Sleep and indolamine alterations induced by thiamine deficiency

Brain Research, 248 (1982) 275-283 Elsevier Biomedical Press

275

Sleep and Indolamine Alterations Induced by Thiamine Deficiency F. CRESPI* and M. JOUVET Department of Experimental Medicine, Claude Bernard University, 69008Lyon (France) (Accepted March l lth, 1982) Key words: thiamine - - pyrithiamine - - sleep - - 5-hydroxytryptamine- - 5-hydroxyindoleaceticacid

Behavioral, polygraphic, biochemical and histological aspects of thiamine deficiencyin rats induced by thiamine-deficient food and pyrithiamine treatment (40 mg/kg daily for 4 days) are described. Behavioral alterations were essentially characterized by ataxia, pilo-erection and paresis. Polygraphic data indicated an increase in slow-wavesleep (SWS) of 33 ~ and decreases in paradoxical sleep (PS) and wakefulness (W), respectively, of 69 ~ and 27 ~. These effects were reversed by complete food and thiamine administration, the reversal including an overshoot in PS. Biochemical assays, performed when the polygraphic data indicated a large effect, demonstrated a significant increase in serotonin (5-HT) and 5-hydroxyindolacetic-acid(5-HIAA). These effects were particularly evident in the raphe system and the locus coeruleus. Histological data from the raphe dorsalis displayed a notable increase in yellowfluorescencein pyrithiamine-treated animals over controls. We conclude from these experiments that a deficiencyin thiamine affects the serotonergic system and that the subsequent effects on sleep are a consequence of this serotonergic change. INTRODUCTION Thiamine-deficient animals have been used as an experimental model to simulate symptoms of Wernicke's encephalopathy and Korsakoff's psychosis resulting from malnutrition or alcoholism. In these diseases, ataxia, myoclonic seizure and loss of retrograde and anterograde memory are the most important neurological findingsS,27, za,aS-41. Post-mortem examinations of brains from patients suffering these conditions have shown symmetrical lesions in the paraventricular regions of the thalamus and hypothalamus, mamillary bodies, the periaqueductal regions of the midbrain, the floor of the fourth ventricle and the anterior lobe of cerebellum (particularly in the vermis) 19. ' M e m o r y alterations' following thiamine deficiency have been also observed together with histological changes in the raphe nuclei, spinal and medullary central grey substances, cerebellum and mamillary nuclei zg. Thiamine deficiency is now established as the cau-

sative factor of most of these symptoms 19,27,a2,aT,41. Further, a loss of indolamine-labeled cerebellar mossy fibers and of parallel fiber-like systems, but not of diffuse axon inputs, has been reported z. These alterations agree with biochemical findings of decreased [3H]5-HT uptake in cerebellar synaptosomal fractions from the thiamine-deficient brain, and it is speculated that these changes in cerebellar indolamines may be related to the generation of ataxia zS. The relationship between 5-HT and myoclonic seizure has already been recognized in man 9,2z,a4, and several studies have also been done in animals. The main results published indicate that thiamine deficiency prevents autoradiographic labeling in almost all indolamine neurons and their processes in the midbrain and medulla. In thiamine-deficient animals only a few dystrophic cells with hypertrophied axons are seen. The periaqueductal regions of spinal cord, medulla, midbrain and diencephalon, the mamillary nuclei, habenular nuclei and cere-

* Present address: Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62 20157 Milano, Italy. 0006-8993/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

276 bellum are the most severely affected 1. Additional correlative autoradiographic studies performed after intraventricular infusion of radioactive 5-HT demonstrated and underlined the loss of labeling of the so called 'synaptic-serotoninergic axons' in the cerebellum and also in the brainstem and diencephalon of thiamine deficient animals1, 2. Pyrithiamine treatment induced a significant increase in the ratio of 5-HIAA to 5-HT in all brain regions with the exception of the cerebral cortex. Part of this increase in 5-HIAA was shown to be due to impairment of active transport of this 5-HT metabolite out of the brain 36. Recent studies 26 with thiamine-deficient rats have also suggested an increase in the in vivo turnover of 5-HT and/or an impaired effiux of 5-HIAA from the brain. Pyrithiamine apparently induced an increase in endogenous 5-HIAA in the medulla and pons simultaneously with the onset of neurological signs, and both parameters could be reversed by thiamine administration 24. Further, a functional role of the serotonergic system in the regulation of the sleep and waking states has been demonstrated TM. The most significant results supporting this hypothesis have been obtained with PCPA-treated animals, which demonstrated a severe insomnia (reversed by injection of D,L-5-HTP), and with electrolytic destruction of the raphe system, resulting in a marked insomnia 12, 13,20.

