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An investigation of the tremorgenic effects of oxotremorine and tremorine after stereotaxic injection into rat brain
Int. J. Neuropharmac., 1969, 8, 291-297
Pergamon Press.
Printed in Gt. Britain.
AN INVESTIGATION OF THE T R E M O R G E N I C EFFECTS OF OXOTREMORINE AND T R E M O R I N E AFTER STEREOTAXIC INJECTION INTO RAT BRAIN B. Cox and D. POTKONJAK Department of Pharmacology, The University, Manchester 13 (Accepted 14 January 1969) Summary--The effect of stereotaxic administration of tremorine and oxotremorine into four brain areas has been investigated using rats. Tremorine produced tremor significantly higher than control after administration into the globus pallidus, caudate nucleus, and substantia nigra. Oxotremorine produced tremor significantly higher than control only after injection into the substantia nigra. Dyflos injected into the caudate nucleus produced tremor. No change in the tremorgenic action of tremorine was observed after the simultaneous intracaudate injections of atropine and tremorine, and dyftos and tremorine. Intraperitoneal atropine reduced the tremorgenic action of an intracaudate injection of tremorine. Cox and POTKONJAK (1969a) have reported a quantitative method for the measurement of tremor in the rat, which was used to investigate the actions of tremorine (1,4-dipyrrolidino2-butyne) and oxotremorine [1,-(2-oxopyrrolidino)-4-pyrrolidino-butyne-2] given by intraperitoneal injection. It was noted that the time course of the tremor did not coincide with the time course of an increase in brain acetylcholine which also occurred. The same workers have shown (1969b) that, although atropine inhibited oxotremorine tremor, dyflos failed to potentiate it. The failure of dyflos to potentiate oxotremorine suggested that the tremor drug was acting directly, rather than indirectly through the release of acetylcholine. Furthermore, HAMMER et al. (1968b) have reported that in atropine pretreated mice there is an increased clearance of oxotremorine from the brain, which may indicate that the antagonism of tremorine and oxotremorine tremor by atropine is not due to an effect at the site of tremor production. In order to investigate these problems further, we have examined the effects of tremorine and oxotremorine administered by stereotaxic injection into selected brain areas and studied the effect of atropine and dyflos on any tremor produced. The brain areas chosen were parts of the extrapyramidal system involved in the control of movement and posture (KAADA, 1963).
METHODS Male Wistar rats weighing 190-210 g were used in all the experiments. Stereotaxic injection Tremor drugs were injected into the globus pallidus, caudate nucleus, substantia nigra and red nucleus by a stereotaxic method similar to that of HORSLEY and CLARKE (1908) and CLARK (1939), using the stereotaxic system of co-ordinates (DE GROOT, 1959). 291
292
B. Cox and D. POTKONJAK
A rat anaesthetised with ether was placed on the stereotaxic instrument and its shaved head immobilised. An incision 2-2.5 cm long was made through the skin in the midline. The site required was located by using the appropriate stereotaxic co-ordinates and a small hole was drilled in the skull to allow access of a No. 16 hypodermic needle. The needle was lowered to the required depth and an injection made from a micrometer syringe. The volume delivered at each site of injection was 0.003 ml. After injection the needle was withdrawn. The skin was drawn together and sutured. Injections were always unilateral, but for any one series were made alternately on the left and right sides. The depth of anaesthesia was adjusted so that the rat recovered consciousness within 3 min of the injection being made. Rats injected with saline by the stereotaxic method into that same four areas as the tremor drugs, and rats injected with the tremor drugs by the intraperitoneal route after ether anaesthesia were used as controls.
Measurement of tremor Tremor was recorded by a method described previously (Cox and POTKONJAK,1969a). The recording was started exactly 2 min after the stereotaxic injection. The rat was suspended in a perspex box (18 × 7 x 7 cm) from a gramophone pick-up head. The movements of the rat were thus converted into electrical energy which was fed into two channels of a Grass Polygraph (Model 7). Channel 1 gave a qualitative record of the tremor. Channel 2, an integrator channel, gave a quantitative record of the tremor. One complete deflection of the integrator pen was given an arbitrary value of 100 tremor units. An automatic zero returned the pen to baseline every 15 sec. Sample traces are shown in Fig. 1. The tremor was recorded for 5 min (twenty 15-sec samples) during a period of tremor, if tremor occurred. If it did not occur then a control period of 5 min was recorded from 3 min to 8 min after injection. The tremor is always presented as the mean of the twenty 15-sec samples recorded in the 5 min (arbitrary units). At the end of the tremor recordings the rat was sacrificed and the brain removed. After 10 ~ formalin fixation the brain was sectioned and the site of the injection confirmed histologically by locating the needle track. Statistics The Mann-Whitney U test (SIEGEL, 1956) was used to determine the statistical significance of the differences between groups. Unless otherwise stated the criterion of a significant difference between means was P~0.05. Drugs All drugs were freshly prepared for injection in 0-9 9/00saline and the concentration expressed as mg/ml of the free base. Atropine sulphate, B.D.H. Ltd. Dyflos (Diisopropylfluorophosphate), Koch-Light Ltd. Oxotremorine as the base. Tremorine dihydrochloride.
