this model was subjected to further scrutiny to determine how it measured up to the criteria outlined above. The results suggest that it may indeed represent an improvement over existing models. From the results with amitriptyline, mianserin, and viloxazine, it can be seen that the pattern of effects of the antidepressant drugs follow a standard trend. Thus the
.
1
10-5
0
10-4
10-S
1
10‘2
NAD [M]
FIGURE 4. lo~concentration effect curves for noradrenaline on the rat isolated anococcygeus muscle for noradrenaline alone (x) and in the presence of either mianserin, 10%-t (O), or amitrip~iine; IO-’ (Cl), lO*M (O), and fO”M (&.
Prediction of Antidepressant
Activity
prediction of antidepressant activity is based on the following properties: The drug must normalize the behavioral changes and reverse the elevated II-OHCS concentrations induced by bulbectomy-without parallel effects occurring in sham-operated animals. Using this protocol, mianserin, which failed to give a positive indication in previously established tests (van Riezen, Behagel and Chafik, 1975), was identified as active. The next question to be answered was therefore: Can antidepressants be differentiated from other centrally acting drugs? In a previous report it has been shown that this is the case. Thus, using the “anxiosoif” test, which is essentially a passive-avoidance paradigm in which thirsty rats learn to avoid an electrified water spout (Rigter et al., 1977), it has been shown that other psychotropic drugs, including major and minor tranquilizers, lithium, and amphetamine (van Riezen et al, 1976; Rigter et al., 1977) can be distinguished from antidepressant drugs. However the anxiosoif experiment is time consuming, involving 4 days of testing and would not fulfill the criteria of being quick and easy to perform. It was for this reason that the two simpler behavioral tests and the one simple biochemical test were introduced. These less complex tests not only fulfill the criteria of simplicity and speed but also exclude other drugs (Cairncross et al., 1977). Thus although the tranquilizers may modify the behavioral changes following bulbectomy, they also cause parallel changes in the sham-operated controls. The next criterion to be considered is that of reproducibility, and from the small standard errors seen in the three sets of data, it can be seen that the tests do give reproducible results. The third criterion-an in vivo test-is also fulfilled by the bulbectomized model and its ability to identify other central nervous system actions is shown by the effects of tranylcypromine. This drug is a monoamineoxidase inhibitor .but also has central stimulant actions (Goodman and Gilman, 1975) and although it normalized some, but not all, of the features of the bulbectomy syndrome it also produced marked changes in controls. Therefore the bulbectomy model would not necessarily indicate selection of this type of antidepressant for further investigation. Elimination of this type of drug may be considered an advantage when the problems associated with the use of the conventional monoamineoxidase inhibitors or CNS stimulant drugs are considered. As to the remaining criteria, the antidepressant drugs are active in the test after chronic administration, a situation which mimics the use of the drug in the clinic, and the test does not presuppose any particular mechanism of action. The major disadvantage of the bulbectomy model is that it requires a surgical operation, which adds to the>complexity of the whole process. However the surgical technique is simple and the rats need little or no postoperative care, so that once a system has been established it is easy to ensure a constant supply of two-week bulbectomized animals. For comparative purposes, the reserpine-reversal and reserpine-prevention tests were also examined. These tests were quick and easy to perform and required no extensive preparation. However, in our hands they demonstrated a number of undesirable features. In the reserpinized rat there appeared to be a great variability in response and even when apparently large changes occurred it was not always possible to demonstrate significant effects. In the case of drug interactions it is