The studies described here were undertaken in order to investigate alterations of the sleep and waking cycle in parallel with variations of 5-HT metabolism after thiamine deficiency. MATERIALS A N D METHODS

Thirty-eight male rats (weight approximately 200 g) of the OFA strain (supplied by I F F A CREDO, France) were used in this study. Thiamine deficiency was produced by i.p. injection ofpyrithiamine (Pyrithiamine-HC1, Sigma) in conjunction with a thiamine-deficient diet (Extralabo, France) and reversed by either complete food or s.c. injection of thiamine (Thiamine, Sigma). Preliminary experiments had shown that a thiamine-deficient diet alone produced no evident

effects even after 2 months. A preliminary group of 10 rats was used to establish the dose-effect relationship for pyrithiamine (ataxia, pilo-erection, paresis of limbs). The optimum dose for these experiments was found to be 40 mg/kg daily for 4 days, and was used for the neurophysiological, biochemical and histological studies in the main experiments.

Neurophysiological and behavioral studies Group I. Five rats were implanted for EEG and E M G evaluation following a method previously described17, a5 and kept under standard conditions (temperature 24 °C, light/dark 12 h/12 h). These rats had received thiamine deficient food only, from 7 days prior to implantation, and this diet was continued. Continuous recordings started 8 days after implantation and records from the following 7 days were used as control. During this period i.p. injections of 0.5 ml saline solution, one each on the third and fourth days, were made to habituate the animal to manipulations. The rats were then treated with pyrithiamine as described above, and recording continued for several days beyond the end of the treatment. When a large effect on waking, SWS and PS was noticable, these rats were sacrificed for biochemical studies (see Fig. 2). Group II. Five animals were implanted and treated with thiamine-deficient food and pyrithiamine in the same way as those in group I, and continuously monitored. In order to observe the reversibility of the effects of thiamine deficiency at the time when the maximum manifestations of thiamine deficiency appeared, feeding with complete food was begun. When EEG values returned to normal levels, a second treatment with pyrithiamine was effected but this time in conjunction with complete food. Since no EEG changes were observed in this condition, animals were again fed with the thiamine deficient food, but another treatment with pyrithiamine was necessary to give the same effects on EEG values as the ones seen after the initial treatment of all animals. The deficiency manifestations were reversed by thiamine (400 mg in 2 ml saline solution, s.c.).

277 Polygraphic data were scored in 30 s epochs according to the classical criteria of sleep stateslT,ZL

Biochemical studies Biochemical assays were performed in pyrithiamine-treated rats previously used for EEG recording (Group I), in 5 pyrithiamine-treated non-implanted rats, and a further 5 non-implanted rats which had received 250 #1 saline i.p. daily for 4 days. All the rats were decapitated and the brains were rapidly removed and frozen. 500 #m frontal sections were made using a Leitz 1310 cryostat, and the brain nuclei were dissected out as previously described 3. TRY, 5-HT and 5HIAA content was evaluated by high-pressure liquid chromatography8, ~3. Histological studies The histological studies were done by a histofluorescence method 7 using 5 rats treated with pyrithiamine (3 animals injected i.p. with saline were used as control.) RESULTS

A. Behavior We investigated the effects of pyrithiamine (administered i.p.) on the following behavioral characteristics: pilo-erection, ataxia, weight loss and paresis. The behavioral effects of pyrithiamine varied with the dose administered. (1) Doses of 25 mg/kg daily for 1 or 2 days did not induce any evident changes in behavior. (2) Doses of 25 mg/kg daily for 4 days induced, 6-7 days after the beginning of treatment, pilo-erection and moderate ataxia. These pathological aspects disappeared after a further 2-4 days. (3) Doses of 40 mg/kg daily for 4 days induced complete prostration accompanied by pilo-erection and paresis (most notably of the hindlimbs) (see Fig. 1). (4) Doses of 50 mg/kg daily for 4 days induced the same behavioral effects but this treatment was lethal after 6-7 days. The dose used in this study was as in (3). B. Polygraphic aspects of thiamine deficiency The first group of 5 rats received pyrithiamine (as

