Neurotransmitter function in thiamine-deficiency encephalopathy

Neuroche~istry International, Vol.4, No. 6, pp. 449 to 464, 1982. Printed in Great Britain.

0197-0186/82/060449-16503.00/0 © 1982PergamonPress Ltd.

REVIEW NEUROTRANSMITTER F U N C T I O N IN THIAMINE-DEFICIENCY ENCEPHALOPATHY ROGER F. BUTTERWORTH Laboratory of Neurochemistry, Clinical Research Institute of Montreal (University of Montreal), Montreal, Quebec, Canada H2W IR7

CRITIQUE by M. K. GAITONDE

Al~tract--The encephalopathy caused by severe thiamine depletion of the mammalian CNS is accompanied by regionally selectivechanges in neurotransmitter function. Thiamine deficiency induced by administration of the central thiamine antagonist, pyrithiamine, causes more widespread lesions and accompanying changes in neurotransmitter function than does the deficiencystate induced by chronic deprivation of the vitamin. There is convincing evidence for a central muscarinic cholinergic lesion in pyrithiamine-treated rats and neuropharmacological studies show that this lesiori is partially responsible for the neurological deficit resulting from this treatment. There is also good evidence to suggest that thiamine deprivation selectively affects cerebellar afferent and efferent systems. Included in these are a loss of serotoninergic mossy fibres and of the functional integrity of glutamatergic granule cells. In addition, abnormalities of both nerve terminals and glial cells are found in lateral vestibular nucleus and it has been proposed that a loss of Purkinje cell terminals and concomitant decreases of pontine GABA may reflect these changes. The selective vulnerability of brain structures to thiamine deprivation is reflected in (i) the turnover rate of total thiamine in these areas and (ii) the selective decreases in activity of the thiamine pyrophosphate dependent enzyme pyruvate dehydrogenase.

Thiamine deficiency in both humans and experimental animals results in characteristic neurological symptoms, most of which are promptly reversed by thiamine administration. This latter finding has led to the general acceptance of the concept of 'the biochemical lesion' first enunciated by Peters (1936). One of the characteristics of thiamine-deficiency encephalopathy is its predilection for specific CNS structures with sparing of neighbouring ones. This selective vulnerability of certain brain regions to thiamine deficiency has been suggested to have a metabolic basis (McCandless and Schenker, 1968; Troncoso, Johnston, Hess, Griffin and Price, 1981). The nature of 'the biochemical lesion' in thiamine deficiency, however~ is unclear. Hypotheses involving a decreased activity in brain of one of the thiamine pyrophosphate (TPP)dependent enzymes or of depletion of cerebral thiamine triphosphate (TTP) leading to disruption in neuronal electrical activity, have been advanced

(McCandless, Curley and Cassidy, 1976; Cooper and Pincus, 1979). A review of the literature surrounding neurotransmitter function in thiamine deficiency reveals, in addition to a great deal of valuable information concerning the vitamin's role in CNS function, a substantial quantity of conflicting data. One reason for this is the lack of uniformity used in the experimental approach to the problem. Some studies have used, as experimental regimen, the chronic feeding of a thiamine-deficient diet to experimental animals, resulting in 35-45 days, in neurological symptoms of thiamine-deficiency. This experimental approach necessitates the inclusion of a control group of animals, 'pair fed' to either equal consumption of thiamine-supplemented diet or to equal weight (with the deficient group) to adequately control for the anorexic effect of long-term thiamine deprivation. Other studies have made use of thiamine antagonists 449

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such as pyriihiamine which are t\)und to deplete CNS thiamine more rapidly than dictar5 restriction resulting in neurological signs of thiamine deficiency in 12 16 days. Most investigators have considered thai this latter approach does not necessitate the inclusion of'pair fed' control animals. After a careful review of the literature, it is the opinion of this reviewer that, although the encephalopathy produced by the two experimental procedures shows many qualitative similarities, there are specific difl'erences of both a neurobehavioural and ncuropathological nature between the two models. For example, when a series of neurobehavioural tests were performed daily on a group of rats chronically deprived of thiamine and compared to at second group treated with pyrithiamine (PT), several important differences were noted (Jolicoeur, Rondeau. Hamel, Butterworth, Barbeau, 1979: Hamel, Butterworth, Jolicoeur and Barbeau, 1980). When tested on the day each rat lost its righting reflex, the PT rats showed additional neurological signs including a severe catalepsy. Ira addition, neuropathological studies have shown that the localisation of lesions due to thiamine deficiency is dependent on the experimental method used to produce the deficiency state. Chronic thiamine deprivation in the rat produced lesions confined mainly to the lateral pontine tegmentum (in particular the lateral vestibular nucleus) and floor of the fourth ventricle (Collins, 1967: Tellez and Terry, 1968: Robertson, Wasan and Skinner. 1968: Troncoso et a/.. 1981). Pyrithiamine treatment, on the other hand, causes in rats. reproducible lesions of a much wider localisation including mammillary bodies, thalamus, inferior olives and cerebellum, in addition to those of the pons (Troncoso et al., 1981; Papp, Tarczy, Takats, Auguszt, Komoty and Toluk, 1981: Takahashi, 1981). In view of these behavioural and neuropathological differences between the effects of chronic thiamine deprivation and pyrithiamine treatment, an attempt has been made in the preparation of this review to describe changes in neurotransmitter function in relation to the experimental regimen used. In addition, since there are certain species differences both with respect to neurological signs and site of lesions in thiamine deficiency encephalopathy, this review will confine itself mainly to a discussion of results obtained in the thiamine deficient rat. It has been suggested thai the encephalopathy due to thiamine deficiency may involve impairment of neurotransmitter function and in the last 15 yr abnormalities of catecholamines, acetylcholine, serotonin, GABA as well as the excitatory dicarboxylic amino acid (ptrtativel neurotransmitters ghitamate and