141
142
K. D. Cairncross et al.
always possible that no significant effect is observed because the timing of the injections are not optimal. Therefore an attempt was made to minimize this possibility by using three different approaches, one involving chronic pretreatment and the other two acute pretreatment. However none of the approaches tried were particularly successful. In contrast to the bulbectomized model, the only antidepressant which the reserpine test conclusively predicted as having antidepressant potential was the monoamineoxidase inhibitor tranylcypromine. Thus we have confirmed the lack of predictive ability of this test for some of the newer antidepressants such as mianserin (van Riezen, 1972). Reversal of reserpine hypothermia in the mouse has been used as a predictive model for antidepressants for a long time (Spencer, 1966). It was not used in this study and so no direct comparisons can be made. However the ability of non-antidepressant drugs to reverse reserpine hypothermia in the mouse is well documented (Whittle, 1967) and therefore, this test apparently does not fulfill the most important criterion for a useful model. The rat isolated anococcygeus muscle was used to determine if it could provide a rapid, reliable, and simple in vitro model for the detection of antidepressants. It was thought that it might give an indirect measure of the inhibition of noradrenaline inactivation by uptake and avoid time consuming and expensive radioactive studies. It failed. In conclusion then, of a number of tests available, the olfactory bulbectomized rat appears worthy of consideration as the test of choice for the screening of compounds for antidepressant activity. Further, if its reliability is confirmed when challenged with a wider range of drugs, it is also possible that analysis of the neurochemical changes which follow bulbectomy could provide some new clues to the biochemical basis of the depressive state and how the antidepressant drugs may work. We wish to thank Organon drugs.
N.V. for generous financial assistance, and S.K.F. and I.C.I.
for gifts of
REFERENCES Barnett A, Taber RI, Roth FE (1969) Activity of antihistamines in laboratory antidepressant tests. Int. J Neuropharmacol
8~73-79
Cairncross KD, Cox B, Forster C, Wren AF (1977) The olfactory bulbectomized rat: a simple model for detecting drugs with antidepressant potential. Br ] Pharmacol 61:497P. Cairncross KD, King MC (1971) Facilitation of avoidance learning in anosmic rats by amitriptyline. Proc Aust Physiol Pharmacol Sot 2:1-2. Garattini S, Ciachetti A, Jori A, Pieri L, Valzelli L (1962) The effect of imipramine, amitriptyline and their monomethyl derivatives on reserpine activity. / Pharm Pharmacol14:509-515.
Garattini S, Jori A (1966) Interactions between imipramine-like drugs and reserpine on body temperature. In Antidepressant Drugs. Eds, S Garattini and MNG Dukes. Excerpta Medica: Amsterdam, pp. 179-194. Gillespie JS (1972) The rat anococcygeus and its response to nerve stimulation some drugs. Br J Pharmaco/45:404-416.
muscle and to
Clowinski J, Axelrod J (1964) Inhibition of tritiated noradrenaline in the intact rat brain by imipramine and structurally related compounds. Nature 204:1318-1319.
Coodlet I, Sugrue M (1974) The effects of a new antidepressant Org GB94 on amine uptake mechanisms. Br.J Pharmacol52:431P.
Prediction of Antidepressant Goodman cological
LS, Gilman A, eds (1975) The PharmaBasis of Therapeutics, Macmillan: New
York. Mattingly D (1962) A simple fluorimetric method for the estimation of free II-hydroxycorticoids in plasma. / C/in Pathol 15:374-379. van Riezen H (1972) Differential central effects of 5-HT antagonists mianserin and cyproheptadine. Arch Int Pharmacodyn
Jher
198:256-269.
van Riezen H, Behagel H, Chafik M (1975) Development of psychotropic drugs. Psychopharmaco/ Bull
ll:lO-14.
van Riezen H, Schnieden H, Wren AF (1976) Behavioural changes following olfactory bulbectomy in rats: a possible model for the detection of antidepressant drugs. BrJ Pharmacol57:426P427P.
Activity
Rigter H, van Riezen H, Wren AF (1977) Pharmacological validation of a new test for the detection of antidepressant drugs. Br J Pharmacol 59:451P-452P. Ross SB, Renyi AL (1967) Inhibition of the uptake of tritiated catecholamines by antidepressants and related agents. Eur J Pharmacol 2:181-186. Spencer PSJ (1966) Antagonism of hypothermia in the mouse by antidepressants. In Antidepressant Drugs. Eds., S Garattini and MNG Dukes. Excerpta Medica: Amsterdam, pp. 195-204. Whittle BA (1967) Reversal of reserpine-induced hypothermia by pharmacological agents other than antidepressants. Nature 216:579-580. Zetler G (1963) The anticataleptic activity of some antidepressives. Arzneimittelforsch. 13:103-109.