Fig. 1. Behavioraleffects of pyrithiamine treatment in rat (40 mg/kg i.p. daily for 4 days). C, control animal injected with saline solution; T, treated animals. Note the pilo-erection, paresis and ataxia.

above) together with thiamine-deficient food ad libiturn. Eight days later, they showed a 63 ~ decrease in PS and a 27 °/o decrease in waking, but in contrast, SWS increased by 3 3 ~ as compared to controls (Fig. 2). The second group of 5 rats, treated until the 9th day in the same way as the first group, showed the same effects on PS, waking and SWS. Complete food given ad libitum from the ninth day was suffi-

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P Fig. 2. Effects o f pyrithiamine (P) injection u p o n vigilance states. SWS, slow wave sleep; W, waking; PS, paradoxical sleep. A m o u n t s o f vigilance states are expressed as ~ o f control (broken horizontal line). Variance analysis : * P < 0.05 ; • * P < 0.01 (n = 5). Pyrithiamine injections are indicated by the arrows. N o t e that pyrithiamine induces an increase in SWS and decreases in PS and W.

C. Biochemical data In order to investigate the possible influence of electrode implantation, the same measures were effected in both implanted and pyrithiamine-treated non-implanted animals. Since no significant differences between these animals were noted, the data obtained were pooled. The biochemical measurements were made at the time when PS decrease and SWS increase were maximum (Fig. 2) Fig. 5 shows the results of these measurements. There was a significant (P < 0.01) increase in both 5-HT and 5-HIAA in the RC (raphe centralis), RD (raphe dorsalis), LC (locus coeruleus) and ST (striata), in 5-HIAA only in RM (raphe magnus) and Hyp. (hypothalamus), and in 5-HT only in SN (substantia nigra). No significant changes in TRY were seen. D. Histological data A strong increase in yellow fluorescence, indicating an increase in 5-HT, was observed in the RD cells of pyrithiamine-treated animals. Cells were intact and their nuclei were well apparent. A diffuse fluorescence was also observed between the cells. In contrast, the green fluorescence (indicating the presence of noradrenaline, NA) of the LC did not reveal any differences between control and treated animals, and would have masked any changes in yellow fluorescence. These results are illustrated in black and white in Fig. 6. DISCUSSION

cient to ensure that sleep quantities returned to control values about 20 days later. PS recovered rapidly and overshot, reaching a value 40 ~ above control values (Fig. 3) 2 days after complete food was given again. This PS recovery only affected the PS in the dark period and subsequently stabilized to control values (Fig. 4). Next, a second treatment with pyrithiamine was administered and no sleep changes were observed. As described above, a subsequent treatment with deficient food plus pyrithiamine was necessary to obtain the effects of thiamine deficiency seen in the Group I, though these effects occurred more rapidly (Fig. 3). Finally, thiamine injection resulted in a rapid reversal of these changes.

Following treatment with pyrithiamine, the decrease in waking and PS and the abnormal behavioral manifestations during wakefulness (ataxia, piloerection, paresis of the limbs) appeared to be the most striking results obtained. Biochemical assays, performed when the behavioral effects were maximum, demonstrated a significant accumulation of 5H T and 5-HIAA in the raphe nuclei. Similar biochemical effects were observed in the terminal regions of the indolaminergic system (and in particular the LC). Although we did not quantify the 5HT-induced fluorescence with microspectrofluorometric analysis, the increase of this fluorescence was notable in each treated rat. Moreover our subjective evaluation of histofluorescence is corroborated by

