144
Brain Research, 114 (1976) 144-151 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Harmaline-induced tremor in the rat: abolition by 3-acetylpyridine destruction of cerebellar climbing fibers
RABI SIMANTOV, SOLOMON H. SNYDER and MARY-LOU OSTER-GRANITE Departments of Pharmacology and Experimental Therapeutics (S.H.S.) and Psychiatry and Behavioral Sciences ( R.S.) and Neurology (M.-L.O.G.), Johns Hopkins University Sehool of Medicine, Baltimore, Md. 21205 (U.S.A.)
(Accepted May 18th, 1976)
The pronounced tremor elicited by harmaline in numerous animal species has been used as a model of neurological diseases such as Parkinsonism. Neurophysiologic studies indicate that harmaline stimulates the firing ofcerebellar climbing fibers which in turn activate Purkinje cell firing in the cerebellum 7,9. It has been postulated that the tremor elicited by harmaline is associated with the enhanced firing rate of the climbing fibers. However, harmaline has a number of metabolic effects, including inhibition of monoamine oxidase, and could conceivably elicit tremor via other mechanisms, such as those involving monoamines, in parts of the brain such as the corpus striatum, which is rich in several monoamines. Recently it has been shown that 3-acetylpyridine elicits a fairly selective destruction of the inferior olivary nucleus in the brain stem which is followed by degeneration of the climbing fiber projection from the olivary nucleus to the Purkinje cells of the cerebellum1, 2. To test whether this neuronal pathway is responsible for harmaline-induced tremor, we have evaluated the influence of 3-acetylpyridine destruction of this pathway upon the tremorigenic actions of harmaline. Experiments were performed on male Sprague-Dawley rats (150-200 g). Harmaline HC1 (Regis Chemical Company); nicotinamide, nicotinic acid and 3-acetylpyridine (Sigma) were injected intraperitoneally in different doses, as indicated in each experiment. The tremor induced by harmaline was scored by an arbitrary scale between 0 = no tremor and 3 = maximal tremor, by an observer blinded to the treatment regimen. Degree 1 of tremor was scored when the rats displayed tremor of the trunk and extremities but for less than 5 0 ~ of a standard 5 rain observation period. Degree 2 was scored when tremor was present 50-100 ~ of the observed time period, and degree 3 represented constant tremor. Forty-eight hours after the injection of 3-acetylpyridine, some rats were perfused through the left ventricle with a mixed aldehyde solution containing 1 ~ glutaraldehyde, 2 ~ paraformaldehyde, and 1 ~ acrolein in 0.1 M cacodylate buffer, pH 7.6 (ref. 11). The cerebellum and brain stem were removed 1 h after perfusion and the inferior olives and cerebellar cortex were dissected away. Slabs of these were placed in fixative
145
Fig. 1. a: an early degenerating climbing fiber (CF) process in the molecular layer 48 h after 3-acetylpyridine contains an increasingly dense cytoplasmic matrix; dense, clustered synaptic vesicles; and intact synaptic contacts with Purkinje cell dendritic spines (s). × 57,000. b: degenerating climbing fiber process which has retracted from the surface ofa Purkinje cell dendrite (D) contains an increased density on the presynaptic surface. Some spine contacts (S) are still intact. Other spines contact parallel fiber processes (PF). × 34,000.