143
K. D. CAIRNCROSS, B. Cox,
CHRISTINE FORSTER, AND ALLISON F. WREN
A new model for the detection of antidepressant drugs has been described and compared with two other tests. This new model depends on the ability of the antidepressant drugs to selectively modify some behavioral and biochemical changes which occur following bilateral ablation of the olfactory bulbs in rats. The bulbectomy syndrome has been characterized as a learning deficit on a stepdown passive-avoidance test; hyper-reactivity; and as an increase in circulating II-hydroxycorticosteroids (II-OHCS). The antidepressants, amitrip~line (10 mg/kg), mianserin (IO mg/kg), and viloxazine (IO mg/kg) administered i.p. daily for 7 days corrected the learning deficit, reduced the hyper-reactivity, and the elevated plasma (II-OHCS) in a reproducible manner. Thus this test indicates the potential antidepressant activity of mianserin, a drug which, although used in the clinic, was not detected by conventional screening tests. The present study not only confirms the apparent lack of activity of mianserin in two other tests (reserpine reversal and potentiation of noradrenaline) but also shows that, in the relatively low doses used in the bulbectomy model, the established antidepressants may also fail to give positive responses in these other tests.
KeyWords:
Olfactory bulbectomy;
Antidepressants;
Screening methods.
INTRODUCTION The problem of using animal models in predicting drugs with antidepressant potential is well known (Zetler, 1963; Barnett, Taber and Roth, 1969; Goodlet and Sugrue, 1974; van Riezen, Behagel and Chafik, 1975). Models currently in use for the detection of drugs with antidepressant activity include reserpine reversal (Garattini et al., 1962; Carattini and Jori, 1966; Spencer, 1966) and inhibition of the neuronal uptake for noradrenaline, which may be measured either directly or indirectly through the potentiation of responses to noradrenaline. All the above tests presuppose an action via neuronal catecholamine systems and are reported to give positive results with the established tricyclic antidepressants (~lowinski and Axelrod, 1964; Ross and Renyi, 1967). However, a new tetracyclic antidepressant (mianserin) has recently been introduced which apparently failed to Received February 3,1978; accepted March 15, 1978. From the Department of Pharmacology, Materia Medica and Therapeutics, Manchester Manchester, U. K. A.F.W. is an SRC (CASE award) Scholar. Address reprint requests to: K. D. Cairncross, School of Biological Sciences, Macquarie North Ryde, N.S.W. 2113, Australia.
University,
University,
131 @Elsevier North-Holland,
Inc., 1978, Journal of Pharmacological
Methods
1,131-143
(1978)