aspartate have been implicated m the pathoph3.siological mechanisms of cerebral dysfunction ;is,,t)Clated with thiamine deficiency. ('ATECHOLAMINFS Noradrenaline concentrations, and to a lesser extent those of adrenaline are reportedly increased m cerebral cortex of thiamine deficient rats (lwata. Watanabe, Nishikawa and Ohashi. 1969}. Subsequent experiments showed that catecholamine turnover was significantly reduced in cerebral cortex, brain stem and cerebellum of thiamine-deprived rats when compared to pair fed controls (lwata, Nishikawa and Baba. 19701. These latter changes were reversed by thiamine administration as were the associated lowered blood catecholamine levels and marked hypotension and bradycardia associated with thiamine deftciency. In addition, recent studies have shown that the concentration of 3-methoxy-4-hydroxyphenyl glycol IMHPG), the primary metabolite of noradrenaline was diminished in lumbar CSF of patients with Korsakoff's Syndrome (McEntee and Mair, 1978). The extent of the metabolite's reduction was significantly correlated with measures of memory impairment of individual patients. It was suggested that the memory disorder of Korsakoff's Syndrome may restrh from damage to ascending noradrenergic pathways by the diencephalic and brain stem lesions associated with the disease. SEROTONIN In recent studies (Plaitakis, Nicklas and Berl, 1978a), the effect of thiamine deprivation (TD) or pyrithiamine treatment (PT) on neural function was examined by studying the high affinity uptake systems for several putative neurotransmitters by synaptosomal preparations isolated from cerebellum, hypothalamus and telencephalon. These studies revealed a selective impairment of serotonin (5HT) uptake by cerebellar and, to a lesser extent, hypothalamic synaptosomes, which correlated with the development of neurological signs of thiamine deficiency. Kinetic studies on the synaptosomal preparation from cerebellum of PT-treated rats revealed abnormalities both of l/maXand K m parameters (Fig. 1). Administration of high doses of thiamine to PT animals reversed the 5HT uptake abnormalities. Furthermore, this reversal coincided with clinical improvement in these animals. Although thiamine administration in i'ir:o caused reversal of the 5HT uptake deficit, administration of either thiamine or its phosphate esters, or pyri-

Review

451

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thiamine in vitro were without significant effect (Plaitakis et al., 1978a). The possibility that 5HT uptake might be uptake by the noradrenaline system was effectively eliminated by experiments using selective inhibitors. Fluoxetine, a selective inhibitor of 5HT uptake, when added to the preincubation medium, inhibited 5HT uptake by cerebellar synaptosomes in control a n d PT rats whereas desmethylimipramine, a selective inhibitor of noradrenaline uptake, did not. Thiamine deficiency has repeatedly been shown to have no effect o n 5HT levels either in whole brain (Itokawa, Schultz and Cooper, 1972) or brain regions (Plaitakis, Van Woert, Hwang and Berl, 1978b). However, P T rats had significantly elevated levels of the 5HT metabolite 5-hydroxyindole acetic acid (5HIAA)

in all of seven brain areas studied (Plaitakis et al., 1978b), (Table 1). These changes in 5HIAA concentrations were particularly significant in striatum and hypothalamus, those in hippocampus a n d cerebellum being less pronounced. To determine whether or not the increased cerebral 5HIAA accompanying thiaminedeficiency encephalopathy resulted from enhanced 5HT turnover in vivo or was a result of impaired efftux of the metabolite, 5HT turnover was measured by studying (i) the rate of 5HT accumulation following m o n o a m i n e oxidase inhibition (using pargyline), (ii) the rate of 5HIAA formation following intracisternal administration of radiolabeled 5HT and (iii) the rate of disappearance of intracisternally administered labeled 5HIAA. Following pargyline administration,

Table 1. Effect of pyrithiamine treatment (PT) on brain levels of serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) Serotonin

5HIAA

Controls PT Cerebellum Medulla-pons Hypothalamus Striatum Midbrain Hippocampus Cortex

0.24 0.38 0.68 0.42 0.62 0.32 0.38

___0.04 + 0.03 --k-_0.07 + 0.03 +_ 0.07 + 0.02 + 0.04

Pair fed 0.24 0.39 0.65 0.40 0.63 0.32 0.37

__ 0.04 + 0.02 + 0.04 + 0.02 +__0.07 ___0.03 + 0.02

Controls Ad lib.

0.21 0.41 0.64 0.37 0.64 0.34 0.33

+ 0.03 + 0.03 _+ 0.02 + 0.02~ + 0.05 + 0.04 + 0.03:~

Pair fed

PT

0.12 + 0.02* 0.75 +__0.09t 0.78 ___0.10+ 0.78 + 0.10t 1.07 ___0.14"t" 0.39 _ 0.05"f" 0.30 + 0.04"I"

0.09 0.55 0.53 0.51 0.78 0.30 0.21

Each value is expressed in #g/g wet wt +_ S.D. (N = 6). Pair fed: pair fed controls on thiamine deficient diet plus thiamine (2.2 mg/100 mg diet). Ad lib.: freely fed controls on regular rat chow. * P < 0.01 PT vs controls. t P < 0.001 PT vs controls. ~; P < 0.05 pair fed vs ad lib. controls (from Plaitakis et al., 1978b).

+ 0.01 ___0.05 ___0.04 ___0.06 + 0.08 _ 0.01 + 0.03

Ad lib.

0.09 0.51 0.49 0.48 0.79 0.30 0.20

___0.01 _ 0.06 + 0.03 _+ 0.03 + 0.04 + 0.04 + 0.05

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increased accumulation of 5HT occurred in lhc brain~, of PT-treated rats indicating an tactcase m svntllesis. Changes of greatest ntagnitude occurred in cerebelhim, h)pothalamus and hippocampus. Following intracisternal injection of [~'~C]5HT, both levels of [IaC]YHIAA and the magnitude of the ratio [ 1'~C]5H IAA,'[ t"~CI51-1T increased significantly. Changes in this latter parameter were most pronounced in cerebellum: therc was no change in this ratio in cerebral cortex. The increased 5HIAA was apparently due. in part, to impairment of the transport of this metabolite as indicated by an increase in its retention following intracisternal administration. An autoradiographic study was undertaken to examine the relationship between thiamine deprivation and the loss of labeled indoleamine axons m rat cerebellum (Chan-Palay. Plaitakis, Nicklas and Berl, 1977). The study made use of light microscopic autoradiography after lengthy intraventricular mfusion of [3H]5HT simultaneously with cold l),L-noradrenaline, experimental conditions which reportedly permit indoleamine structures to take up [~H]YHT selectively. Autoradiograms, obtained under such conditions, of cerebellar cortex and deep nuclei of normal animals revealed plexuses of labeled indoleamine axons. The plexus to the cortex included mossy fibre rosettes and parallel fibre-like axons. This finding supports the suggestion of Bloom, Hoffer, Siggins, Barker and Nicoll {1972), who postulated tas a result of experiments involving micro-iontophoretic application) that some terminals of mossy fibres ma) be serotoninergic. In addition to mossy fibres, all deep cerebellar nuclei and Deiter's nucleus were found by autoradiographic studies to be richly 5HT-innervated (Chan-Palay et al., 1977). Autoradiograms from comparable regions of cerebella of thiamine deprived rats showed a dramatic decrease in all indoleaminelabeled structures including a marked loss of mossy fibre rosettes and parallel fibre-like axons. Similar autoradiographic studies on PT-animats were not performed. The findings of a dramatic loss of labeling of 5HT axons in the cerebellum of TD rats by autoradiograpby after intraventricular infusion of [3H]YHT, coupled with the observed decreased l:m,~ for high affinity 5HT uptake by cerebellar synaptosomes are consistent with the hypothesis that thiamine deficiency may be accompanied by a selective loss of serotoninergic (mossy and parallel) fibres. How the findings of increased turnover of 5HT, in thiaminedeficiency are related to this hypothesis, however, are not clear, at this time. Certain behavioural correlates with these 5HT ab-