RESULTS 1. The ability of intraperitoneal tremorine and oxotremorine to produce tremor after ether anaesthesia Sample traces of the tremor recording from six rats, three without previous administration of ether, and three after ether anaesthesia, are shown in Fig. 1. The mean background
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aomp/k’d
AFTER fTMR
Alf4fslHfsl*
FIG. I. Nemo.
f.p. 292
Stereotaxic injection of oxotremorine and trernorine
293
activity r e c o r d e d after saline injection into an u n a n a e s t h e t i s e d rat was 40 units. This value c o m p a r e d well with a m e a n value o f 42 units o b t a i n e d previously with the same r e c o r d i n g system ( C o x and POTKONJAK, 1969a). A rat, injected with saline after e t h e r anaesthesia, showed at the beginning o f the r e c o r d i n g large s p a s m o d i c r e s p i r a t o r y movements, which resulted in a higher b a c k g r o u n d activity. O x o t r e m o r i n e (1 mg/kg) in an u n a n a e s t h e t i s e d rat gave a mean 15 sec t r e m o r o f 130 units. In a similar rat after ether anaesthesia the t r e m o r recorded was 109 units. T r e m o r i n e 20 m g / k g injected into an u n a n a e s t h e t i s e d rat gave a mean 15 sec t r e m o r o f 137 units which was increased to 158 units in the anaesthetised rat. As it was shown t h a t t r e m o r could be both o b s e r v e d a n d r e c o r d e d after ether anaesthesia, the effect o f saline, t r e m o r i n e and o x o t r e m o r i n e was investigated by injecting t h e m stereotaxically into the brains o f rats anaesthetised with ether.
2.
The effect of stereotaxic injection of tremor drugs
P r e l i m i n a r y experiments with o x o t r e m o r i n e i n d i c a t e d t h a t the a r e a which gave the most consistent t r e m o r after injection was the s u b s t a n t i a nigra. Therefore, a range o f solutions 0.125, 0.25, 0"5, 1"0 and 2-0 m g / m l were injected into this a r e a in individual rats and gave t r e m o r values o f 62, 72, 78, 85 and 76 units respectively. The c o n c e n t r a t i o n selected for further study was 1 mg/ml. T r e m o r i n e 20 mg/ml had a c o m p a r a b l e effect to oxotl'emorine I mg/ml and this concentration was therefore used.
Saline The effect o f saline injected by the stereotaxic m e t h o d is shown in Table 1. N o n e o f these injections p r o d u c e d visible tremor. The mean values o b t a i n e d after injection into the globus TABLE 1. THE MEAN 15 s e c TREMOR READING AFTER STEREOTAXIC INJECTION OF SALINE INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
1
30
48
30
73
2 3 4 5
52 46 55 36
65 35 33 25
45 24 58 48
67 60 67 86
Mean tremor
44
41
41
71~
All tremor values in arbitrary units. *=Rats in which tremor was clearly visible (none in this Table). t = Mean value significantly higher than other saline means. GP =Globus pallidus; CN =caudate nucleus; SN--substantia n igra; RN =red nucleus. pallidus, c a u d a t e nucleus a n d s u b s t a n t i a nigra were n o t significantly different f r o m each other. The m e a n t r e m o r value o b t a i n e d after injection into the red nucleus was higher ( P = 0 . 0 0 4 ) t h a n the results f r o m the o t h e r three areas.
294
B. Cox and D. POTKONJAK
Tremorine T h e effect of" s t e r e o t a x i c injection o f t r e m o r i n e 20 m g / m l is s h o w n in T a b l e 2. I n j e c t i o n i n t o all f o u r a r e a s p r o d u c e d o b s e r v a b l e t r e m o r in each rat tested. T h e r e was n o significant TABLE 2. THE MEAN 15 s e c TREMOR READING AFTER STEREOrAXIC INJECTION OF TREMORINE ( 2 0 mg,'ml) INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
2 3 4 5 6
82* 98* 75* 123" 98* t08"
84* 124" 67 ':: 83* 116" 141"
104" 76* 59* 71" 93*
61" 179" 96* 23* 66*
Mean tremor
97~
103t
81*
85
I
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. -~ Mean values significantly higher than corresponding saline controls. GP :Globus pallidus; CN -- caudate nucleus : SN-- substant ia nigra: R N - - r e d nucleus. difference b e t w e e n the t r e m o r r e c o r d e d f r o m any o f t h e f o u r g r o u p s . H o w e v e r , the t r e m o r r e c o r d e d a f t e r i n j e c t i o n i n t o the g l o b u s pallidus, c a u d a t e n u c l e u s a n d s u b s t a n t i a n i g r a was significantly h i g h e r ( P = 0 . 0 0 4 ) t h a n the c o r r e s p o n d i n g saline c o n t r o l s . T h e r e was n o significant difference ( P ~ 0 . 3 4 5 ) b e t w e e n t h e m e a n t r e m o r r e a d i n g s after t r e m o r i n e o r saline i n j e c t i o n i n t o t h e red nucleus.