0160.5402/7W@X11-0131/$01.75
132
K. D. Cairncross et al.
give positive results in the established tests (van Riezen, 1972). The significance of this finding was twofold. Firstly, it indicated that the established models for the detection of drugs with antidepressant potential were indeed unreliable. Secondly, as these tests imply an interaction with central noradrenergic systems, it cast doubt on the accepted theories for the mode of action of the existing antidepressants. In the present study a new model, the olfactory bulbectomized rat, is compared with two other tests (reserpine reversal and potentiation of noradrenaline) in an attempt to determine if it provides a more reliable model for the prediction of antidepressant potential. METHODS Male Sprague-Dawley rats weighing 200-250 g were used throughout the experiments, which were carried out at the same time each day at an ambient temperature of 20 Zk 1°C. Olfactory Bulbectomy A rat was anesthetized with an i.p. injection of Equithesin (3.3 ml/kg) and its head immobilized in a David Kopf stereotaxic frame. After shaving the head a longitudinal incision was made through the skin and periosteum which extended at least 1 cm anterior and 1 cm posterior to the bregma. The skin and periosteum were retracted and any superficial bleeding points sealed off with bone wax (Ethicon Ltd). Using calipers, two points were marked on the skull surface which were 5 mm anterior to the bregma and 2 mm lateral to the midline (Fig. 1, top). Two holes, 2 mm in diameter, were then drilled through the skull at the marked points, care being taken not to penetrate the dura. After drilling, the area was cleaned with sterile saline and dried with sterile gauze swabs. The dura was then perforated and cut away using the point of a sterile 27 SWG hypodermic needle. If the rat was destined to become a sham-operated control, then at this point the holes were packed with Sterispon absorbable gelatin sponge (Allen and Hanburys Ltd). If the rat was to be bulbectomized, then the olfactory bulbs were severed using a small curved knife inserted through the holes (Fig 1, bottom). At this point care is necessary to avoid damage to the frontal cortex. The excised bulbs were then aspirated through a sterile Pasteur pipette using a water suction pump. After aspiration it was usual to see some bleeding and the excess blood was removed by means of swabs before continuing. The holes were then packed with absorbable gelatin. From this point the bulbectomized and sham-operated rats were treated identically. The retractors were removed from the skin and the wound closed with Ethicon sutures. Penicillin dusting powder was available but as wound infection did not frequently occur, it was not used. The duration of the operation was usually less than 15 min and recovery from the anesthetic commenced within 30 min. Postoperatively the rats were housed 5 per cage. Bulbectomized and sham-operated rats were not mixed together. Both groups were then left for 7 days to recover from the operation. On Day 8 and all following days, the rats were subjected to an identical standardized handling procedure involving their removal from the home cage in a constant order so that they become acclimatized to being removed from
Prediction of Antidepressant
Activity
the cage and to the experimenter. On Day 15 drug pretreatment began, arranged to coincide with the handling procedure. Drugs were given as one i.p. injection daily for a minimum of 7 days and then the rats were subjected to three standard tests. Two behavioral and one biochemical test was performed. The reactivity and step-down passive-avoidance tests took place between 9 AM and 1 PM for each individual rat. Rats were killed and blood collected between 2 and 4 PM. Bulbectomy
Syndrome
Tests
1. Reactivity A rat was removed from its home cage and placed on a demarcated area of bench. Reactivity was assessed on the basis of the rat’s response to two novel
I .
curved knife
FIGURE 1. Top: Exposed dorsal surface of rat skull showing position of burr holes overlying the olfactory bulbs. Bottom: Rat brain in saggital section showing correct placement of curved knife.
133
134
K. D. Cairncross et al. TABLE 1
Scoring System for Reactivity Test RESPONSE
SCORES 0
No response
1
Slight
jump
and moves
2
Slight
jump
and freezes
3
Rat jumps
and freezes
for 2-15 set
4
Rat jumps
and freezes
for > 15
“Maximum
away for O-2 set
score for the two novel stimuli
stimuli: (1) a puff of air blown whilst it was facing away from and in front of the rat’s nose. that of King (1958), based on (Table 1).
was 8.
sharply on to the rat’s back from a distance of IO cm the observer; and (2) a loud clap delivered close to An arbitrary scoring system was devised, similar to the freezing response of the rat to each stimulus
2. Passive Avoidance A perspex box with a 55-cm2 stainless steel grid floor, the bars of which were 1.5 cm apart and connected to a stimulator (SRI Model 6051) was used. A scrambled shock of 1.5 MA (7Ov, 6 ms, 6 Hz) was delivered to the grid. The rat’s paws were dampened with a wet cloth to improve electrical conductivity and placed on a central wooden platform (19 cm2). The latency time for the rat to step off the platform with all four paws was measured. The procedure was repeated with an intertrial interval of 1 min until the rat remained on the platform for at least 1 min. The number of trials needed by each rat to reach this learning criterion was recorded. 3. II-Hydroxycotficosteroid Assay Immediately after the completion of behavioral testing the rat was returned to its home cage. One hour afterwards it was removed again, ensuring that the established handling procedure was used. It was taken into an adjacent laboratory, sacrificed, and exsanguinated within 2 min. This prevented the parameter of novelty from affecting the concentration of the circulating II-hydroxycorticosteroids (II-OHCS). Blood was collected in heparinized tubes and centrifuged to obtain plasma. The plasma was then either assayed immediately to determine the ll-hydroxycorticosterone concentration (Mattingly, 1962), or stored at -20°C for assay at a later date.