normalities have been suggested, tt)r cx~unpic, bt i~as been proposed that the hypothermia obsc!xed ii1 thiamine deiiciency may b c a result of altered hypolhalamic 5HT function since this monoaminL' ha> been implicated in hypothalamic nlccharll~,nls of thermoregulation in rats {Myers, 19781 and patients with Wernick's Encephalopath? reportcdly develop thermoregutatory disturbances (Lipton, Payne, Garza and Rosenberg, 1978: Plaitakis, Nicklas. Van Woerl, Hwang and Berl. 1981). In addition it has been suggested that the amnestic syndrome seen m patients with Korsakoff's Psychosis may be related to changes in the 5HT system (Plaitakis et al., 1981l. ;'-AMINOBUTYRI(" ACID Chronic thiamine deprivation in rats leads to a decrease in the concentration of ;,-aminobutyric acid (GABA) in whole brain of symptomatic animals (Ferraft, 1957: Gubler. Adams, Hammond, Yuan, Guo and Bennion, 1974). Similarly, studies making use of pyrithiamine treatment reveal significant decreases in whole brain GABA (Gubler, Adams, Hammond, Yuan, Guo and Bennion, 1974: Gaitonde, Fayein and Johnson, 19751. In order to further elucidate the role of GABA in the neurological manifestations of thiamine deficiency, more recent studies were undertaken in which re qional cerebral GABA concentrations were measured in thiamine deprived rats both prior to and following the onset of neurological symptoms (Butterworth, Hamel and Barbeau. 1978: Hamel, Butterworth and Barbeau, 1979; Butterworth, 1981). In a parallel series of experiments, in which thiamine deficiency was induced by pyrithiamine treatment, regional GABA concentrations were again measured in symptomatic animals (Butterworth, Hamel, Landreville and Barbeau, 19791. Cerebellar GABA levels were significantly diminished in symptomatic thiamine deprived (TDt and pyrithiaminetreated (PT) rats. Furthermore, GABA concentrations in cerebella from TD rats were found to be significantly diminished in concentration prior to the appearance of neurological symptoms (Table 2). No changes in GABA levels were found in any of the other six brain regions {medulla-pons. midbrain. hypothalamus, hippocampus, stria/urn or cerebral cortex) of TD rats either before or following the appearance of neurological signs of thiamine deficiency. In PT rats. GABA concentrations were significantly diminished in medulla-pons, midbrain and cerebral cortex {in addition to cerebellum) (Butterworth et al., 1979). A subsequent study, however, revealed no changes in GABA in either cerebellum or medulla-

Review

453

Table 2. Effect of thiamine deprivation (TD) or pyrithiamine treatment (PT) on GABA concentrations in cerebellum Cerebellar GABA concentration (/~mol/g wet wt, S.E.M.)

Treatment group

2.48 + 2.48 + 1.70 + 1.84 _ 1.64 +

1. Ad libitum fed 2. Pair fed 3. Thiamine-deprived (TD), 14 days presymptomatic 4. Thiamine-deprived (TD), 40 days, symptomatic 5. Pyrithiamine-treated (PT), symptomatic

0.14 0.19 0.16" 0.14" 0.20*

* P < 0.01 compared to pair fed controls (data from Butterworth et al., 1978, 1979 and Hamel et al., 1979).

in particularly high concentration in Purkinje cells (Rindi, Patrini, Comincioli and Reggiani, 1980). These inhibitory GABAergic neurons make up the major efferents from cerebellum (see Fig. 3) and Purkinje cell abnormalities have been observed in thiaminedeficiency (Victor, Adams and Collins, 1971; Mesulam and Van Hoesen, 1976). In addition, loss of nerve terminals in the lateral vestibular (Deiter's) nucleus and in fastigeal nucleus are constant findings in TD and PT rat brain (Tellez and Terry, 1968; Pena and Felter, 1973; Troncoso et al., 1981). Since these nuclei contain Purkinje cell nerve endings (Fonnum, StormMathisen and Warberg, 1970), selective loss of the latter could be responsible for the observed GABA changes in medulla-pons (Butterworth et al., 1978; Hamel et al., 1979) in thiamine deficiency. An alternative explanation might involve the selective early loss of glial cell integrity frequently noted in lateral vestibular nucleus in thiamine deficiency encephalopathy (Collins, 1967; Robertson et al., 1968). There is a convincing body of evidence now that glial cells are inti-

pons of PT rats (Plaitakis, Nicklas and Berl, 1979). One possible reason for the discrepancy could be the fact that biochemical studies in the latter report were done somewhat earlier (after 12 days of pyrithiamine treatment rather than 18 days used in the former study). Decreased incorporation of [~4CJglucose into amino acids in brain has been reported in pyrithiamine-treated rats (Gaitonde et al., 1975; Plaitakis et al., 1979). Furthermore, studies of [14C]labeled amino acids and organic acids from rat brain following intraventricular injection of [14C]~t-ketoglutarate revealed that metabolism of the keto acid through both the tricarboxylic acid (TCA) cycle and the GABA shunt pathways were reduced in both TD and PT rats (glucose-GABA conversion by brain is shown in Fig. 2). One possible explanation for the observed regional GABA changes might involve selective GABAergic neuronal dysfunction in thiamine deficiency. Histochemical studies have shown that thiamine is present

