BRAIN RESEARCH

E L S E V I E R Brain Research 656 (1994) 257-262

Research report

Effect of administration of 3-acetylpyridine followed by niacinamide injection on survival, extent of the inferior olivary complex lesion,

and response to harmaline in the young rat

N. Jones, N. Le Marec, T. Stelz, J. Caston * Unit,ersitd de Rouen, Laboratoire de Neurophysiologie sensorielle, Facultd des Sciences, 76821 Mont-Saint-Aignan Cedex. France

Accepted 31 May 1994

Abstract

In the 15 day-old DA/HAN strained rat, i.p. injection of 3-acetylpyridine (3-AP) 50, 65 or 95 mg" kg 1 was followed 2 to 4 hours later by administration of niacinamide (300 mg. kg- i). The percentage of survival and the extent of the inferior olivary complex (IOC) lesion, determined histologically, were correlated with both the dose of 3-AP administered and the time delay between 3-AP and niacinamide injections. Moreover, the tremor elicited by harmaline was also correlated with the extent of the IOC lesion. The results show that it is more advantageous to administer 95 rag. kg- 1 3-AP and to delay niacinamide injection by 2h30 or less to get the higher percentage of survival (about 90%) and a reasonably high percentage of totally IOC lesioned rats (more than 30%). They also demonstrate that the harmaline test is not sufficient to acutely judge of the extent of the IOC lesion and that, in all cases, histological controls have to be done. The results are discussed in terms of interrelationships of the variable studied.

Key words: Inferior olivary complex; 3-Acetylpyridine; Harmaline; Rat

I. Introduct ion

Lesion of the climbing fibers contacting the Purkinje cells of the cerebellar cortex is achieved by destroying the soma from which these fibers arise, soma which are located in the inferior olivary complex (IOC). Such a destruction can be carried out either electrolytically or systemically, but the systemic lesion is easier than the electrolytical one especially in very young animals. For this purpose, ip or iv injection of 3-acetylpyridine (3-AP) is done. A lot of studies using 3-AP to destroy the IOC in the rat have been reported in the literature, the aim of these studies being mainly to observe the behavior of the animals after the lesion [5,12,16,17], to record the activity of Purkinje cells in the cerebellar cortex in climbing fiber deafferented animals [4,5,6,8,9,10,11] or to look for the extent of the lesion by histological methods [1,2,8,14]. A survey of these different studies shows that many factors can influence the action of 3-AP on both the IOC lesion and the percentage of

* Corresponding author. Fax: (33) 35-14 63 49.

0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00671-X

survival: the dose of 3-AP administered, the age of the animals, the fact that 3-AP injection is followed or not by administration of nicotinamide or niacinamide (two potent inhibitors of the action of 3-AP [3,6,7]) and the time delay between injections of 3-AP and niaci- namide. The protocols used in the above-mentioned studies being different and in a number of papers the exact experimental protocol and the histological con- trols being lacking, it is therefore difficult, and most often impossible, to compare these different studies and to correlate the percentage of survival and the extent of the IOC lesion to the dose of 3-AP adminis- tered. In order to study the effect of the IOC lesion on learning and memory in the rat, which is the aim of a subsequent paper, it is necessary to produce lesions which are similar in all animals of a given group. For this reason, we have undertaken the present prelimi- nary experiment to correlate the percentage of survival and the extent of the IOC lesion with both the dose of 3-AP administered and the time delay between 3-AP and niacinamide injections. Moreover, we have also correlated the response to harmaline, a drug that acts upon the olivary neurons and elicits tremor, with the

258 A' ,Ion e.s el a / . / Brain Research 056 (1094) 25 7-262

extent of the IOC lesion in order to know, during the experiment, the probability to get a complete or a partial lesion of the IOC.

2. Materials and methods

2.1. Animals

The experiments were carried out on DA/HAN strain rats (pigmented rats) 15 days old at the beginning of the experiments. The young rats were housed with their mother in standard condi- tions: 12 h light-12 h dark, 22°C, food and water ad libitum.