Reserpine
Pretreatment
1. Reserpine
Reversal
(Acute)
Rats were housed in groups of five. At time zero a rat received 2.5 mg/kg reserpine i.p. and 0.5 hr later it was injected with one of the following: mianserin, IO mg/kg; amitriptyline, IO mg/kg; viloxazine, 5 mg/kg; tranylcypromine, IO mg/kg; saline. All drugs were given by the i.p. route. At 1.5 hr after reserpine, the rat was placed on an LKB Animex activity meter (maximum sensitivity 40 PA) in the dark for 20 min in
Prediction of Antidepressant Activity a cage (floor area 1,000 cm2). The meter was connected to an external counter and the activity recorded every 5 min. The experiment was carried out between 11 AM and 4 PM. 2. Reserpine Prevention (Acute) In this variation the antidepressants were injected at time zero. One hour later the rat received an i.p. injection of reserpine, 2.5 mg/kg. After a further hour the locomotor activity was recorded for 20 min in a manner identical to that described above. 3. Reserpine Prevention (Chronic) Five groups of rats received either one of the four antidepressant drugs or saline injected daily i.p. for a total of 8 days. On Day 6 each rat received a saline injection 1 hr prior to the recording of locomotor activity. On Day 7 at 1 hr after either antidepressant or saline injection, each rat received an injection of reserpine 2.5 mg/kg i.p., and after a further hour locomotor activity was recorded as above. On Day 8, twenty-four hours after the reserpine treatment, a second period of locomotor activity recording was made. Isolated Rat Anococcygeus
Preparation
The isolated rat anococcygeus preparation (Gillespie, 1972) was set up in Krebs solution maintained at 37” ? 0.5”C aerated with 95% 02/5% CO, mixture, for the recording of isometric contractions. Log concentration effect curves were obtained to exogenously applied noradrenaline using a drug contact time of 60 sec. The log concentration effect curves were then repeated on some tissues in the absence of, and on other tissues in the presence of, concentrations of either amitriptyline (lO+M, lO-‘jM, IO-‘M) or mianserin (10P6M). The preincubation period with the modifying drugs present in the Krebs solution was 30 min. RESULTS Olfactory
Bulbectomy
The effect in rats of sham operation or olfactory bulbectomy on passive-avoidance behavior, reactivity, and the plasma concentrations of II-OHCS is shown in Fig. 2. The bulbectomized rat demonstrated a significant deficit in the acquisition of a passive-avoidance response and a significant increase in reactivity and plasma llOHCS levels when compared to the sham-operated rat. Chronic pretreatment with amitriptyline, mianserin, and viloxazine significantly reduced the reactivity, the elevated II-OHCS concentrations, and the number of trials required by bulbectomized rats to acquire the learning criterion. With the exception of the effect of mianserin on reactivity, these pretreatment schedules had no effect on the shamoperated controls. Tranylcypromine produced a different pattern of responses in the bulbectomized rat. The passive-avoidance deficit was exaggerated, there was no effect on reactivity but the plasma II-OHCS levels were significantly reduced. Tranylcypromine also modified the response of the sham-operated rats as the number of trials to acquire the step-down response were significantly increased, as was the reactivity and plasma II-OHCS concentrations.