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mately involved in neuronal regulation, particularly in the case of GABAergic and glutamatergic synapses. Selective glial cell changes in lateral vestibular nucleus then might be expected to interfere with GABA regulation in this nucleus. Finally, evidence for a disorder in GABAergic function is supported by the finding that presymptomatic thiamine-deprived rats were more susceptible to seizures induced by the GABA antagonist picrotoxm than were pair fed controls (Table 3i. Seizure incidence increased from 25 to 100% following administration of picrotoxin (4 mg/kg, sc). GLUTAMA]'E AN[) ASPARTAI[! Glutamate and aspartate meet many of the attatomical, neurochemical and physiological criteria for neurotransmitter status in the C N S : they open sodium and potassium ionic channels and cause a rapid, powerful, excitatory response. Both amino acids are present in all brain cells due to their involvement in multiple metabolic events. A high afl-init~ uptake system exists tk)r glutamate and aspartate in

t h e t"dhc-, t:q vildltgc:- ~

]'able 3. Efli:ct of thiamine deptlx, ation c,n suscepiib~ht3 to picrotoxna induced convulsions Treatment

Pair fed controls Thiamine-deprived, 14 days presymptomatic

N

Incidence

i2

3 12

12

il!-

hlgti allimt 5 ilptakc lvllowing iL.:<,,i,, <~ ni:in,,c~ ! spccllic g l t l ! a h l d l c r g l c a l n t 'as['~itlI,A[clglL lil>it.w, tiax. v bcLn I,lv, i)oscd, i l l c l u d i l l g liic ctUtlcT(,Mll~G.J; ,.llad cmorhinal hipr, ocampa! pathx~si3~, a, ~cll a, d~, c~.~bcllar r)alalicl librcs (McGc~.l. I celt:, and \ I t ( i c e ! 19178). Whole {ar;illil COflcentra[ioll~ Of glutamate ha,. c ~epealedl S been found to b,_- decieascd m S}llllHorllatic duamme-deprived rats (Ferlarl, 1957: (iubl,.z c; o1. i974, (iiatonde Nixey and Sl'i,ulmali. 19741 aiR1 ill ~,m, iieated with tlm~mine anlagcmists iGublcr ,'r ~d.. 1974a Gaitonde ~'t <;I., 1975). Similur rcsuh.,, haxc hccn rcpoited in the mouse (Holt)v, aL-h, Kauflruain lkoss,. Thomas and McDougal. 190.8!. More tL-cent sludles on regional couccntration.~ ot glutamate lU t i l i a l n l n c deficieucy er~cwphalopath5 re'+calcd substantmll> dill fercnl resuhb betaeen thiamine deprived (1 Di rats and thnsc animals t/cared v, itfi p~rithitunmc {PIL (Bt_itterworth c1 at.. 1978: Hamcl et M.. 1979: Buttci~urth. 1982)Ccrebellar ghitainute, for example, was lound to be significantly decreased m symptotmttic (but not presymptomatic) T D rats but was found to be unchanged m ~,ymptomati~ animals made thiamine ddicient by pyrlthiamine treatment IPT)(Table 41. The Iinding of unchanged cerebellm glmaniate m Pq lais ,aas subsequently confirmed by othc)>, (Plaitakls et al.. 1979). Glutanmtc levels m medull,t-pon,,, were ieportcd)y decreased in thmmine-deptixed rats (Hamel e~ a/. 19791 and unchanged {Buttm-v~(~rth ct ,_tl.. 1979) or decreased (Piaitakis el a/. 1979) Ul pylithiamine treated animals. Glutamate le'~els m midD l a i n Y, e r c l u t u l d to be decreased as a r e s u l t o f either thiamine deprivatum or pyrithiamuae treatment (Butlerworth ,_'t ,tl.. 1978: Hamel et a l 1979: P,uttc~w(~rdh cl a/., 1979i. hicurporatiou of [l"C]label fi cma [i ~(']ghic:o.',e into h,ain ghltamale has been found to bc dccre,tsed in rats lnade thiamine deficient by thiamine deprivation i I l ) ) (Takahashi. 1981) or b) pyrithiaminc trcamient (PT) (Gaitondc ctal.. 1975). In the case ol the latter btudy. COlD,el siOlt of [11-14(']glucose llito gltitarnate

Seizure charactetis~ics Or~set mmal Duration (mm) 1915 ± 285

1212 26.50 ± 3.22 .....................................................

38.50 ± ! 50 45.67 ± 16.¢,8

455

Review Table 4. Effect of thiamine deprivation (TD) or pyrithiamine treatment (PT) on glutamate concentrations in cerebellum Treatment group 1. Ad libitum controls 2. Pair fed controls 3. Thiamine-deprived (TD), 14 days, asymptomatic 4. Thiamine-deprived (TD), 40 days, symptomatic 5. Pyrithiamine-treated (PT), symptomatic

Cerebellar glutamate concentration (#mol/g wet wt _+ S.E.M.) 9.86 + 0.20 10.47 _+ 0.16 10.08 ___0.31 7.57 __+0.20* 9.09 + 0.22

* P < 0.001 compared to pair fed controls (data from Butterworth et al., 1978, 1979 and Hamel et al., 1979).

was decreased only in the brain of s y m p t o m a t i c PT animals. In TD rats, conversion of intraventricularly injected [~4C]~-ketoglutarate into glutamate was found to be significantly decreased (Gubler et al., 1974). The findings of decreased incorporation of [14C]glucose into amino acids in thiamine-deficient brain may reflect disordered neurotransmitter function. Several lines of evidence suggest that glucose labels the neurotransmitter pools of glutamate, aspartate and GABA. Glucose incorporation into these amino acids is extremely sensitive to variations in physiological function whereas incorporation from other precursors is not (Gibson, Peterson and Sansone, 1981). Anaesthetic levels of sodium pentobarbitone depress the incorporation of [U-14C]glucose but not that of [1-14C]acetate or [1-~4C]butyrate into glutamate, glutamine and aspartate (Cremer and Lucas, 1971). In addition, electrical stimulation of guinea pig cerebral cortical slices leads to selective release of [14C]glutamate, [14C]aspartate and [~4C]GABA synthesised from [~4C]glucose (Potashner, 1976), this release being calcium dependent and inhibited by the sodium channel blocker tetrodotoxin (Potashner, 1978). There is good evidence to suggest that glucose is metabolised in brain within the large glutamate pool located in neurons (Van den Berg and Garfinkel, 1971; Balazs, Patel and Richter, 1973) and the highest rate of synthesis of amino acids derived from glucose occurs in nerve endings (Rose, 1972). Uptake of glutamate was significantly enhanced in synaptosomal preparations isolated from cerebella from both TD and PT rats as compared to controls. However, glutamate uptake by synaptosomal preparations from medulla-pons and telencephalon was not significantly different. In the PT group, increased high affinity uptake of glutamate was maximal shortly after the onset of neurological abnormalities (after 12 days of pyrithiamine treatment); this increase gradually diminished as animals became more severely af-