at 4 °C overnight. After postfixation in 1 ~ OsO4 in 0.1 M cacodylate buffer, p H 7.6, containing 10 ~ sucrose, blocks were processed conventionally in Epon with en bloc staining with 1 ~ uranyl acetate in absolute ethanol for 1 h. Sections were cut on a Reichert O M U - 2 ultramicrotome with a diamond kifen and stained with lead citrate and uranyl acetate. The stained sections were examined in a Philips 200 electron microscope. As reported earlied,2, 5 after 3-acetylpyridine few, if any, intact climbing fibers remain in the sampled areas of lobules adjacent to the primary fissure. Degenerating climbing fiber terminals occur in scattered regions of the molecular layer. An increased density of presynaptic terminals (Fig. 1a) is followed by condensation and detachment of the degenerating climbing fiber processes from the Purkinje cell dendrites (Fig. lb). Glial processes sequester the detached boutons and subsequent degeneration progresses rapidly, with little debris remaining at 48 h. The cortex is striking in its absence of climbing fiber processes and synapses with parallel fiber dilatations containing synaptic vesicles appearing more numerous. No alterations are detected in the dendritic stems, the nuclei, or the somata of Purkinje cells except for the absence of en passant climbing fiber synapses. The deafferented spines appear normal except that they lack
146 residual postsynaptic specialization (Fig. 2). All other synaptic types in the cortex are normal in both appearance and distribution. In the internal granular layer climbing fiber glomerular en marron contacts with Golgi cells (Fig. 3) appear normal and are easily found. Both climbing fiber and mossy fiber glomeruli are present with no evidence of degeneration. These anatomical findings concur with those of Llin~ts and coworkers 5 who found rapid degeneration and removal of climbing fiber endings following 3-acetylpyridine. The few climbing fibers remaining at 48 h may not represent olivary climbing fibers, but instead may arise from the locus coerulus, the raphe nuclei, or even the vestibular nuclei. With the brief degeneration process, the postsynaptic surface remains free of any residual specialization, unlike parallel fiber deafferentation in adult rats following thiophen 3, in which spine processes of Purkinj e cell often contain or continue to develop postsynaptic specializations 11. The augmentation of parallel fiber terminals may result from sprouting as after parallel fiber lesions 10, or in the absence of the climbing fiber endings, the parallel fiber processes may just become more noticeable. In normal rats harmaline, in doses between 5 and 25 mg/kg, produces a marked fine tremor with a frequency of about 8-12/sec, involving all extremities and the entire trunk. Although the intensity of the tremor appears to be similar at all doses, the latency before the initiation of tremor is shorter at higher doses, being about 3 min at 25 mg/kg (Fig. 4). This latency period gradually increases to about 12 min at 5 mg/kg. The total duration of the tremor is about 2-3 h. These findings resemble numerous previous studies describing harmaline-induced tremor 6-9. In preliminary studies in our rats, a dose of 78 mg/kg represents the LD~0 for 3-acetylpyridine. Death occurs 6 h to 2 weeks after injection. Survivors show some ataxia but otherwise eat, drink and are active for months. At 78 mg/kg, 3-acetylpyridine abolishes tremor production by harmaline (1.25 mg/kg) (Fig. 4). However, harmaline elicits some locomotor activation with animals moving about fairly randomly with some increase in ataxia. Complete abolition of the tremor response to harmaline is apparent by about 8 h after 3-acetylpyridine, with a marked reduction at 6 h and a minimal reduction at 4 h (Fig. 4). Interestingly, marked degeneration of olivary cells and climbing fibers is first apparent about 7 h after 3-acetylpyridine administration, which is also the time when behavioral evidence of cerebellar function impairment is first evident 1,2. Complete abolition of the harmaline-induced tremor requires a dose of 78 mg/kg of 3-acety!pyridine, which is also the LD50 dose and the dose required for complete destruction of climbing fibers1, 2. At doses of 65 and 72 mg/kg tremor is partially prevented, while 50 mg/kg of 3-acetylpyridine fails to affect the harmaline-induced tremor. Oxotremorine produces a marked tremor, presumably by stimulating muscarinic cholinergic receptor sites in the brain. In our rats 0.75 or 1.5 mg/kg of oxotremorine elicit a pronounced tremor of all extremities and the trunk. However, in marked contrast to the influences of harmaline, the oxotremorine tremor persists with no reduction in intensity in rats surviving 48 h after treatment with 78 mg/kg of 3-acetylpyridine.
Fig. 2. Abundant parallel fiber processes (PF) near a Purkinje cell dendrite which contains spines (S) lacking any postsynaptic specialization. Spine contacts to parallel fibers (*) are normal, but no contacts to climbing fibers are found. × 33,000.
Fig. 3. A typical climbing fiber (CF) en marron contact to a Golgi cell (Go) contains the usual arrangement of vesicles and mitochondria in the glomerular rosette: Despite degeneration of climbing fiber processes in the molecular layer, climbing fiber glomerular collaterals were normal in appearance throughout the internal granular layer 48 h after 3-acetylpyridine. × 23,000.