2.2. Experimental procedure

Three hundred and seventy-eight rats were administered 3-AP (50, 65 or 95 mg.kg- l ) by i.p. injection followed by i.p. administra- tion of niacinamide (300 mg.kg-1). Therefore, according to both the dose of 3-AP administered and the time delay between 3-AP and niacinamide injections, the animals were divided into 15 groups. All the animals were weighed every day to control for their growth. In each group, the number of animals which died was noted. Seven days after 3-AP administration, that is when the rats were 22-day-old, they were administered harmaline (20 mg-kg-1) by i.p. injection and the tremor elicited by the drug was recorded for about 1 h. Control (non-treated) rats were also injected with harmaline and their tremor recorded.

2.3. Recording o f tremor

Tremor was recorded in order to quantify the response to harma- line. For this purpose, just after the injection, the animal was placed for 10 minutes on the membrane of a loud-speaker connected to a strip-chart recorded. The latency and the frequency of tremor could be measured from the records. The total duration of tremor was also measured during the second five minutes. The amplitude of tremor could not be measured since it depended on the position of the animal onto the loud-speaker membrane.

2. 4. Histological control

As a whole, the brains of 128 animals were examined histologi- cally when they were about 1.5 month old. The rats were overdosed with pentobarbital sodium and perfused intracardially with a mixture of 4% paraformaldehyde and 0.3% glutaraldehyde in phosphate buffer. Then, the brains were removed and placed in 10% formalin for several days. Frozen sections t5 /~m thick stained with Cresyl violet were examined under the light microscope.

2.5. Statistical treatment o f the results

Relevant comparisons were made according to the X 2 test (dr = 1; P = 0.95 or 0.99).

3. Results

o01 L;

IIIIflHHIHIIIIIIIIIII[UtlIIItltlIIIII 0 ~ 9 5 ~'~"

~2.5 ~-2.5 ="3.5 ~3.5

time delay between 3-AP and niacinamide

Fig. 1. Percentage of survival according to both 3-AP dose and time delay (in hours) between 3-AP and niacinamide injections.

which were still alive after one week surviving until they die a natural death. The mortality was greatly reduced when administration of 3-AP was followed by injection of niacinamide. As a whole, among the 378 rats which were administered 3-AP (50-95 mg-kg-1 ) and after a 2h-4h delay, niacinamide (300 mg" kg-1), 194 animals (51.3%) survived. Examination of Fig. t shows that the percentage of survival depends on both the dose of 3-AP administered and the time delay between 3-AP and niacinamide injections. Whatever the dose of 3-AP injected to the animals (50, 65 or 95 mg" kg-~), it is clear that the shorter the time delay the greater the percentage of survival. Besides, when the time delay between 3-AP and niacinamide injec- tions was shorter than or equal to 2h30, more than 90% of survival was reached and increasing the dose of 3-AP from 50 to 95 mg. kg -1 did not significantly decrease survival. When the time delay between the two injections was longer than 3h30, the percentage of survival was small (between 10.0 and 3715%) and was not systematically enhanced when the dose of 3-AP administered decreased. When the time interval was longer than 2h30 and shorter than 3h30, the percent- age of survival was significantly reduced when the dose of 3-AP injected was increased (between 50 and 95 mg" kg -1, g 2 = 21.6; P = 0.99).