fected. In the TD rats, changes in glutamate uptake were less than in the PT group, first appearing 4 weeks after the start of dietary thiamine deprivation when animals showed mild neurological signs. Maximal increases in glutamate high affinity uptake were found after 5 weeks on the diet but, after 7-9 weeks, when animals were at an advanced stage of chronic dietary thiamine deficiency, the glutamate uptake abnormality had disappeared (Plaitakis et al., 1979). Kinetic studies of glutamate uptake by cerebellar synaptosomes from the PT group during the stage of maximal uptake changes revealed Vmax parameters increased by 100~o without significant changes in Kin. The selective alterations of high affinity glutamate uptake in cerebellum, when taken in conjunction with the findings of decreased concentrations of glutamate in this same region of brain in TD rats, at first sight suggests loss of glutamatergic neurons in cerebellum in thiamine deficiency. However, loss of glutamatergic nerve terminals in cerebellum would be expected to be accompanied by decreased high affinity uptake of glutamate by synaptosomal preparations rather than the substantial (albeit transient) increases observed in the aforementioned studies. An alternative explanation might involve glial cell abnormalities in thiamine deficiency. Gliosomes are a contaminant of synaptosomal preparations (Plaitakis et al., 1979) and, since glia, in addition to neurons, possess a high affinity uptake system for glutamate (Roberts and Keen, 1974), it is possible that the enhanced glutamate uptake, and associated Vm~xchanges, were a result of proliferated glial cells. In fact, it has been reported that, in symptomatic TD rats, following the initial stages when glial cell cytoplasm is swollen (Collins, 1967; Robertson et al., 1968), there is an increase in the number of glial cells within the area of involvement (Collins, 1967). A more recent study revealed enhanced high affinity glutamate uptake following kainic acid lesions at a stage when marked glial proliferation was apparent (Foster and Roberts, 1980).

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Fig. 4. Simplified diagram of major cerebellar afferent fibre systems of mammalian brain (PF: parallel fibres, MF: mossy fibres. CF: climbing fibres).

The high affinity glutamate uptake abnormality observed in TD rat cerebellum, then, could reflect the pattern of cellular abnormalities involved, starting with glial cell changes (swelling of cytoplasm, vacuolation etc.) proceeding to the loss of nerve terminals described in several studies (Tellez and Terry, 1968; Pena and Felter, 1973). Early increases in glutamate uptake might reflect the early glial cell changes whereas the (later) d e c r e a s e s in high affinity uptake of glutamate uptake might be the result of loss of integrity of glutamatergic nerve terminals. Further work is clearly required to more fully elucidate the abnormalities of cerebetlar glutamate in thiamine deficiency suggested by the studies described, as well as their relationship to both glial and neuronal dysfunction. If the cerebellar glutamate changes are, in fact, due to neuronal abnormalities, the cerebeltar neuron most likely to be affected is the granule cell. Association of glutamate with cerebellar granule cells has been suggested by studies on mutants and following certain viral infections which selectively deplete these cerebellar interneurons and reduce high affinity glutamate uptake. Ataxic 'staggerer' and 'weaver' mutant mice, both of which show selective loss of granule cells are found to have diminished concentrations of glutamate in cerebellum (McBride, Aprison and Kusano, 1976). Viral infections in hamsters that destroy granule cells bring about reduced endogenous cerebellar glutamate and reduced high affinity glutamate uptake (Young, Oster-Granite, Herndon and Snyder, 1974). Selective degeneration of cerebellar granule cells has been reported in thiamine-deficient monkeys (Mesulam and Van Hoesen, 1976). These granule cell changes were reportedly only revealed by Fink-Heimer stain-

ing {routine stares did not reveal thc patholog3i and tt ~as suggested that the granule cell lesion ma\ bc t>t greater importance than prex.iously thought. P:+lh:,r or cerebellar white matter has recently been des~.:ribcd in PT rats (Takahashi, 1981). 111 addition, grant+it cell dysfunction resulting from the selective loss of serotoninergic mossy tibres has been suggested tPhtitakis ¢'t al.. 1981). A simplified schematic representation of major cerebeltar afferent fibre connection is shown in Fig. 4, Decreased cerebral a.spartate has repeatedly been shown to accompany thiamine deficiency encephalopathy (Gaitonde el al., 1975; Butterworth el al.. 1978: Plaitakis et al., 19791. Aspartate levels were found to be significantly diminished in medulla-pons, cerebellum and midbrain of symptomatic thiamine-deprived rats tHamel et al., 1979). Furthermore the changes in medulla-pons were found to precede the development of neurological signs of thiamine deprivation (Butterworth, 1981). Decreased aspartate was later reported in medulla-pons, midbrain and cerebral cortex of symptomatic pyrithiamine-treated rats iButterworth et al.. 1979; Ptaitakis er al., 19791 and mice {Brosemer, Grammer and Gurusiddaiah, 1981tJ High affinity uptake of aspartate by cerebellar synaptosomes was found to be increased m symptomatic thiamine-deprived and pyrithiamine-treated rats (Plaitakis el al., 1979) and this uptake abnormality showed an onset and magnitude which correlated well with the onset and degree of neurological impairment. As was the case with the glutamate uptake defect in cerebellum of thiamine deficient rats, measurement of the kinetics of aspartate uptake by cerebellar synaptosomes revealed abnormalities of Vm~,,but not of K m parameters. The cause of observed abnormalities of aspartate land possibly a further cause of glutamate changes) in the cerebellum and medulla of thiamine deficient rats may be related to the reported histological changes in the inferior olive nucleus. In a recent study, all animals in a group treated with pyrithiamine and sacrificed after development of neurological manifestations of thiamine deficiency, showed evidence of lesions in the inferior olives (Troncoso eta/., 1981). In addition, it has been shown autoradiographically that thiaminedeprivation markedly affects the serotoninergic terminals in the inferior olive (Chan-Palay et el., 1977}. The inferior olive is the major source of climbing fibre afferents to cerebellum (Desclin, 1974) and recent studies suggest that aspartate (Perry, MacLean, Perry and Hansen, 1976; Nadi, Kanter, McBride and Aprison, 1977) or glutamate (Butterworth, Hamel, Landreville and Barbeau, 1978) may be the excitatory neuro-