149
~H
A
rt0 I.d OC I'-
nQ Oi
0
50
70
4
90
3-ACETYLPYRIDINE DOSE ( m g / k g )
B
12
24
48
INTERVAL BETWEEN 3-ACETYLPYRIDINE AND HARMALINE INdECTIONS (hrs)
Fig. 4. Inhibition of harmaline-induced tremor by 3-acetylpyridine. A: different doses of 3-acetylpyridine were injected and rats that survived after 48 h received 12.5 mg/kg harmaline. B: 3-acetylpyridine (78 mg/kg) was injected at different times before harmaline (12.5 mg/kg). In both experiments the extent of tremor was scored 15 min after harmaline injection and each point on the figures represents the mean value from 10-15 rats :5 S.E.M. Some influences o f 3-acetylpyridine u p o n b r a i n function a p p e a r to result f r o m the c o m p e t i t i o n o f 3-acetylpyridine with n i c o t i n a m i d e for i n c o r p o r a t i o n into N A D 4. I f prevention o f the h a r m a l i n e - i n d u c e d t r e m o r is a t t r i b u t a b l e to this m e t a b o l i c action, then large doses o f n i c o t i n a m i d e might prevent this effect. By contrast, nicotinic acid, which c a n n o t substitute for n i c o t i n a m i d e in the exchange reaction with N A D 4, o u g h t n o t to p r o t e c t against 3-acetylpyridine. R a t s t r e a t e d with n i c o t i n a m i d e (500 mg/kg) i m m e d i a t e l y after 3-acetylpyridine a n d a d m i n i s t e r e d h a r m a l i n e 48 h later show the same intensity o f t r e m o r as c o n t r o l animals injected with h a r m a l i n e with neither 3-acetylp y r i d i n e n o r n i c o t i n a m i d e t r e a t m e n t . Besides preventing the influence o f 3-acetylpyridine on h a r m a l i n e - i n d u c e d t r e m o r , n i c o t i n a m i d e also prevents the lethal effects o f 3-acetylpyridine. Thus, while only 50 ~ o f rats t r e a t e d with 3-acetylpyridine alone survive after 48 h, there are no d e a t h s in g r o u p s o f rats receiving n i c o t i n a m i d e i m m e d i a t e l y TABLE I Effect o f nicotinamide and nicotinic acid on animal survival and harmaline-induced tremor
Groups of 15 rats were injected (i.p.) with 500 mg/kg of nicotinamide or nicotinic acid immediately after receiving 78 mg/kg 3-acetylpyridine. Tremor induction by harmaline (12.5 mg/kg) and animal survival were determined 48 h later. Data are expressed as mean values :5 S.E.M. for tremor and for animal survival as the ratio of the number of rats alive 48 h after 3-acetylpyridine to the total number in each group. Analogue treatment
None Nicotinamide Nicotinic acid
Control Rats
3-A cetylpyridine-treated rats
Animals sur rived
Degree o f tremor
Animals survived
Degree o f tremor
15/15 15/15 15/15
2.7:5 0.3 2.8 ± 0.3 2.6:5 0.3
8/15 15/15 6/15
0.2:5 0.1 2.7:5 0.3 0.2:5 0.1
150 3-
/p.-.~
~-Nicotinomide
r,. 0
n. o I--
2-
6
,=.,
o
n. (.9 ta.i E)
,=,, r~ t9
w
E3
1-
1-
~ -Nicotinic
o-
~
0
I()
[-, 25
DOSE (mg/kg)
Acid
O-
,~--4 50 500
o
~
a
12
24
4a
INTERVAL BETWEEN 3-ACETYLPYRIDINE AND NICOTINAMIDE INJECTIONS (hrs)
Fig. 5. Effectof nicotinamide and nicotinic acid on the harmaline-inducedtremor as a function of treatment with 3-acetylpyridine.A: harmaline (12.5 mg/kg) was injected 48 h after injection of different doses of nicotinamide or nicotinic acid. B: harmaline (12.5 mg/kg) was injected at different times after injection of 500 mg/kg nicotinamide. In both experiments (A and B) each point is the mean value from 8-12 rats :[ S.E.M. after the 3-acetylpyridine. In marked contrast, nicotinic acid administration (500 mg/kg) provides no protection against the lethal effects of 3-acetylpyridine or its effect on harmaline-induced tremor (Table 1). The protective action of nicotinamide occurs at fairly low doses. As little as 10 mg/kg ofnicotinamide prevents the lethal effects of 3-acetylpyridine and its influences on harmaline-induced tremor, while 4 mg/kg has a partial effect (Fig. 5). By contrast, at 4 doses between 5 and 500 mg/kg, nicotinic acid fails to prevent the effects of 3-acetylpyridine. The protective influence