Oxotremorine T h e effect o f s t e r e o t a x i c i n j e c t i o n o f o x o t r e m o r i n e 1 m g / m l , is s h o w n in T a b l e 3. O n l y a f t e r i n j e c t i o n o f o x o t r e m o r i n e i n t o t h e s u b s t a n t i a n i g r a was the m e a n t r e m o r r e a d i n g significantly ]"ABLE3. THE MEAN 15 sec TREMOR READING AFTER STEREOTAXIC INJECTION OF OXOTREMORINE (1 mg/ml) INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
2 3 4 5
23 53 68 40 105"
45 85 15 78 59
77* 135" 89* 63* 78*
152" 63* 123" 148"
Mean tremor
58
56
88~
107
1
49*
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. t = M e a n value significantly higher than equivalent saline control. GP = Globus pallidus; CN = caudate nucleus; SN = substantia nigra; R N = red nucleus.
295
Stereotaxic injection of oxotremorine and tremorine
higher (P=0-004) than the equivalent saline control value. During the recording period no tremor could be observed visually in the group of rats receiving oxotremorine into the caudate nucleus. One rat out of five exhibited tremor symptoms after injection into the globus pallidus, but tremor was observed visually in all the rats where injections were made into the area of the substantia nigra and the red nucleus. The mean tremor readings after the injection of oxotremorine (1 mg/ml) into the globus pallidus and the caudate nucleus were significantly lower (P -0.048) than the corresponding mean values after the injection of tremorine 20 mg/ml. The mean values after injection into the other areas were not significantly different from the corresponding means after tremorine.
Atropine The effect of atropine pretreatment on the tremor produced by intracaudate injection of tremorine 20 mg/ml is shown in Table 4. TABLE 4. THE
EFFECT OF ATROPINE
AND
DYFLOS PRETREATMENT
ON TREMOR PRODUCED
BY I N T R A C A U D A T E
INJECTION OF TREMORINE
(20 mg/ml) Drug
Rat Nos.
Intracaudate
Intraperitoneal
injection (mg/ml)
injection (mg/kg)
1
2
2
4
5
Mean
Atropine 10
-Atropine 10
61 31
77 63
90 44
58 186
47 40
67t 73
Tremorine 20
tremor
Atropine 10
57
106
98
74
45
76
Atropine 10+ tremorine 20
m
112"
141"
138"
106"
88*
117
Dyflos 1
m
19
131"
81"
112"
83*
85
108"
103"
153"
93*
69*
105
Dyflos 1 + tremorine 20
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. t = Mean tremor value significantly higher than equivalent saline control.
Atropine 10 mg/ml injected into the caudate nucleus gave a mean tremor value which was significantly higher than the value recorded after intracaudate injection of saline (Table 1). It was not significantly different from the mean value recorded after an intraperitoneal injection of atropine 10 mg/kg. Intracaudate injection of tremorine 20 mg/ml into rats pretreated with atropine 10 mg/kg i.p. gave a mean value of 76 units similar to the mean of 73 units obtained from the rats receiving only the pretreatment dose. It was lower than the mean of 103 units recorded from rats receiving intracaudate tremorine (Table 2), but the difference between means did not achieve the accepted level of statistical significance (P = 0.063). When atropine and tremorine were injected together into the caudate nucleus the mean tremor recorded was not significantly different from the mean obtained after tremorine alone, but it was significantly higher (P =0.008) than the mean for intracaudate injection of atropine. G
296
B. Cox and D. POTKON,IAK
It was also significantly higher (P =0.028) than the tremor recorded from the rats receiving atropine by the intraperitoneal route prior to an intracaudate injection of tremorine. Dyflos The effect of intracaudate injection of dyflos 1 mg/ml on the trelnor produced by intracaudate injection of tremorine 20 mg/ml is shown in Table 4. Stereotaxic injection of dyflos gave a mean tremor value of 85 units. Only one rat out of five failed to show visual evidence of tremor. Simultaneous injection of tremorine and dyflos gave a mean tremor value of 105 units which was not significantly different from the 103 units recorded after tremorine alone (Table 2). It was not significantly higher than the tremor recorded from rats receiving dyflos alone.