Review transmitter of climbing fibres. These latter conclusions led directly from studies using 3-acetyl pyridine, a neurotoxin shown histologically to cause selective degeneration of inferior olive with ensuing destruction of cerebellar climbing fibres (Desclin and Escubi, 1974). 3-Acetyl pyridine treated rats show permanent cerebellar ataxia, consistent with disturbed olivocerebellar function (Jolicoeur et al., 1979). ACETYLCHOLINE Although impaired synthesis of acetylcholine by thiamine-deficient brain was first reported in 1940 (Mann and Quastel, 1940) and despite numerous subsequent studies, the functional significance of this cholinergic lesion remains controversial. Recent technological improvements have helped to clarify some of the confusion surrounding thiamine deficiency's effect on cholinergic transmission. Conflicting reports of brain acetylcholine decreases (Cheney, Gubler and Jaussi, 1969; Heinrich, Stadler and Weiser, 1973; Vorbees, Schmidt and Barrett, 1978) and of no changes (Speeg, Chen, McCandless and Schenker, 1970; Reynolds and Blass, 1975) in thiamine deficiency were probably related to the use of different experimental methods of acetylcholine assay, to variable degrees of post-mortem acetylcholine hydrolysis and to lack of knowledge of compartmentation of the metabolism of choline used as a precursor (Freeman and Jenden, 1976). A combination of gas chromatography and mass spectrometry allows sensitive, specific measurement of choline and acetylcholine (Jenden, Choi, Silverman, Steinborn, Roch and Booth, 1974) and sacrifice by microwave irradiation has been shown to rapidly inactivate cerebral choline acetyltransferase and cholinesterase (Stravinoha, Weintraub and Modak, 1973). Problems due to compartmentation can be minimised by estimation of the rate of acetylcholine synthesis using a double-labelling technique in which incorporation of both 1-14C]glucose and [2H4]choline is measured (Gibson, Blass and Jenden, 1978a; Gibson, Shimada, Blass, 1978a). Using such techniques, acetylcholine levels were found to be unchanged after 13 days of pyrithiamine treatment (PT) at which time animals showed marked neurological impairment (Barclay, Gibson and Blass, 1981a). Acetylcholine synthesis, on the other hand, was markedly diminished whether it was measured by incorporation of glucose or choline; compared to pair fed controls, incorporation of [U-14C]glucose into acetylcholine was found to be decreased by 51~o in PT rats. Incorporation of deuterated choline into acetylcholine was reduced by 36~o. These results con-

457

firmed the previous study of Vorhees et al. (1978) who measured acetyicholine turnover by the hemicholinium method in which rats were (appropriately) sacrificed by focussed microwave irradiation. In the latter experiment, symptomatic pyrithiamine-treated rats showed reductions in acetylcholine turnover in only midbrain and hippocampus. A previous study by the same authors had revealed a selective acetylcholine turnover reduction in midbrain of symptomatic thiamine-deprived (TD) rats (Vorhees, Schmidt, Barrett and Schenker, 1977). Administration of the central acetylcholinesterase inhibitor physostigmine has repeatedly been shown to prolong survival time in PT rats (Cheney et al., 1969; Barclay et al., 1981a). Furthermore, physostigmine was found to partially reverse the impairment in scores in a sensitive 'string-test' that detects neurological abnormalities in thiamine-deficient rats (Barclay et al., 1981b). Neostigmine, a peripheral-acting acetylcholinesterase inhibitor had no effect on string test performance. In addition, since the effect of physostigmine was blocked by atropine but not by methatropine (Fig. 5) which does not cross the bloodbrain barrier, impaired string-test performance has been suggested to be a central cholinergic effect. The fact that atropine is primarily a muscarinic blocker, suggests that the impaired string-test performance in PT rats is a muscarinic phenomenon. Further evidence for such a contention was provided by experiments using arecoline, a direct muscarinic agonist which was found to be as effective as physostigmine in restoring string-test performance whereas nicotine

a

8 r ~ NORMALSCORE 6 ~" |

m

[ ] BEFORE [ ] AFTER ¢rP
T,

•

2 m INE

2

PHY$OSTIGNINE

ATROPINE RHYSO-

STIGt41NE + METHATROPINE

Fig. 5. Effect of cholinergic drugs on string test score in pyrithiamine treated rats. Scores were determined before and after i.p. injection of saline, thiamine (100mg/kg), physostigmine (0.05-0.5 mg/kg), neostigmine (0.2mg/kg), physostigmine + methatropine (2.0mg/kg) or physostigmine + atropine (2.0mg/kg). Bars represent S.E.M. (from Barclay et al., 1981a).

45£

I~t~(il R t

I~1 [ i l R \ \ { f l ~ II]

GLUCOSE l

NAD+

1

PYRUVATE~

C°2U

--

7"- ~ACETYLCoA CoASH CAT

~ CHOLN IE

ACETYLCHOLINE Fig. 6. Possible relationship between oxidative metabolism of glucose and acetylcholine synthesis in mammalian brain showing the key role of the pyruvate dehydrogenase complex. E~: pyruvate dehydrogenase (EC 4.1.1,1), TPP dependent. E2: lipoate acetyltransferase (EC 2.3.1.12) E3: lipoamide dehydrogenase IEC 1.6.4.3). CAT: choline acetyltransferase.

was without effect (Barclay et al., 1981a). Furthermore, mecamylamine, a central nicotinic blocker does not alter the effect of physostigmine, adding further credence to the notion that impairment in string-test performance associated with pyrithiamine induced thiamine deficiency is the result of a central muscarinic cholinergic lesion.