of nicotinamide is evident whether administered simultaneously with or up to 3 h after 3-acetylpyridine. When given 4 h after 3-acetylpyridine, nicotinamide partially protects, while if administered 6 h or more after the 3-acetylpyridine, there is little protection. Since the neuropathologic effects of 3-acetylpyridine are first apparent at 6-8 h, it appears that the metabolic actions of 3-acetylpyridine upon susceptible neurons are subject to reversal by nicotinamide almost until the time they are evident by microscopic examination. The observations of this study support other evidence that tremor induced by harmaline is mediated by the olivo-cerebellar pathway via climbing fiber projections to the Purkinje cells 6,7,9. The neuropathologic effects of 3-acetylpyridine are largely restricted to the lower brain steml, 2. Besides the inferior olivary nucleus 3-acetylpyridine damages the nucleus of the facial nerve, the nucleus ambiguus and the hypoglossal nucleus 1,2. However, these other nuclei are less severely affected than the inferior olivary nucleus. The selectivity of 3-acetylpyridine effects is supported by its failure to alter tremor induced by oxotremorine. Recent neuroanatomical evidence indicates that the only cerebellar input affected by 3-acetylpyridine is the climbing fiber projection to the Purkinje cells 1,2. Thus, 3acetylpyridine treatment affords a selective means of destroying this neuronal pathway, providing a tool to evaluate unique biochemical or physiological properties of climbing fibers. Abolition of harmaline-induced tremor may be a simple technique to screen for destruction of the olivo-cerebellar pathway by 3-acetylpyridine.
151 Th e a u th o r s w o u l d like to t h a n k J. F i r m a n for his technical assistance. This research was s u p p o r t e d in p a r t by U S P H S G r a n t s NS-08997 (to R . M . H . ) , MH-18501, R S D A M H - 3 3 1 2 8 (to S.H.S.) and an E l e a n o r R o o s e v e l t fellowship
(to R.S.).
1 Desclin, J. C., Histological evidence supporting the inferior olive as the major source of cerebellar climbing fibers in the rat, Brain Research, 77 (1974) 365-384. 2 Desclin, J. C. and Escubi, J., Effects of 3-acetylpyridine on the central nervous system of the rat as demonstrable by silver methods, Brain Research, 77 (1974) 349-364. 3 Herndon, R. M., Thiophen induced granule cell necrosis in the rat cerebellum. An electronmicroscopic study, Exp. Brain Res., 6 (1968) 49-68. 4 Kaplan, N. O., Goldin, A., Humphreys, S. R., Ciotti, M. M. and Venditti, M., Significance of enzymatically catalyzed exchange reactions in chemotherapy, Science, 120 (1954) 432440. 5 Sotelo, C., Hillman, D. E., Zamora, A. J. and Llinhs, R., Climbing fiber differentiation: its action on Purkinje cell dendritic spines, Brain Research, 68 (1975) 574-581. 6 Llinfis, R. and Volkind, R. A., Repetitive climbing fiber activation of Purkinje cells in the cat cerebellum following the administration of harmaline, Fed. Proc., 31 (1972) 377a. 7 Llingts, R. and Volkind, R. A., The olivo-cerebral system: functional properties as revealed by harmaline-induced tremor, Exp. Brain Res., 18 (1973) 69-87. 8 Mao, C. C., Guidotti, A. and Costa, E., Inhibition by diazepam of the tremor and the increase of cerebellar cGMP content elicited by harmaline, Brain Research, 83 (1975) 516-519. 9 Montigny, C. De and Lamarre, Y., Rhythmic activity induced by harmaline in the olivo-cerebellobulbar system of cat, Brain Research, 53 (1973) 81-95. 10 Mouren-Mathieu, A. M. and Colonnier, M., The molecular layer of the adult cat cerebellar cortex after lesions of the parallel fibers: an optic and electron microscopic study, Brain Research, 1 (1969) 307-323. 11 Oster-Granite, M.-L. and Herndon, R. M., The pathogenesis of parvovirus-induced cerebellat hypoplasia in the Syrian hamster, Mesocricetus auratus. Fluorescent antibody, foliation, cytoarchitectonic, Golgi, and electron microscopic studies, J. camp. Neurol, in press.