135
136
K. D. Cairncross et al. TABLE 2
Effects of Antidepressant
Drugs on Reserpine (2.5 mg/kg)
MEAN CUMULATIVE LOCOMOTORACTIVIN SCORE RESERPINE” REVERSAL
RESERPINE~ PREVENTION
DRUG
N
SALINE
Saline Amitriptyline
7 4
688 1111
28 133
29 102
(IO mg/kg) Mianserin
5
1415”
237
104
(IO mg/kg) Viloxazine
4
1055
48
13
(5 mg/kg) Tranylcypromine
4
985
1861’
1841c
(10 mg/kg) a Antidepressant drugs given 4 hr after reserpine injection. b Antidepressant drugs given 1 hr prior to reserpine injection. c Indicates significant difference from corresponding control, P < 0.05 by Mann Whitney U-test.
At the completion of this study, each rat brain was examined to determine the success or failure of the bulbectomy operation. A successful bulbectomy was accepted if more than 70% of the olfactory bulb tissue had been removed with no evidence of cortical damage (Fig. 3, top). Results from animals in which either less than 70% of the bulbs had been removed (Fig. 3, middle) or macroscopic examination revealed damage to the frontal cortex (Fig. 3, bottom) were discarded. Reserpine
Pretreatment
Saline pretreated animals responded to acute antidepressant treatment by an apparent increase in locomotor activity, as shown by the group means being higher than in the control group (Table 2). However, because of the wide variability in the individual responses, the increase in locomotor activity rarely achieved the accepted level of statistical significance when tested by the nonparametric Mann Whitney U-test. In animals pretreated with reserpine (2.5 mg/kg) and receiving a saline injection 0.5 hr later, there was a significant loss of locomotor activity when compared to the appropriate saline control (Table 2). Of the antidepressants tested, only an acute injection of tranylcypromine significantly increased the locomotor activity when compared with that recorded after reserpine alone (Table 2). The results of injecting a single dose of the antidepressant drugs 1 hr prior to the reserpine treatment are also shown in Table 2. Reserpine administration following saline produced a significant decrease in locomotor activity. Again, of the antidepressants tested, only tranylcypromine prevented the reserpine-induced changes. The effects of chronic pretreatment with the antidepressants on reserpine-induced loss of locomotor activity is shown in Table 3. In this situation all three antidepressants caused a significant increase in the activity of the saline controls, measured 6 days after treatment began. Reserpine injection brought about a significant loss of locomotor activity both at 1 hr and 24 hr after injection. Although
Prediction of Antidepressant
Activity
137
6
1
41?
.E
z
% 2z” o-
7
x x
i3 IIYizl;c x
x
x
MI
lo
IIAN
10
VIL
5
‘CP
10
FIGURE 2. Effect of antidepressant drugs on passive avoidance (top), reactivity (middle), and plasma ll-OHCS concentrations of sham-operated (open columns) and olfactory bulbectomized (hatched columns) rats (bottom). Each column represents the mean 2 SE (except reactivity score where the Mann Whitney U-test was used for statistical comparisons) for groups of at least 10 rats. (O), significant difference between saline pretreated sham operated and bulbectomized rats. (x), significant change induced by drug pretreatment when compared with appropriate control. P < 0.05.
138
K. D. Cairncross et al.
Prediction of Antidepressant TABLE 3 Effects of Chronic 8-Day Pretreatment Reserpine (2.5 mg/kg)
with Antidepressant
Activity
Drugs on
MEAN CUMULATIVE LOCOMOTORACTIVITY SCORE DAY 7 (RESERPINE,1 DAY 8 (RESERPINE, 24 HR) HR)
DRUG
N
DAY 6 (SALINE)
Saline Amitriptyline
7 5
668 987”
28 964
30 69
(IO mg/kg) Mianserin
5
1460a
65
264
(IO n-g/kg) Viloxazine
5
1128”
624
1 54a
(5 mg/kg) a Indicates significant difference U-test.