Several theories for the impaired synthesis of acetylcholine in thiamine deficiency have been proposed. all stemming from the role of thiamine as a coenzyme in carbohydrate metabolism (Vorhees et al., 1977). Firstly, it has been suggested that a reduction in acetylcholine synthesis could be the result of a reduction in the availability of high energy phosphates produced by tricarboxylic acid cycle metabolism. However, the content and turnover of cerebral ATP, ADP and AMP in brainstem and cortex have been found to be unaffected by thiamine deficiency (McCandless and Cassidy, 1976). A second possible cause of the decreased acetylcholine synthesis associated with thiamine deficiency might be a reduction of acetyl CoA resulting from impaired pyruvate dehydrogenase complex activity (see Fig. 6) which is thiamine pyrophosphate (TPP) dependent. Data regarding acetyl CoA levels in thiamine deficiency arc conflicting. In addition regional levels of acetyl CoA in thiamine deficiency have not been studied. However, it has been observed that although the amount of pyruvate ultimately leading to acetylcholine constitutes tess than 1'I,, of that resulting in CO: produc-

tlon. pyruvate dehydrogenase (PDH() inhfl~ition causes a measurable impairment of acetylchohnc s~nthesis {Gibson, Jope and Blass, 1975L Several studies have addressed the question ol abnormalities of pyruvate dehydrogenase acti'~il',, in regions of the brain of thiamine dcprivcd rats. l;nfortunately, little if any attention has been paid to the study of the enzyme's activity in pyrithiamine treated rats so that comparisons between the two experimental situations is not possible. PDHC activities m the brain stem (including pons) of symptomatic TD rats have repeatedly been found to be significantly decreased, by up to 60",, (Dreyfus and Hauscr, 1965: McCandless and Schenker. 1968: McCandless and Cassidy. 1976: Pincus and Wells. 1972). In two of these studies, cerebellar PDHC activity was found to be similarly decreased (McCandless and Schmlker, 1968; Pincus and Wells, 1972}, whereas the enzyme's activity in cerebral cortex, midbrain or spinal cord showed no reproducible changes. These diminished activities of PDHC in brain stem and cerebellum have been considered by some to be of too small a magnitude to play an important role m the encephalopathy due to thiamine-deficiency (Dreyfus and Hauser. 1975: Pincus and Wells. 1972). However, a more recently published study has provided evidence that mammalian brain may possess very' little reserve capacity of PDHC over that required to maintain normal pyruvate flux. Furthermore, PDHC appears to be distributed non-uniformly in brain (Reynolds and Blass, 1976) suggesting that certain brain structures may be particularly vulnerable to acquired or inherited disorders of the enzyme. Indeed, other suggestive evidence in support of the hypothesis that impaired PDHC may play a key rote in thiamine de[iciency's effects on the central nervous system, comes from recent studies on an inherited neurological disease m

200

HEPATIC PDHC ACTIVITY IO0 (PERCENT CHANGE)

~

CONTROL! CONTROL2 CONTROL3 PATIENT

NO ADDITIONS

+ATP

+ATP, +M92+,CO2"

Fig. 7. PDHC activation in a liver biopsy sample of a patient with L e i g h ' s Subacute Necrotising Encephalomyelopathy.

Review which PDHC is defective. Studies from three laboratories have recently revealed an abnormality of PDHC in Leigh Disease (Leigh's Subacute Necrotising Encephalomyelopathy) (Devivo, Haymond, Obert, Nelson and Pagliare, 1979; Butterworth and Melanqon, 1980, unpublished; Sorbi and Blass, 1982). PDHC activity in liver, cultured skin fibroblasts and brain samples from such patients shows a defective activation (see Fig. 7). Of particular interest is the report that the neuropathological findings in Leigh Disease, notably the symmetrical necrotic lesions of thalamus and pons resemble closely those found in Wernick's Encephalopathy associated with thiamine deficiency in man (Montpetit, Anderman, Carpenter, Fawcett, Aborowski-Suis and Giberson, 1971). SELECTIVE VULNERABILITY OF THE CNS IN THIAMINE DEFICIENCY The mammalian central nervous system is not a homogeneous organ from the point of view of its histology and biochemistry and differences in the composition of the tissue undoubtedly correlate with metabolic and biochemical differences on the one hand, and the vulnerability of the tissue to metabolic insult on the other. Metabolic differences and variations in enzymatic activity noted in various areas of brain may be due to differences in vasculature, in comparative density of neurons and glial cells and in the degree of myelination. The biochemical characteristics of certain neuronal populations or specific brain nuclei must also play a key role in the phenomenon of selective vulnerability in thiamine deficiency. It has been suggested that the CNS structures having the highest content of TPP tend to be affected earlier and to a greater extent than others by thiamine deficiency (Dreyfus, 1976). Total thiamine content in regions of the nervous system of the rat has been determined under steady state conditions using [triazole-2-14C]thiamine as tracer. Using an appropriate model of compartmental analysis, influx and efflux fractional rate constants, turnover times and turnover rates were calculated (Rindi et al., 1980). Thiamine turnover rates are shown in Table 5. Thus, cerebellum, medulla and pons were the regions having the greatest thiamine turnover rate and cerebral cortex the least. These findings are in good agreement with the sensitivity of the regions of the rat brain to thiamine deficiency. Total thiamine depletion caused by thiamine-deprivation (TD) is more marked in cerebellum and pons (Dreyfus, 1961; Pincus and Grove, 1970) than in cerebral cortex and

459

midbrain (Pincus and Grove, 1970). Furthermore, in TD rats, the reduction in thiamine pyrophosphate (TPP) is selectively greater in pons than in other brain regions and this reduction is found to precede the development of neurological signs of thiamine deficiency (Pincus and Wells, 1972). In symptomatic TD rats, pontine TPP levels fall to 26~o of control levels, far in excess of the decreases observed in other brain regions and addition of TPP to homogenates of brain from TD rats has been shown to cause an increase in the activity of pyruvate dehydrogenase in pons but not in other regions. At the cellular level there is evidence to suggest that the ratio of glial cells to neurons may be important in determining vulnerability of the anatomical site in thiamine deficiency. There is a convincing body of information from neuropathological studies to show that swelling and vacuolation of glial cells is a consistent finding in early thiamine deprivation in the rat. Furthermore, a comparative study of the effect of thiamine deficiency on energy metabolites and enzyme activities in C-6 glioma and C-13 neuroblastoma cell lines revealed that the glioma cells showed marked changes in ATP, phosphocreatine and in the activities of thiamine dependent enzymes, changes not found in the neuroblastoma cells (Schwartz and McCandless, 1976). A subsequent study revealed impaired lipid and fatty acid synthesis in C-6 glioma cells, consistent with reduced acetyl CoA synthesis resulting from impaired pyruvate oxidation (Volpe and Marasa, 1978). The neuroblastoma cell line exhibited little or no reduction in fatty acid synthesis under similar conditions of thiamine deficiency. Regional metabolic variations and susceptibilities among neurons are well established. Certain neuronal types require perineuronal glia for effective synaptic regulations; examples of such neurons are those utilising GABA or glutamate as neurotransmitter. It would Table 5. Turnover rates of total thiamine in regions of the rat central nervous system (from Rindi et al.. 1980) Thiamine turnover rate Brain region Cerebellum Medulla Pons Spinal cord Hypothalamus Midbrain Striatum Cerebral cortex