Brain Research, 114 (1976) 144-151 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Harmaline-induced tremor in the rat: abolition by 3-acetylpyridine destruction of cerebellar climbing fibers
RABI SIMANTOV, SOLOMON H. SNYDER and MARY-LOU OSTER-GRANITE Departments of Pharmacology and Experimental Therapeutics (S.H.S.) and Psychiatry and Behavioral Sciences ( R.S.) and Neurology (M.-L.O.G.), Johns Hopkins University Sehool of Medicine, Baltimore, Md. 21205 (U.S.A.)
(Accepted May 18th, 1976)
The pronounced tremor elicited by harmaline in numerous animal species has been used as a model of neurological diseases such as Parkinsonism. Neurophysiologic studies indicate that harmaline stimulates the firing ofcerebellar climbing fibers which in turn activate Purkinje cell firing in the cerebellum 7,9. It has been postulated that the tremor elicited by harmaline is associated with the enhanced firing rate of the climbing fibers. However, harmaline has a number of metabolic effects, including inhibition of monoamine oxidase, and could conceivably elicit tremor via other mechanisms, such as those involving monoamines, in parts of the brain such as the corpus striatum, which is rich in several monoamines. Recently it has been shown that 3-acetylpyridine elicits a fairly selective destruction of the inferior olivary nucleus in the brain stem which is followed by degeneration of the climbing fiber projection from the olivary nucleus to the Purkinje cells of the cerebellum1, 2. To test whether this neuronal pathway is responsible for harmaline-induced tremor, we have evaluated the influence of 3-acetylpyridine destruction of this pathway upon the tremorigenic actions of harmaline. Experiments were performed on male Sprague-Dawley rats (150-200 g). Harmaline HC1 (Regis Chemical Company); nicotinamide, nicotinic acid and 3-acetylpyridine (Sigma) were injected intraperitoneally in different doses, as indicated in each experiment. The tremor induced by harmaline was scored by an arbitrary scale between 0 = no tremor and 3 = maximal tremor, by an observer blinded to the treatment regimen. Degree 1 of tremor was scored when the rats displayed tremor of the trunk and extremities but for less than 5 0 ~ of a standard 5 rain observation period. Degree 2 was scored when tremor was present 50-100 ~ of the observed time period, and degree 3 represented constant tremor. Forty-eight hours after the injection of 3-acetylpyridine, some rats were perfused through the left ventricle with a mixed aldehyde solution containing 1 ~ glutaraldehyde, 2 ~ paraformaldehyde, and 1 ~ acrolein in 0.1 M cacodylate buffer, pH 7.6 (ref. 11). The cerebellum and brain stem were removed 1 h after perfusion and the inferior olives and cerebellar cortex were dissected away. Slabs of these were placed in fixative
145
Fig. 1. a: an early degenerating climbing fiber (CF) process in the molecular layer 48 h after 3-acetylpyridine contains an increasingly dense cytoplasmic matrix; dense, clustered synaptic vesicles; and intact synaptic contacts with Purkinje cell dendritic spines (s). × 57,000. b: degenerating climbing fiber process which has retracted from the surface ofa Purkinje cell dendrite (D) contains an increased density on the presynaptic surface. Some spine contacts (S) are still intact. Other spines contact parallel fiber processes (PF). × 34,000.