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thiamine-deficient food and thiamine. The animals (n = 5) were monitored during 55 days. The amounts of SWS, W and PS are expressed as % of control (broken horizontal line). For other abbreviations see Fig. 2. Note that the effects obtained by pyrithiamine treatment (10 days) are followed by a PS overshoot (10-15 days). Pyrithiamine together with complete food did not induce significant effects, neither did deficient food alone. Pyrithiamine effects are antagonized by thiamine (* P < 0.01). the biochemical observations; the 5-HT and 5H I A A increases may therefore follow f r o m an increase in their synthesis. Indeed, this result agrees with both the in vivo increase in the turnover of 5H T and/or the impairment of ettlux of 5-HIAA f r o m the brain observed by others26, 36. In contrast, after histological treatment for catecholamines, the green fluorescence of LC was not changed. This result is in agreement with the work of Iwata et al. 10, and may indicate that the levels of LC catecholamines are not affected by thiamine deficiency. Since in the LC, N A neurones outnumber the 5-HT neurones by 100 to 16,83, the green fluorescence would mask any change in yellow fluorescence. Thus, our biochemical and histochemical data, in agreement with previous studies, show that the thiamine deficiency affects the serotonergic system.

Our behavioral data are also in agreement with previous reports 19,24,26,27,a2,3e,37,41 in which it has been shown that thiamine seems to be a causative factor in behavioral impairment. However, to our knowledge, no work concerning the effects of pyrithiamine upon the vigilance cycle has been carried out. The increase of SWS observed is correlated to the 5-HT synthesis increase. This finding is in accordance with some pharmacological alterations of 5H T levels in relation to sleep: in PCPA-treated rats a significant decrease in cerebral 5-HT is accompanied by an insomnia whereas an increase in 5-HT levels following 5-HTP administration restored the SWStl,z0. The overshoot of PS that appeared when normal food or thiamine was given may correspond to the

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suppression o f the tonic i n h i b i t i o n o f 5 - H T on the L C neurons. Such a p h e n o m e n o n has a l r e a d y been r e p o r t e d when PS s u p p r e s s i o n h a d been achieved in artificial situations4,14,15,2z,4L T h e significantly larger ( P < 0.01) p e r c e n t a g e o f increase in PS d u r i n g the d a r k t h a n the light p e r i o d seen in o u r experiments reflects the lower a c t u a l q u a n t i t y o f PS n o r m a l l y occurring d u r i n g the d a r k period. O u r results s h o w t h a t the a c t u a l time a b o v e c o n t r o l spent in PS d u r i n g the o v e r s h o o t p h a s e is slightly greater d u r i n g the d a r k t h a n the light period. effects of the pyrithiamine on sleep state was observed (see Fig. 2). C, control; TRY, tryptophan; 5oHT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxy-indolacetic acid; Locus-C, locus coertdeus; S-Nigra, substantia nigra; hypothal., hypothalamus. Results are expressed in ng/mg of tissue. Variance analysis: * P < 0.01; (C: n = 5; treated: n = 10). For other abbreviations see Figs. 2 and 3. Note the significant increases of 5-HIAA in the raphe nuclei, Locus C., Hypothal. and Striata.

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Fig. 6. Histology (fluorescence technique following Falck and Hillarp, 1962) of dorsal raphe nucleus (RD) and locus coeruleus (LC) after pyrithiamin¢ treatment. FLM, fasciculus longitudinalis medialis. Note the increase in fluorescence in the RD of treated animals over controls. In contrast no difference is obvious in the LC. (Magnification: RD x 150; LC × 240). (See Results section D).

H o w e v e r , with these small samples the difference is n o t significant. Interestingly, the largest effect on 5 - H T or 5H I A A as m e a s u r e d b y high-pressure liquid c h r o m a t o g r a p h y was in the L C : this r e g i o n is a n a t o m i c a l l y

c o n n e c t e d to the r a p h e system a n d receives a 5 - H T i n n e r v a t i o n f r o m this latter areal6, al. F u r t h e r m o r e , the 5 - H T n e u r o n s p r o j e c t i n g to the L C are t h o u g h t to exert an i n h i b i t o r y role on L C n e u r o n a l function 2s. Thus an increase in 5 - H T t u r n o v e r w o u l d

282 i n d u c e a d e c r e a s e in L C n e u r o n a l a c t i v i t y al o r in N A

C N R S ( L A 1 6 2 ) a n d D . R . E . T . ( G r a n t 80-175). F . C .

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help with the English language.

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J a c q u e s M o u r e t f o r his o r i g i n a l c o n t r i b u t i o n in this ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y I N S E R M

work. (U52),

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