#g/g wet wt/h

~0 Cerebellar rate

0.551 0.543 0.454 0.389 0.357 0.294 0.268 0.159

100 98 82 70 65 53 48 29

,~-(]0

R()(i~ R t

t~I I I I I t \ \ ' l l R

III

Thiamine Deficiency

I

Decreased Thiamine Iand TPP in pons and cerebellum

[ Neuronal PDHC d e f i c i t

Glial PDHC ci~t (and energy) defi

Decreased synthesis of

Decreased synthesis of I

Acetylcholi ne

Glutamate and GABA

I

GABAand Glutamatergic neuronal dysfunctionj Fig. 8. Possible mechanisms involved in the cholinergic and amino acid neurotransmitter dysfunction m thiamine-deficiency encephalopathy.

not be unreasonable, therefore, to advance the hypothesis that abnormal glial metabolism forms part of the basis of the selective vulnerability of certain brain structures in thiamine deficiency. Glutamate and GABA concentrations are selectively reduced in medulta-pons and cerebellum of TD rats and acetylcholine synthesis is reduced in medulla. In addition, there is a convincing body of evidence that pyruvate dehydrogenase, the rate-limiting enzyme in glucose oxidation by brain shows preferentially decreased activity in these regions of brain in thiamine deprivation. Possible mechanisms involved in these cholinergic and amino acid neurotransmitter abnormalities are shown in Fig. 8. Thus, dietary thiamine deprivation may lead to selective TPP decreases in pons and cerebellum giving rise to diminished activity of PDH(' and reduction in synthesis of acetylcholine, GABA and glutamate with ensuing neuronal dysfunction. In the casc of the amino acids, glial energy metabolism abnormalities could represent an alternative or additional cause of neuronal dysfunction. There are, however, certain shortcomings of such a hypothesis. For example, diminished PDHC activity would be expected to be accompanied by diminished activity of the tricarboxylic acid cycle with resultant decreased ATP produclion. However, ATP levels are found to be maintained in brain stem at a time when PDHC activity is decreased (McCandless and Cassidy. 1976). Further-

more, PDHC deficiency would fail to explain the marked changes in serotoninergic function described in thiamine-deprived rat brain. It is therefore likely that more than one mechanism is involved in the pathogenesis of neurotransmitter abnormalities and ensuing neurological dysfunction accompanying thiamine deficiency. Not only is thiamine, in the form of thiamine pyrophosphate (TPP), an essential cofactor for the pyruvate and :~-ketoglutarate complexes and for transketolase, but it has also been proposed that thiamine triphosphate ITTPI is intimately associated with the propagation of the neuronal electrical potential. TTP is released from intact nerve preparations (Cooper, Roth and Kini, 1963) and is selectively localised in nerve membranes (Barchi and Braun, 1972). Electrical stimulation and potassium depolarisation however, produced no changes in TTP uptake, synthesis or release (Berman and Fishman, 1975). Thus, although a neurophysiologically active form of thiamine may well exist, its precise role in disorders of neurotransmitter function in thiamine deficiency encephalopathy remains to be established. CONCLUSIONS The encephalopathy associated with thiamine depletion in the CNS is preceded by regionally-selective changes in neurotransmitter function suggesting a

Review causal relationship between these changes and the development of neurological signs of thiamine deficiency. A review of these neurochemical changes reveals specific differences both in nature, degree and topography of neurotransmitter function depending on the experimental regimen used to induce thiamine deficiency. For example, whereas chronic thiamine deprivation leads to amino acid changes in medullapons and cerebellum, pyrithiamine treatment (PT) is accompanied by more widespread changes. The localisation of these amino acid abnormalities reflects, to some degree, the more widespread localisation of lesions in PT rat brain. In view of these and other similar findings, it may be appropriate to caution against future extrapolation of data from one experimental situation to the other, an occurence which has led, in the past, to considerable confusion of the literature surrounding thiamine-deficiency and its effect on the CNS. Evidence for a central muscarinic cholinergic lesion has been presented in thiamine deficiency and behavioural neuropharmacological studies have shown that this lesion is partially responsible for the neurological symptoms of thiamine deficiency induced by pyrithiamine treatment. Many studies reviewed suggest that thiamine deficiency in rats interferes with cerebellar afferent and efferent systems. Thiamine deprivation appears to be associated with a loss of (serotoninergic) cerebellar afferent mossy fibres and it was suggested that this fibre loss could provide one possible explanation for the granule cell dysfunction observed in thiamine deficiency. In addition, since the granule cell neurotransmitter is probably glutamate, loss of granule cell function could explain the glutamate changes in cerebellum of TD rats. Cerebella from symptomatic PT rats contain decreased concentrations of aspartate which has been suggested to reflect the lesions of inferior olive nucleus described following pyrithiamine treatment. Previous studies have shown that the inferior olive nucleus is the major source of cerebellar climbing fibres which are reportedly aspartatergic and/or glutamatergic in nature. Both TD and PT rats are found to exhibit reproducible lesions (both neuronal and glial in nature) of the lateral vestibular nucleus (LVN). There is some evidence to suggest that the reported GABA changes in medulla pons may reflect loss of Purkinje cell nerve terminals in this nucleus. A recent study (McCandless, 1982) reported abnormalities of energy metabolism in LVN in thiamine deficiency. Additional microanalytical studies of this kind, on isolated nuclei or distinct neuronal populations would be of considerable assistance to our

461

understanding of metabolic and neurotransmitter changes in thiamine deficiency. Future studies will b e required to account for, not only the selective vulnerability of certain brain regions to a lack of thiamine, but also the observed neuronal and glial changes in these regions. Neurochemical techniques are now available using marker enzymes and the differential labeling patterns of certain metabolites in neuronal and glial 'compartments' and application of these techniques could be of substantial value in future attempts to elucidate the neurochemical mechanisms involved in thiaminedeficiency encephalopathy. Acknowledgment--Work accomplished in the author's laboratory was funded by the Medical Research Council of Canada. The author wishes to express his gratitude to Ms France Landreville and Sylvie de Bellefeuillefor their capable assistance.

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