at 4 °C overnight. After postfixation in 1 ~ OsO4 in 0.1 M cacodylate buffer, p H 7.6, containing 10 ~ sucrose, blocks were processed conventionally in Epon with en bloc staining with 1 ~ uranyl acetate in absolute ethanol for 1 h. Sections were cut on a Reichert O M U - 2 ultramicrotome with a diamond kifen and stained with lead citrate and uranyl acetate. The stained sections were examined in a Philips 200 electron microscope. As reported earlied,2, 5 after 3-acetylpyridine few, if any, intact climbing fibers remain in the sampled areas of lobules adjacent to the primary fissure. Degenerating climbing fiber terminals occur in scattered regions of the molecular layer. An increased density of presynaptic terminals (Fig. 1a) is followed by condensation and detachment of the degenerating climbing fiber processes from the Purkinje cell dendrites (Fig. lb). Glial processes sequester the detached boutons and subsequent degeneration progresses rapidly, with little debris remaining at 48 h. The cortex is striking in its absence of climbing fiber processes and synapses with parallel fiber dilatations containing synaptic vesicles appearing more numerous. No alterations are detected in the dendritic stems, the nuclei, or the somata of Purkinje cells except for the absence of en passant climbing fiber synapses. The deafferented spines appear normal except that they lack
146 residual postsynaptic specialization (Fig. 2). All other synaptic types in the cortex are normal in both appearance and distribution. In the internal granular layer climbing fiber glomerular en marron contacts with Golgi cells (Fig. 3) appear normal and are easily found. Both climbing fiber and mossy fiber glomeruli are present with no evidence of degeneration. These anatomical findings concur with those of Llin~ts and coworkers 5 who found rapid degeneration and removal of climbing fiber endings following 3-acetylpyridine. The few climbing fibers remaining at 48 h may not represent olivary climbing fibers, but instead may arise from the locus coerulus, the raphe nuclei, or even the vestibular nuclei. With the brief degeneration process, the postsynaptic surface remains free of any residual specialization, unlike parallel fiber deafferentation in adult rats following thiophen 3, in which spine processes of Purkinj e cell often contain or continue to develop postsynaptic specializations 11. The augmentation of parallel fiber terminals may result from sprouting as after parallel fiber lesions 10, or in the absence of the climbing fiber endings, the parallel fiber processes may just become more noticeable. In normal rats harmaline, in doses between 5 and 25 mg/kg, produces a marked fine tremor with a frequency of about 8-12/sec, involving all extremities and the entire trunk. Although the intensity of the tremor appears to be similar at all doses, the latency before the initiation of tremor is shorter at higher doses, being about 3 min at 25 mg/kg (Fig. 4). This latency period gradually increases to about 12 min at 5 mg/kg. The total duration of the tremor is about 2-3 h. These findings resemble numerous previous studies describing harmaline-induced tremor 6-9. In preliminary studies in our rats, a dose of 78 mg/kg represents the LD~0 for 3-acetylpyridine. Death occurs 6 h to 2 weeks after injection. Survivors show some ataxia but otherwise eat, drink and are active for months. At 78 mg/kg, 3-acetylpyridine abolishes tremor production by harmaline (1.25 mg/kg) (Fig. 4). However, harmaline elicits some locomotor activation with animals moving about fairly randomly with some increase in ataxia. Complete abolition of the tremor response to harmaline is apparent by about 8 h after 3-acetylpyridine, with a marked reduction at 6 h and a minimal reduction at 4 h (Fig. 4). Interestingly, marked degeneration of olivary cells and climbing fibers is first apparent about 7 h after 3-acetylpyridine administration, which is also the time when behavioral evidence of cerebellar function impairment is first evident 1,2. Complete abolition of the harmaline-induced tremor requires a dose of 78 mg/kg of 3-acety!pyridine, which is also the LD50 dose and the dose required for complete destruction of climbing fibers1, 2. At doses of 65 and 72 mg/kg tremor is partially prevented, while 50 mg/kg of 3-acetylpyridine fails to affect the harmaline-induced tremor. Oxotremorine produces a marked tremor, presumably by stimulating muscarinic cholinergic receptor sites in the brain. In our rats 0.75 or 1.5 mg/kg of oxotremorine elicit a pronounced tremor of all extremities and the trunk. However, in marked contrast to the influences of harmaline, the oxotremorine tremor persists with no reduction in intensity in rats surviving 48 h after treatment with 78 mg/kg of 3-acetylpyridine.
Fig. 2. Abundant parallel fiber processes (PF) near a Purkinje cell dendrite which contains spines (S) lacking any postsynaptic specialization. Spine contacts to parallel fibers (*) are normal, but no contacts to climbing fibers are found. × 33,000.
Fig. 3. A typical climbing fiber (CF) en marron contact to a Golgi cell (Go) contains the usual arrangement of vesicles and mitochondria in the glomerular rosette: Despite degeneration of climbing fiber processes in the molecular layer, climbing fiber glomerular collaterals were normal in appearance throughout the internal granular layer 48 h after 3-acetylpyridine. × 23,000.