DISCUSSION Preliminary experiments showed that intraperitoneal oxotremorine and tremorine could produce tremor after ether anaesthesia. Therefore the stereotaxic experiments were not invalidated by the use of this anaesthetic. In spite of the relatively high saline readings significant differences could be obtained between these values and the values obtained when the animals were exhibiting tremor symptoms. The red nucleus gave the highest mean value after saline injection. This nucleus is small compared with the other areas and lies deep in the brain near the mid-sagittal line. With the stereotaxic co-ordinates used it is possible that the higher background activity was due to the trauma in some area more superficial than the site injection. The higher values obtained were not as a result of tremor, but could be seen to be due to an increase in spontaneous activity of the rats. Whilst the possibility that an injection volume of 0.003 ml could affect brain areas adjacent to the site of injection (e.g. by diffusion) cannot be discounted, a central rather than peripheral action of these drugs is suggested by the absence of any peripheral symptoms after tremorine or oxotremorine injection by the stereotaxic method. Furthermore, when a volume of 0'003 ml of I mg/ml oxotremorine was injected intraperitoneally, neither tremor nor peripheral symptoms were observed. Although the evidence for specific tremor sites within the central nervous system is not conclusive, oxotremorine injected into the substantia nigra and the red nucleus did produce signs of tremor. In our experiments oxotremorine did not prove as effective as tremorine in producing tremor. This is perhaps surprising in that oxotremorine is reported to be the active metabolite of tremorine and to be responsible for producing the tremor symptoms (CHo et al., 1961). CONNOR et al. (1967) have reported tremor after intracaudate carbachol injection, but in our hands the potent muscarinic agonist oxotremorine (CHO et al., 1962) was most effective in the substantia nigra. The three other areas including the caudate nucleus gave recorded values similar to those of the saline controls. It is possible that the ineffectiveness of oxotremorine was due to its rapid clearance and metabolism in the rat (HAMMER et al., 1968b). In this connection it has also been shown (Cox and POTKONJAK,1969a)that maximum oxotremorine tremor occurs within 5 min of an intraperitoneal injection. Thus, it is also possible that the maximum tremor stimulus due to stereotaxic injection of oxotremorine occurred in the first 3 min after the injection whilst the rat was still anaesthetised. The activity of tremorine after stereotaxic injection suggests either that the brain tissue is capable of metabolising tremorine to oxotremorine, or that tremorine can also act directly in its own right.
Stereotaxic injection of oxotremorine and tremorine
297
T h e a t r o p i n e experiments showed t h a t a t r o p i n e injected by the i n t r a p e r i t o n e a l route was an effective a n t a g o n i s t o f t r e m o r i n e t r e m o r , b u t t h a t i n t r a c a u d a t e a t r o p i n e was not a n effective a n t a g o n i s t in the c o n c e n t r a t i o n used. It w o u l d seem possible therefore t h a t the a t r o p i n e does n o t a n t a g o n i s e t r e m o r i n e at its site o f action, but either affects its m e t a b o l i s m (HAMMER et al., 1968a), or inhibits a n o t h e r site r e m o t e f r o m that o f injection where the t r a n s m i s s i o n is m e d i a t e d via a t r o p i n e sensitive receptors. I n t r a c a u d a t e injection o f dyflos p r o d u c e d t r e m o r on its own, s u p p o r t i n g the t h e o r y t h a t the t r e m o r site in this a r e a is acetylcholine sensitive. H o w e v e r , dyflos did not p o t e n t i a t e t r e m o r i n e t r e m o r , suggesting that the t r e m o r i n e is not acting via acetylcholine release. N e i t h e r was there evidence o f a d d i t i o n o f the t r e m o r responses p r o d u c e d by the two drugs. This confirms the previous findings ( C o x and POTKONJAK, 1969b) that i n t r a p e r i t o n e a l dyflos did not increase the t r e m o r readings after i n t r a p e r i t o n e a l o x o t r e m o r i n e . F u r t h e r , in this latter study, no p o t e n t i a t i o n o f low or high doses o f o x o t r e m o r i n e was observed. T h e r e f o r e the inability to detect p o t e n t i a t i o n was n o t due to the fact that the t r e m o r r e c o r d e d to either d r u g alone was a l r e a d y the m a x i m u m possible tremor. This investigation s u p p o r t s the t h e o r y that t r e m o r i n e t r e m o r is a result o f a direct action o f the drug, or its active m e t a b o l i t e o x o t r e m o r i n e , on certain sites within the central nervous system. The action w o u l d seem to be o f a cholinergic nature, but not involving acetylcholine release. Aeknowledgements--Oxotremorine was obtained as a gift from two sources: Dr. A. K. CHO, Dept. of Pharmacology, U.C.L.A., Los Angeles, California, U.S.A. and Dr. R. W. BRIMBLECOMBE,Ministry of Defence Chemical Defence Establishment, Porton Down, Wiltshire. Tremorine was obtained as a gift from Lilly Research Laboratories Ltd. and May & Baker Ltd.
REFERENCES CHO, A. K., I-IASLErT, W. L. and JENDEN,D. J. (1961). The identification of an active metabolite of tremorine. Biochem. biophys. Res. Commun. 5: 276-279. CHO, A. K., HASLETT,W. L. and JENDEN, D. J (1962) The peripheral actions of oxotremorine, a metabolite of tremorine. J. Pharmac. exp. Ther. 138: 249-257. CLARK,G. (1939). The use of the Horsley-Clarke instrument on the rat. Science 90: 92. CONNOR, J. D., Rossh G. V. and BAKER,W. W. (1967). Antagonism of intracaudate carbachol tremor by local injections of catecholamines. J. Pharmac. exp. Ther. 155: 545-551. Cox, B. and POTKONJAK,D. (1969a). The relationship between tremor and change in brain acetylcholine concentrationproducedbyinjectionoftremorineoroxotremorineintherat. Br. J. Pharmac. (Inpress). Cox, B. and POTKONJAK,D. (1969b). The effects of atropine and dyflos on the tremor and increase in whole brain acetylcholine produced by injection of oxotremorine in the rat. Br. J. Pharmac. (In press). DE GROOT, J. (1959). The rat forebrain in stereotaxic co-ordinates. In Verhandelingen Der Kononklijke, Nederlandse Academic Van V/etenschappen, Afd. Natuurkunde, Tweede Reeks, Deel LII, No. 4, N.V. Noord-Hollandsche Uttgevers Maatschappi, Amsterdam. HAMMER,W., KARLEN,B.~ RANE, A. and SJ(~QVIST,F. (1968a). Rate of metabolism of tremorine and oxotremorine in rats and mice. Life Sci. 7: 197-204. HAMMER,W., KARLEN,B. and SJ()QVIST,F. (1968b). Drug induced changes in brain levels of oxotremorine in mice. Life Sci. 7: 205-211. HORSLEV, V. and CLARKE,R. H. (1908). The structure and functions of the cerebellum examined by a new method. Brain 31: 45-124. KAADA,B. R. (1963). The pathophysiology of Parkinsonian tremor, rigidity and hypokinesia. Acta neurol. scand. 39 Suppl. 4: 39-51. SIEGEL, S. (1956). Nonparamerric Statistics for the Behavioral Sciences. McGraw-Hill, London.