from corresponding
control, P < 0.05, by Mann Whitney
the loss of activity in rats pretreated with the antidepressants appeared not to be as marked as in the controls, only one value (that for viloxazine at 24 hr after reserpine) achieved the accepted level of statistical significance. No results were obtained for tranylcypromine because the combination of chronic pretreatment with this drug and reserpinization caused death in the majority of the group of rats under test. Rat Isolated Anococcygeus
Preparation
The effects of amitriptyline and mianserin on the response of the rat anococcygeus preparation to noradrenaline is shown in Fig. 4. Neither amitriptyline nor mianserin potentiated the response to noradrenaline. Mianserin (10e6M) had no significant effect and with amitriptyline there was a depression of the maximum response, which was particularly evident at a concentration of 10m5M. DISCUSSION A general dissatisfaction with methods currently in use for the prediction of antidepressant activity (see Introduction) has led to a search for new models. It was decided that any new model should fulfill the following criteria. Firstly, it should identify antidepressant drugs and differentiate between them and other psychotropic drugs. Secondly, it should be capable of giving reproducible results with data which are quick and easy to obtain. Thirdly, it should be an in vivo test so that drugs which do not penetrate the central nervous system can be eliminated or that other central nervous system actions (e.g., stimulation or depression) can be simultaneously assessed. Fourthly, to mimic the clinical situation, it would be useful
FIGURE 3. Rat brains following bulbectomy operations. Top, successful bulbectomy; Middle, incomplete bulbectomy; Bottom, successful operation but slight damage to frontal cortex (arrow).
139
140 K. D. Cairncrosset al.
if the antidepressants were active when administered chronically. Finally, as there is still doubt about the underlying biochemical disorder in depressive illness, the test should not presuppose any particular mechanism of action. Of a number of animal models considered, the olfactory bulbectomized rat seemed worthy of further investigation. Some years ago Cairncross and King (1971) had shown that amitriptyline reversed the deficit in acquisition of a one-way avoidance test exhibited by bulbectomized rats. Consequently, this model was subjected to further scrutiny to determine how it measured up to the criteria outlined above. The results suggest that it may indeed represent an improvement over existing models. From the results with amitriptyline, mianserin, and viloxazine, it can be seen that the pattern of effects of the antidepressant drugs follow a standard trend. Thus the
.
1
10-5
0
10-4
10-S
1
10‘2
NAD [M]
FIGURE 4. lo~concentration effect curves for noradrenaline on the rat isolated anococcygeus muscle for noradrenaline alone (x) and in the presence of either mianserin, 10%-t (O), or amitrip~iine; IO-’ (Cl), lO*M (O), and fO”M (&.
Prediction of Antidepressant
Activity
prediction of antidepressant activity is based on the following properties: The drug must normalize the behavioral changes and reverse the elevated II-OHCS concentrations induced by bulbectomy-without parallel effects occurring in sham-operated animals. Using this protocol, mianserin, which failed to give a positive indication in previously established tests (van Riezen, Behagel and Chafik, 1975), was identified as active. The next question to be answered was therefore: Can antidepressants be differentiated from other centrally acting drugs? In a previous report it has been shown that this is the case. Thus, using the “anxiosoif” test, which is essentially a passive-avoidance paradigm in which thirsty rats learn to avoid an electrified water spout (Rigter et al., 1977), it has been shown that other psychotropic drugs, including major and minor tranquilizers, lithium, and amphetamine (van Riezen et al, 1976; Rigter et al., 1977) can be distinguished from antidepressant drugs. However the anxiosoif experiment is time consuming, involving 4 days of testing and would not fulfill the criteria of being quick and easy to perform. It was for this reason that the two simpler behavioral tests and the one simple biochemical test were introduced. These less complex tests not only fulfill the criteria of simplicity and speed but also exclude other drugs (Cairncross et al., 1977). Thus although the tranquilizers may modify the behavioral changes following bulbectomy, they also cause parallel changes in the sham-operated controls. The next criterion to be considered is that of reproducibility, and from the small standard errors seen in the three sets of data, it can be seen that the tests do give