149
~H
A
rt0 I.d OC I'-
nQ Oi
0
50
70
4
90
3-ACETYLPYRIDINE DOSE ( m g / k g )
B
12
24
48
INTERVAL BETWEEN 3-ACETYLPYRIDINE AND HARMALINE INdECTIONS (hrs)
Fig. 4. Inhibition of harmaline-induced tremor by 3-acetylpyridine. A: different doses of 3-acetylpyridine were injected and rats that survived after 48 h received 12.5 mg/kg harmaline. B: 3-acetylpyridine (78 mg/kg) was injected at different times before harmaline (12.5 mg/kg). In both experiments the extent of tremor was scored 15 min after harmaline injection and each point on the figures represents the mean value from 10-15 rats :5 S.E.M. Some influences o f 3-acetylpyridine u p o n b r a i n function a p p e a r to result f r o m the c o m p e t i t i o n o f 3-acetylpyridine with n i c o t i n a m i d e for i n c o r p o r a t i o n into N A D 4. I f prevention o f the h a r m a l i n e - i n d u c e d t r e m o r is a t t r i b u t a b l e to this m e t a b o l i c action, then large doses o f n i c o t i n a m i d e might prevent this effect. By contrast, nicotinic acid, which c a n n o t substitute for n i c o t i n a m i d e in the exchange reaction with N A D 4, o u g h t n o t to p r o t e c t against 3-acetylpyridine. R a t s t r e a t e d with n i c o t i n a m i d e (500 mg/kg) i m m e d i a t e l y after 3-acetylpyridine a n d a d m i n i s t e r e d h a r m a l i n e 48 h later show the same intensity o f t r e m o r as c o n t r o l animals injected with h a r m a l i n e with neither 3-acetylp y r i d i n e n o r n i c o t i n a m i d e t r e a t m e n t . Besides preventing the influence o f 3-acetylpyridine on h a r m a l i n e - i n d u c e d t r e m o r , n i c o t i n a m i d e also prevents the lethal effects o f 3-acetylpyridine. Thus, while only 50 ~ o f rats t r e a t e d with 3-acetylpyridine alone survive after 48 h, there are no d e a t h s in g r o u p s o f rats receiving n i c o t i n a m i d e i m m e d i a t e l y TABLE I Effect o f nicotinamide and nicotinic acid on animal survival and harmaline-induced tremor
Groups of 15 rats were injected (i.p.) with 500 mg/kg of nicotinamide or nicotinic acid immediately after receiving 78 mg/kg 3-acetylpyridine. Tremor induction by harmaline (12.5 mg/kg) and animal survival were determined 48 h later. Data are expressed as mean values :5 S.E.M. for tremor and for animal survival as the ratio of the number of rats alive 48 h after 3-acetylpyridine to the total number in each group. Analogue treatment
None Nicotinamide Nicotinic acid
Control Rats
3-A cetylpyridine-treated rats
Animals sur rived
Degree o f tremor
Animals survived
Degree o f tremor
15/15 15/15 15/15
2.7:5 0.3 2.8 ± 0.3 2.6:5 0.3
8/15 15/15 6/15
0.2:5 0.1 2.7:5 0.3 0.2:5 0.1
150 3-
/p.-.~
~-Nicotinomide
r,. 0
n. o I--
2-
6
,=.,
o
n. (.9 ta.i E)
,=,, r~ t9
w
E3
1-
1-
~ -Nicotinic
o-
~
0
I()
[-, 25
DOSE (mg/kg)
Acid
O-
,~--4 50 500
o
~
a
12
24
4a
INTERVAL BETWEEN 3-ACETYLPYRIDINE AND NICOTINAMIDE INJECTIONS (hrs)
Fig. 5. Effectof nicotinamide and nicotinic acid on the harmaline-inducedtremor as a function of treatment with 3-acetylpyridine.A: harmaline (12.5 mg/kg) was injected 48 h after injection of different doses of nicotinamide or nicotinic acid. B: harmaline (12.5 mg/kg) was injected at different times after injection of 500 mg/kg nicotinamide. In both experiments (A and B) each point is the mean value from 8-12 rats :[ S.E.M. after the 3-acetylpyridine. In marked contrast, nicotinic acid administration (500 mg/kg) provides no protection against the lethal effects of 3-acetylpyridine or its effect on harmaline-induced tremor (Table 1). The protective action of nicotinamide occurs at fairly low doses. As little as 10 mg/kg ofnicotinamide prevents the lethal effects of 3-acetylpyridine and its influences on harmaline-induced tremor, while 4 mg/kg has a partial effect (Fig. 5). By contrast, at 4 doses between 5 and 500 mg/kg, nicotinic acid fails to prevent the effects of 3-acetylpyridine. The protective influence