3.1. Percentage of survival according to both 3-AP dose and time delay between 3-AP and niacinamide injections

3.2. Correlation between the extent of the lesion of the IOC and the experimental protocol

When administered 3-AP, a number of rats died during the week following the injection, the animals

As a whole, 128 brains were examined histologically when the rats were about 1.5 month old to observe the

N. Jones et al. / Brain Research 656 (1994) 257-262 259

extent of the IOC lesion; these observations were com- pared to those obtained from non-treated (control) rats (Fig. 2A,D). According to the extent of the lesion, the animals were split into two groups:

(1) Intact IOC (I); the IOC appeared intact (similar to that observed in controls: Fig. 2A,D) or almost intact (scattered degeneration of a few neurons were observed in all or some subdivisions of the IOC);

I

4 1 F ~

t l

o

o

J B

I I

100 5O p,.. Fig. 2. Transverse section of the inferior olivary complex (IOC) at low ( A - C ) and high ( D - F ) magnification. A and D: control rat, the IOC is intact; B and E: partially lesioned rat, clusters of neurons are seen throughout the IOC; C and F: totally lesioned rat, only a few scattered neurons can be observed throughout the IOC.

260 N. Jones ctal./Bratn Research 6.56 (1994) 257-262

(2) Total or subtotal lesioned IOC (T): no neuron or only a few scattered neurons could be observed in the IOC (total lesion: Fig. 2C,F) or clusters of cells were essentially intact (subtotal lesion: Fig. 2B,E).

Fig. 3 depicts the percentage of T rats according to both the dose of 3-AP administered and the time delay between 3-AP and niacinamide injections. In order to get a reasonable number of observations in each class, only two time delays ( ~ 2h30 and > 2h30) were con- sidered. It can be seen that when the time between 3-AP and niacinamide injections was delayed by more than 2h30, the percentage of T rats was about 55% whatever the dose of 3-AP injected. When the time delay was equal to or shorter than 2h30, the percent- age of T rats increased when the dose of 3-AP adminis- tered increased (however, the differences are not sig- nificant: between 50 and 95 rag. kg -J, g 2= 2.2; N.S.) and for the two lowest doses (50 and 65 m g . k g 1) is significantly lower than for longer time delays (g 2 = 5.2; P = 0.95 and X 2= 5.1; P = 0.95, respectively).

Therefore, the time delay between 3-AP and niaci- namide injections seems to be crucial in inducing IOC lesions as it is sufficient to delay the niacinamide injection by more than 2h30 (about 3 h) to get more than 50% of rats bearing total or subtotal IOC lesions.

3.3. Correlation between the responses to harmaline and the extent of the IOC lesion

The latency and the frequency (about 12 cycles per second) of t remor did not differ among the treated animals and the responses of the animals to the drug were divided into four groups according to the dura- tion of t remor during the second 5 rain.

- group + + +: duration of t remor greater than 95% of that of the observed period and similar to that of controls;

I--

= - 2.5 ~ 2.5 t ime d e l a y b e t w e e n

3 -AP and n i a c i n a m i d e

Fig. 3. Correlation between the percentage of total or subtotal IOC lesioned rats (T) and the experimental protocol (dose of 3-AP administered and time delay (in hours) between 3-AP and niaci- namide injections).

"O i-

O o~

I !

Fig. 4. Correlation between the response to harmaline and the extent of the IOC lesion. I: intact or almost intact IOC rats: T: total or subtotal IOC lesioned rats. + + +: duration of tremor greater than 95% of the duration of the observed period (5 rain) and similar to that of controls; + + : duration of tremor between 70 and 80%; + : duration of tremor between 10 and 20%; 0: duration of tremor lower than 10% or no tremor.

- group + + : duration of t remor between 70% and 80%;

- group + : duration of t remor between 10% and 20%;

- group 0: duration of t remor lower than 10% or no tremor.

Fig. 4 gives the percentage of I and T rats respond- ing maximally ( + + + ), near maximally ( + + ), poorly ( + ) and eliciting no response (0) to harmaline. It can be seen that a high percentage (90.9%) of T animals responded minimally ( + ) or did not respond at all to harmaline, while a significant lower percentage (X 2 = 73.6; P = 0.99) responded near maximally ( + + ), no animal responding maximally ( + + + ): On the con- trary, a high percentage (67.2%) of I rats responded maximally ( + + + ) or near maximally ( + + ) to harma- line while a significantly lower percentage (X 2= 16.2; P = 0.99) responded minimally ( + ) or did not respond at all. It has previously been mentioned that I rats were in fact a goup of non-lesioned rats and of rats with very small and scattered IOC lesions. It is important to note that I rats which did not respond to harmaline were never intact and were always poorly and scattered lesioned animals. Therefore, it is clear that a rat re- sponding + + + to harmaline cannot be a completely lesioned one and that an animal which did not respond to harmaline cannot be completely intact.