Pergamon Press.
Printed in Gt. Britain.
AN INVESTIGATION OF THE T R E M O R G E N I C EFFECTS OF OXOTREMORINE AND T R E M O R I N E AFTER STEREOTAXIC INJECTION INTO RAT BRAIN B. Cox and D. POTKONJAK Department of Pharmacology, The University, Manchester 13 (Accepted 14 January 1969) Summary--The effect of stereotaxic administration of tremorine and oxotremorine into four brain areas has been investigated using rats. Tremorine produced tremor significantly higher than control after administration into the globus pallidus, caudate nucleus, and substantia nigra. Oxotremorine produced tremor significantly higher than control only after injection into the substantia nigra. Dyflos injected into the caudate nucleus produced tremor. No change in the tremorgenic action of tremorine was observed after the simultaneous intracaudate injections of atropine and tremorine, and dyftos and tremorine. Intraperitoneal atropine reduced the tremorgenic action of an intracaudate injection of tremorine. Cox and POTKONJAK (1969a) have reported a quantitative method for the measurement of tremor in the rat, which was used to investigate the actions of tremorine (1,4-dipyrrolidino2-butyne) and oxotremorine [1,-(2-oxopyrrolidino)-4-pyrrolidino-butyne-2] given by intraperitoneal injection. It was noted that the time course of the tremor did not coincide with the time course of an increase in brain acetylcholine which also occurred. The same workers have shown (1969b) that, although atropine inhibited oxotremorine tremor, dyflos failed to potentiate it. The failure of dyflos to potentiate oxotremorine suggested that the tremor drug was acting directly, rather than indirectly through the release of acetylcholine. Furthermore, HAMMER et al. (1968b) have reported that in atropine pretreated mice there is an increased clearance of oxotremorine from the brain, which may indicate that the antagonism of tremorine and oxotremorine tremor by atropine is not due to an effect at the site of tremor production. In order to investigate these problems further, we have examined the effects of tremorine and oxotremorine administered by stereotaxic injection into selected brain areas and studied the effect of atropine and dyflos on any tremor produced. The brain areas chosen were parts of the extrapyramidal system involved in the control of movement and posture (KAADA, 1963).
METHODS Male Wistar rats weighing 190-210 g were used in all the experiments. Stereotaxic injection Tremor drugs were injected into the globus pallidus, caudate nucleus, substantia nigra and red nucleus by a stereotaxic method similar to that of HORSLEY and CLARKE (1908) and CLARK (1939), using the stereotaxic system of co-ordinates (DE GROOT, 1959). 291
292
B. Cox and D. POTKONJAK
A rat anaesthetised with ether was placed on the stereotaxic instrument and its shaved head immobilised. An incision 2-2.5 cm long was made through the skin in the midline. The site required was located by using the appropriate stereotaxic co-ordinates and a small hole was drilled in the skull to allow access of a No. 16 hypodermic needle. The needle was lowered to the required depth and an injection made from a micrometer syringe. The volume delivered at each site of injection was 0.003 ml. After injection the needle was withdrawn. The skin was drawn together and sutured. Injections were always unilateral, but for any one series were made alternately on the left and right sides. The depth of anaesthesia was adjusted so that the rat recovered consciousness within 3 min of the injection being made. Rats injected with saline by the stereotaxic method into that same four areas as the tremor drugs, and rats injected with the tremor drugs by the intraperitoneal route after ether anaesthesia were used as controls.