of nicotinamide is evident whether administered simultaneously with or up to 3 h after 3-acetylpyridine. When given 4 h after 3-acetylpyridine, nicotinamide partially protects, while if administered 6 h or more after the 3-acetylpyridine, there is little protection. Since the neuropathologic effects of 3-acetylpyridine are first apparent at 6-8 h, it appears that the metabolic actions of 3-acetylpyridine upon susceptible neurons are subject to reversal by nicotinamide almost until the time they are evident by microscopic examination. The observations of this study support other evidence that tremor induced by harmaline is mediated by the olivo-cerebellar pathway via climbing fiber projections to the Purkinje cells 6,7,9. The neuropathologic effects of 3-acetylpyridine are largely restricted to the lower brain steml, 2. Besides the inferior olivary nucleus 3-acetylpyridine damages the nucleus of the facial nerve, the nucleus ambiguus and the hypoglossal nucleus 1,2. However, these other nuclei are less severely affected than the inferior olivary nucleus. The selectivity of 3-acetylpyridine effects is supported by its failure to alter tremor induced by oxotremorine. Recent neuroanatomical evidence indicates that the only cerebellar input affected by 3-acetylpyridine is the climbing fiber projection to the Purkinje cells 1,2. Thus, 3acetylpyridine treatment affords a selective means of destroying this neuronal pathway, providing a tool to evaluate unique biochemical or physiological properties of climbing fibers. Abolition of harmaline-induced tremor may be a simple technique to screen for destruction of the olivo-cerebellar pathway by 3-acetylpyridine.
151 Th e a u th o r s w o u l d like to t h a n k J. F i r m a n for his technical assistance. This research was s u p p o r t e d in p a r t by U S P H S G r a n t s NS-08997 (to R . M . H . ) , MH-18501, R S D A M H - 3 3 1 2 8 (to S.H.S.) and an E l e a n o r R o o s e v e l t fellowship
(to R.S.).
1 Desclin, J. C., Histological evidence supporting the inferior olive as the major source of cerebellar climbing fibers in the rat, Brain Research, 77 (1974) 365-384. 2 Desclin, J. C. and Escubi, J., Effects of 3-acetylpyridine on the central nervous system of the rat as demonstrable by silver methods, Brain Research, 77 (1974) 349-364. 3 Herndon, R. M., Thiophen induced granule cell necrosis in the rat cerebellum. An electronmicroscopic study, Exp. Brain Res., 6 (1968) 49-68. 4 Kaplan, N. O., Goldin, A., Humphreys, S. R., Ciotti, M. M. and Venditti, M., Significance of enzymatically catalyzed exchange reactions in chemotherapy, Science, 120 (1954) 432440. 5 Sotelo, C., Hillman, D. E., Zamora, A. J. and Llinhs, R., Climbing fiber differentiation: its action on Purkinje cell dendritic spines, Brain Research, 68 (1975) 574-581. 6 Llinfis, R. and Volkind, R. A., Repetitive climbing fiber activation of Purkinje cells in the cat cerebellum following the administration of harmaline, Fed. Proc., 31 (1972) 377a. 7 Llingts, R. and Volkind, R. A., The olivo-cerebral system: functional properties as revealed by harmaline-induced tremor, Exp. Brain Res., 18 (1973) 69-87. 8 Mao, C. C., Guidotti, A. and Costa, E., Inhibition by diazepam of the tremor and the increase of cerebellar cGMP content elicited by harmaline, Brain Research, 83 (1975) 516-519. 9 Montigny, C. De and Lamarre, Y., Rhythmic activity induced by harmaline in the olivo-cerebellobulbar system of cat, Brain Research, 53 (1973) 81-95. 10 Mouren-Mathieu, A. M. and Colonnier, M., The molecular layer of the adult cat cerebellar cortex after lesions of the parallel fibers: an optic and electron microscopic study, Brain Research, 1 (1969) 307-323. 11 Oster-Granite, M.-L. and Herndon, R. M., The pathogenesis of parvovirus-induced cerebellat hypoplasia in the Syrian hamster, Mesocricetus auratus. Fluorescent antibody, foliation, cytoarchitectonic, Golgi, and electron microscopic studies, J. camp. Neurol, in press.