reproducible results. The third criterion-an in vivo test-is also fulfilled by the bulbectomized model and its ability to identify other central nervous system actions is shown by the effects of tranylcypromine. This drug is a monoamineoxidase inhibitor .but also has central stimulant actions (Goodman and Gilman, 1975) and although it normalized some, but not all, of the features of the bulbectomy syndrome it also produced marked changes in controls. Therefore the bulbectomy model would not necessarily indicate selection of this type of antidepressant for further investigation. Elimination of this type of drug may be considered an advantage when the problems associated with the use of the conventional monoamineoxidase inhibitors or CNS stimulant drugs are considered. As to the remaining criteria, the antidepressant drugs are active in the test after chronic administration, a situation which mimics the use of the drug in the clinic, and the test does not presuppose any particular mechanism of action. The major disadvantage of the bulbectomy model is that it requires a surgical operation, which adds to the>complexity of the whole process. However the surgical technique is simple and the rats need little or no postoperative care, so that once a system has been established it is easy to ensure a constant supply of two-week bulbectomized animals. For comparative purposes, the reserpine-reversal and reserpine-prevention tests were also examined. These tests were quick and easy to perform and required no extensive preparation. However, in our hands they demonstrated a number of undesirable features. In the reserpinized rat there appeared to be a great variability in response and even when apparently large changes occurred it was not always possible to demonstrate significant effects. In the case of drug interactions it is
141
142
K. D. Cairncross et al.
always possible that no significant effect is observed because the timing of the injections are not optimal. Therefore an attempt was made to minimize this possibility by using three different approaches, one involving chronic pretreatment and the other two acute pretreatment. However none of the approaches tried were particularly successful. In contrast to the bulbectomized model, the only antidepressant which the reserpine test conclusively predicted as having antidepressant potential was the monoamineoxidase inhibitor tranylcypromine. Thus we have confirmed the lack of predictive ability of this test for some of the newer antidepressants such as mianserin (van Riezen, 1972). Reversal of reserpine hypothermia in the mouse has been used as a predictive model for antidepressants for a long time (Spencer, 1966). It was not used in this study and so no direct comparisons can be made. However the ability of non-antidepressant drugs to reverse reserpine hypothermia in the mouse is well documented (Whittle, 1967) and therefore, this test apparently does not fulfill the most important criterion for a useful model. The rat isolated anococcygeus muscle was used to determine if it could provide a rapid, reliable, and simple in vitro model for the detection of antidepressants. It was thought that it might give an indirect measure of the inhibition of noradrenaline inactivation by uptake and avoid time consuming and expensive radioactive studies. It failed. In conclusion then, of a number of tests available, the olfactory bulbectomized rat appears worthy of consideration as the test of choice for the screening of compounds for antidepressant activity. Further, if its reliability is confirmed when challenged with a wider range of drugs, it is also possible that analysis of the neurochemical changes which follow bulbectomy could provide some new clues to the biochemical basis of the depressive state and how the antidepressant drugs may work. We wish to thank Organon drugs.
N.V. for generous financial assistance, and S.K.F. and I.C.I.
for gifts of
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Activity
Rigter H, van Riezen H, Wren AF (1977) Pharmacological validation of a new test for the detection of antidepressant drugs. Br J Pharmacol 59:451P-452P. Ross SB, Renyi AL (1967) Inhibition of the uptake of tritiated catecholamines by antidepressants and related agents. Eur J Pharmacol 2:181-186. Spencer PSJ (1966) Antagonism of hypothermia in the mouse by antidepressants. In Antidepressant Drugs. Eds., S Garattini and MNG Dukes. Excerpta Medica: Amsterdam, pp. 195-204. Whittle BA (1967) Reversal of reserpine-induced hypothermia by pharmacological agents other than antidepressants. Nature 216:579-580. Zetler G (1963) The anticataleptic activity of some antidepressives. Arzneimittelforsch. 13:103-109.
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