Measurement of tremor Tremor was recorded by a method described previously (Cox and POTKONJAK,1969a). The recording was started exactly 2 min after the stereotaxic injection. The rat was suspended in a perspex box (18 × 7 x 7 cm) from a gramophone pick-up head. The movements of the rat were thus converted into electrical energy which was fed into two channels of a Grass Polygraph (Model 7). Channel 1 gave a qualitative record of the tremor. Channel 2, an integrator channel, gave a quantitative record of the tremor. One complete deflection of the integrator pen was given an arbitrary value of 100 tremor units. An automatic zero returned the pen to baseline every 15 sec. Sample traces are shown in Fig. 1. The tremor was recorded for 5 min (twenty 15-sec samples) during a period of tremor, if tremor occurred. If it did not occur then a control period of 5 min was recorded from 3 min to 8 min after injection. The tremor is always presented as the mean of the twenty 15-sec samples recorded in the 5 min (arbitrary units). At the end of the tremor recordings the rat was sacrificed and the brain removed. After 10 ~ formalin fixation the brain was sectioned and the site of the injection confirmed histologically by locating the needle track. Statistics The Mann-Whitney U test (SIEGEL, 1956) was used to determine the statistical significance of the differences between groups. Unless otherwise stated the criterion of a significant difference between means was P~0.05. Drugs All drugs were freshly prepared for injection in 0-9 9/00saline and the concentration expressed as mg/ml of the free base. Atropine sulphate, B.D.H. Ltd. Dyflos (Diisopropylfluorophosphate), Koch-Light Ltd. Oxotremorine as the base. Tremorine dihydrochloride.
RESULTS 1. The ability of intraperitoneal tremorine and oxotremorine to produce tremor after ether anaesthesia Sample traces of the tremor recording from six rats, three without previous administration of ether, and three after ether anaesthesia, are shown in Fig. 1. The mean background
TRfMORlNf
TRfMORlRf *.m.fk,
aomp/k’d
AFTER fTMR
Alf4fslHfsl*
FIG. I. Nemo.
f.p. 292
Stereotaxic injection of oxotremorine and trernorine
293
activity r e c o r d e d after saline injection into an u n a n a e s t h e t i s e d rat was 40 units. This value c o m p a r e d well with a m e a n value o f 42 units o b t a i n e d previously with the same r e c o r d i n g system ( C o x and POTKONJAK, 1969a). A rat, injected with saline after e t h e r anaesthesia, showed at the beginning o f the r e c o r d i n g large s p a s m o d i c r e s p i r a t o r y movements, which resulted in a higher b a c k g r o u n d activity. O x o t r e m o r i n e (1 mg/kg) in an u n a n a e s t h e t i s e d rat gave a mean 15 sec t r e m o r o f 130 units. In a similar rat after ether anaesthesia the t r e m o r recorded was 109 units. T r e m o r i n e 20 m g / k g injected into an u n a n a e s t h e t i s e d rat gave a mean 15 sec t r e m o r o f 137 units which was increased to 158 units in the anaesthetised rat. As it was shown t h a t t r e m o r could be both o b s e r v e d a n d r e c o r d e d after ether anaesthesia, the effect o f saline, t r e m o r i n e and o x o t r e m o r i n e was investigated by injecting t h e m stereotaxically into the brains o f rats anaesthetised with ether.
2.
The effect of stereotaxic injection of tremor drugs
P r e l i m i n a r y experiments with o x o t r e m o r i n e i n d i c a t e d t h a t the a r e a which gave the most consistent t r e m o r after injection was the s u b s t a n t i a nigra. Therefore, a range o f solutions 0.125, 0.25, 0"5, 1"0 and 2-0 m g / m l were injected into this a r e a in individual rats and gave t r e m o r values o f 62, 72, 78, 85 and 76 units respectively. The c o n c e n t r a t i o n selected for further study was 1 mg/ml. T r e m o r i n e 20 mg/ml had a c o m p a r a b l e effect to oxotl'emorine I mg/ml and this concentration was therefore used.
Saline The effect o f saline injected by the stereotaxic m e t h o d is shown in Table 1. N o n e o f these injections p r o d u c e d visible tremor. The mean values o b t a i n e d after injection into the globus TABLE 1. THE MEAN 15 s e c TREMOR READING AFTER STEREOTAXIC INJECTION OF SALINE INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
1
30
48
30
73
2 3 4 5
52 46 55 36
65 35 33 25
45 24 58 48
67 60 67 86
Mean tremor
44
41
41
71~
All tremor values in arbitrary units. *=Rats in which tremor was clearly visible (none in this Table). t = Mean value significantly higher than other saline means. GP =Globus pallidus; CN =caudate nucleus; SN--substantia n igra; RN =red nucleus. pallidus, c a u d a t e nucleus a n d s u b s t a n t i a nigra were n o t significantly different f r o m each other. The m e a n t r e m o r value o b t a i n e d after injection into the red nucleus was higher ( P = 0 . 0 0 4 ) t h a n the results f r o m the o t h e r three areas.