4. Discussion

From the results of the literature it appears that in 12-21 day-old rats administration of 3-AP at the dose of 65 mg. kg-~ without any subsequent niacinamide injection is followed by a percentage of survival that extends from 0 to 30% and by a partial or a total IOC lesion [10,17]. However, the percentage of rats which were partially or totally lesioned is not given. The

N. Jones et al. /Brain Research 656 (1994) 257-262 261

results of the present study demonstrated that when the animals were administered the same dose of 3-AP (65 m g . k g i), a 10% survival was obtained when niacinamide administration was delayed by more than 3h30 and a much greater percentage when the delay between administrations of 3-AP and niacinamide was shorter. Therefore, it can be concluded that the antag- onistic effect of niacinamide on the neurotoxicity of 3-AP is efficient when both injections are delayed by less than 3h30. The data presented here agree with those of Woodhams et al. [17] showing that, in young rats (age non specified), 40-65 mg. kg 1 of 3-AP fol- lowed by 300 mg" k g - I of niacinamide with no delay between both injections led to an increase in the sur- vival length. Indeed, when the delay between 3-AP and niacinamide injections was shortened, the percentage of survival greatly increased (a delay shorter than 2h30 led t o a 9 2 . 3 % ( 6 5 m g . k g ~ o f3 -AP ) o r e v e n a 100% (50 rag" kg i of 3-AP) survival.

The major result of this study is the good correlation that was demonstrated between the dose of 3-AP ad- ministered plus the time delay between 3-AP and niacinamide injections and the extent (seen histologi- cally) of the IOC lesion. The second interesting result is the correlation between the extent of the IOC lesion and the response to harmaline. Such correlations are not surprising while a systematic study including a separate variation of each paramete r had never been undertaken before.

3-AP is a potent neurotoxic which antagonizes nicotinamide for incorporation into NAD [14,15] and the symptoms elicited by its administration resemble those induced by a nicotinic acid deficiency [1]. Since 3-AP is a competitive antagonist of nicotinamide, its action can be blocked by high doses of nicotinamide or of a similar substance, niacinamide [17]. It is not ex- cluded that the action of 3-AP would be to induce the synthesis of abnormal nucleotides or to alter the glu- cose metabolism in the central nervous system [1]. Whatever the action of 3-AP, its neurotoxicity is great and when the drug action was not antagonized by niacinamide or when the antagonistic drug was admin- istered too late, the percentage of surviving animals was very low. In that way, especially when high doses of 3-AP were administered, not only the IOC was lesioned but also other parts of the central nervous system could be damaged too, especially the hippocam- pus and the hypoglossal nuclei. When the time delay between 3-AP and niacinamide injections was short- ened, the lesion was confined to the IOC, which con- firms the fairly selective sensitivity of this structure for the drug [13,14]. The protective action of niacinamide on both the extent of IOC lesion and survival is similar to that of nicotinamide [16]. However, since the pro- tecting effect is only evident when niacinamide was administered less than 3h30 after 3-AP, it can be

concluded that the neurotoxicity of 3-AP, which is apparent about 6 -8 h after its injection, can be re- versed only when the metabolic damages are not too great.

The reasons of the high affinity of 3-AP for the rat 's IOC neurons are not known but such a high specificity of the drug is demonstrated by the fact that only the IOC neurons were damaged and by the decrease or even the absence of t remor following harmaline injec- tion. However, as demonstrated here, the harmaline test is not sufficient to acutely judge of the extent of the IOC lesion and in all cases histological controls have to be done.

Acknowledgements

This work was partly supported by M R E Grant 92.C.0756.

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