294
B. Cox and D. POTKONJAK
Tremorine T h e effect of" s t e r e o t a x i c injection o f t r e m o r i n e 20 m g / m l is s h o w n in T a b l e 2. I n j e c t i o n i n t o all f o u r a r e a s p r o d u c e d o b s e r v a b l e t r e m o r in each rat tested. T h e r e was n o significant TABLE 2. THE MEAN 15 s e c TREMOR READING AFTER STEREOrAXIC INJECTION OF TREMORINE ( 2 0 mg,'ml) INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
2 3 4 5 6
82* 98* 75* 123" 98* t08"
84* 124" 67 ':: 83* 116" 141"
104" 76* 59* 71" 93*
61" 179" 96* 23* 66*
Mean tremor
97~
103t
81*
85
I
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. -~ Mean values significantly higher than corresponding saline controls. GP :Globus pallidus; CN -- caudate nucleus : SN-- substant ia nigra: R N - - r e d nucleus. difference b e t w e e n the t r e m o r r e c o r d e d f r o m any o f t h e f o u r g r o u p s . H o w e v e r , the t r e m o r r e c o r d e d a f t e r i n j e c t i o n i n t o the g l o b u s pallidus, c a u d a t e n u c l e u s a n d s u b s t a n t i a n i g r a was significantly h i g h e r ( P = 0 . 0 0 4 ) t h a n the c o r r e s p o n d i n g saline c o n t r o l s . T h e r e was n o significant difference ( P ~ 0 . 3 4 5 ) b e t w e e n t h e m e a n t r e m o r r e a d i n g s after t r e m o r i n e o r saline i n j e c t i o n i n t o t h e red nucleus.
Oxotremorine T h e effect o f s t e r e o t a x i c i n j e c t i o n o f o x o t r e m o r i n e 1 m g / m l , is s h o w n in T a b l e 3. O n l y a f t e r i n j e c t i o n o f o x o t r e m o r i n e i n t o t h e s u b s t a n t i a n i g r a was the m e a n t r e m o r r e a d i n g significantly ]"ABLE3. THE MEAN 15 sec TREMOR READING AFTER STEREOTAXIC INJECTION OF OXOTREMORINE (1 mg/ml) INTO FOUR BRAIN AREAS
Mean 15 sec tremor reading Rat
GP
CN
SN
RN
2 3 4 5
23 53 68 40 105"
45 85 15 78 59
77* 135" 89* 63* 78*
152" 63* 123" 148"
Mean tremor
58
56
88~
107
1
49*
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. t = M e a n value significantly higher than equivalent saline control. GP = Globus pallidus; CN = caudate nucleus; SN = substantia nigra; R N = red nucleus.
295
Stereotaxic injection of oxotremorine and tremorine
higher (P=0-004) than the equivalent saline control value. During the recording period no tremor could be observed visually in the group of rats receiving oxotremorine into the caudate nucleus. One rat out of five exhibited tremor symptoms after injection into the globus pallidus, but tremor was observed visually in all the rats where injections were made into the area of the substantia nigra and the red nucleus. The mean tremor readings after the injection of oxotremorine (1 mg/ml) into the globus pallidus and the caudate nucleus were significantly lower (P -0.048) than the corresponding mean values after the injection of tremorine 20 mg/ml. The mean values after injection into the other areas were not significantly different from the corresponding means after tremorine.
Atropine The effect of atropine pretreatment on the tremor produced by intracaudate injection of tremorine 20 mg/ml is shown in Table 4. TABLE 4. THE
EFFECT OF ATROPINE
AND
DYFLOS PRETREATMENT
ON TREMOR PRODUCED
BY I N T R A C A U D A T E
INJECTION OF TREMORINE
(20 mg/ml) Drug
Rat Nos.
Intracaudate
Intraperitoneal
injection (mg/ml)
injection (mg/kg)
1
2
2
4
5
Mean
Atropine 10
-Atropine 10
61 31
77 63
90 44
58 186
47 40
67t 73
Tremorine 20
tremor
Atropine 10
57
106
98
74
45
76
Atropine 10+ tremorine 20
m
112"
141"
138"
106"
88*
117
Dyflos 1
m
19
131"
81"
112"
83*
85
108"
103"
153"
93*
69*
105
Dyflos 1 + tremorine 20
All tremor values in arbitrary units. * = Rats in which tremor was clearly visible. t = Mean tremor value significantly higher than equivalent saline control.
Atropine 10 mg/ml injected into the caudate nucleus gave a mean tremor value which was significantly higher than the value recorded after intracaudate injection of saline (Table 1). It was not significantly different from the mean value recorded after an intraperitoneal injection of atropine 10 mg/kg. Intracaudate injection of tremorine 20 mg/ml into rats pretreated with atropine 10 mg/kg i.p. gave a mean value of 76 units similar to the mean of 73 units obtained from the rats receiving only the pretreatment dose. It was lower than the mean of 103 units recorded from rats receiving intracaudate tremorine (Table 2), but the difference between means did not achieve the accepted level of statistical significance (P = 0.063). When atropine and tremorine were injected together into the caudate nucleus the mean tremor recorded was not significantly different from the mean obtained after tremorine alone, but it was significantly higher (P =0.008) than the mean for intracaudate injection of atropine. G
296
B. Cox and D. POTKON,IAK
It was also significantly higher (P =0.028) than the tremor recorded from the rats receiving atropine by the intraperitoneal route prior to an intracaudate injection of tremorine. Dyflos The effect of intracaudate injection of dyflos 1 mg/ml on the trelnor produced by intracaudate injection of tremorine 20 mg/ml is shown in Table 4. Stereotaxic injection of dyflos gave a mean tremor value of 85 units. Only one rat out of five failed to show visual evidence of tremor. Simultaneous injection of tremorine and dyflos gave a mean tremor value of 105 units which was not significantly different from the 103 units recorded after tremorine alone (Table 2). It was not significantly higher than the tremor recorded from rats receiving dyflos alone.
DISCUSSION Preliminary experiments showed that intraperitoneal oxotremorine and tremorine could produce tremor after ether anaesthesia. Therefore the stereotaxic experiments were not invalidated by the use of this anaesthetic. In spite of the relatively high saline readings significant differences could be obtained between these values and the values obtained when the animals were exhibiting tremor symptoms. The red nucleus gave the highest mean value after saline injection. This nucleus is small compared with the other areas and lies deep in the brain near the mid-sagittal line. With the stereotaxic co-ordinates used it is possible that the higher background activity was due to the trauma in some area more superficial than the site injection. The higher values obtained were not as a result of tremor, but could be seen to be due to an increase in spontaneous activity of the rats. Whilst the possibility that an injection volume of 0.003 ml could affect brain areas adjacent to the site of injection (e.g. by diffusion) cannot be discounted, a central rather than peripheral action of these drugs is suggested by the absence of any peripheral symptoms after tremorine or oxotremorine injection by the stereotaxic method. Furthermore, when a volume of 0'003 ml of I mg/ml oxotremorine was injected intraperitoneally, neither tremor nor peripheral symptoms were observed. Although the evidence for specific tremor sites within the central nervous system is not conclusive, oxotremorine injected into the substantia nigra and the red nucleus did produce signs of tremor. In our experiments oxotremorine did not prove as effective as tremorine in producing tremor. This is perhaps surprising in that oxotremorine is reported to be the active metabolite of tremorine and to be responsible for producing the tremor symptoms (CHo et al., 1961). CONNOR et al. (1967) have reported tremor after intracaudate carbachol injection, but in our hands the potent muscarinic agonist oxotremorine (CHO et al., 1962) was most effective in the substantia nigra. The three other areas including the caudate nucleus gave recorded values similar to those of the saline controls. It is possible that the ineffectiveness of oxotremorine was due to its rapid clearance and metabolism in the rat (HAMMER et al., 1968b). In this connection it has also been shown (Cox and POTKONJAK,1969a)that maximum oxotremorine tremor occurs within 5 min of an intraperitoneal injection. Thus, it is also possible that the maximum tremor stimulus due to stereotaxic injection of oxotremorine occurred in the first 3 min after the injection whilst the rat was still anaesthetised. The activity of tremorine after stereotaxic injection suggests either that the brain tissue is capable of metabolising tremorine to oxotremorine, or that tremorine can also act directly in its own right.
Stereotaxic injection of oxotremorine and tremorine
297
T h e a t r o p i n e experiments showed t h a t a t r o p i n e injected by the i n t r a p e r i t o n e a l route was an effective a n t a g o n i s t o f t r e m o r i n e t r e m o r , b u t t h a t i n t r a c a u d a t e a t r o p i n e was not a n effective a n t a g o n i s t in the c o n c e n t r a t i o n used. It w o u l d seem possible therefore t h a t the a t r o p i n e does n o t a n t a g o n i s e t r e m o r i n e at its site o f action, but either affects its m e t a b o l i s m (HAMMER et al., 1968a), or inhibits a n o t h e r site r e m o t e f r o m that o f injection where the t r a n s m i s s i o n is m e d i a t e d via a t r o p i n e sensitive receptors. I n t r a c a u d a t e injection o f dyflos p r o d u c e d t r e m o r on its own, s u p p o r t i n g the t h e o r y t h a t the t r e m o r site in this a r e a is acetylcholine sensitive. H o w e v e r , dyflos did not p o t e n t i a t e t r e m o r i n e t r e m o r , suggesting that the t r e m o r i n e is not acting via acetylcholine release. N e i t h e r was there evidence o f a d d i t i o n o f the t r e m o r responses p r o d u c e d by the two drugs. This confirms the previous findings ( C o x and POTKONJAK, 1969b) that i n t r a p e r i t o n e a l dyflos did not increase the t r e m o r readings after i n t r a p e r i t o n e a l o x o t r e m o r i n e . F u r t h e r , in this latter study, no p o t e n t i a t i o n o f low or high doses o f o x o t r e m o r i n e was observed. T h e r e f o r e the inability to detect p o t e n t i a t i o n was n o t due to the fact that the t r e m o r r e c o r d e d to either d r u g alone was a l r e a d y the m a x i m u m possible tremor. This investigation s u p p o r t s the t h e o r y that t r e m o r i n e t r e m o r is a result o f a direct action o f the drug, or its active m e t a b o l i t e o x o t r e m o r i n e , on certain sites within the central nervous system. The action w o u l d seem to be o f a cholinergic nature, but not involving acetylcholine release. Aeknowledgements--Oxotremorine was obtained as a gift from two sources: Dr. A. K. CHO, Dept. of Pharmacology, U.C.L.A., Los Angeles, California, U.S.A. and Dr. R. W. BRIMBLECOMBE,Ministry of Defence Chemical Defence Establishment, Porton Down, Wiltshire. Tremorine was obtained as a gift from Lilly Research Laboratories Ltd. and May & Baker Ltd.
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