Thiamine content and turnover rates of some rat nervous regions, using labeled thiamine as a tracer

Brain Research, 181 (1980) 36%380 ?t~ Elsevier/North-Holland Biomedical Press

369

T H I A M I N E C O N T E N T A N D T U R N O V E R RATES OF SOME RAT NERVOUS REGIONS, U S I N G L A B E L E D T H I A M I N E AS A T R A C E R

GIANGUIDO R[NDI, CESARE PATR1NI, VALERIANO COMINCIOLI and CARLO REGGIANI Institutes (~f Human Physiology and Mathematics, University of Pavia, 27100 Pavia (Italy)

(Accepted May 24th, 1979) Key words':

thiamine - - turnover - - mathematical model - - rat

SUMMARY The content of total thiamine radioactivity in some nervous structures and liver of the rat was determined in a steady state condition, using [thiazole-2-t4C]thiamine as a tracer. The contents were analyzed by a mamillary type compartmental model which enabled us to calculate the influx and efflux fractional rate constants, turnover times, turnover rates and relative accuracy. Total thiamine turnover rates of the central nervous system regions were found to be ordered in the following sequence: cerebellum (0.55 #g/g.h) > medulla and p o n s > spinal cord and hypothalamus > midbrain (plus thalamic area) and corpus striatum > cerebral cortex (0.16 #g/g.h). Sciatic nerve turnover rate was 0.58 #g/g.h. The turnover times were mainly between 5 and 10 h (range 2.4-16.4 h). The influx rate constants could be ordered as follows: cerebellum > hypothalamus, pons and medulla > corpus striatum, spinal cord, midbrain (plus thalamic area) and sciatic nerve > cerebral cortex. The results show in general a good agreement between turnover rate values and brain regional sensitivity to thiamine deficiency, the most vulnerable areas to thiamine depletion being those with the highest turnover rates.

INTRODUCTION The problem of thiamine function in the nervous system is yet unresolved 12. A useful approach could be the study of the thiamine turnover rate in nervous structures, especially those selectively affected by thiamine deficiency la. This could also contribute towards the explanation of their particular vulnerability to avitaminosisZa, a.5. Dreyfus 12 speculated that the parts of the nervous system having the highest content of TPP*

* Abbreviations used: TPP, thiamine-pyrophosphate; TCA, trichloroacetic acid.

370 tend to be affected earlier and to a greater extent than others by thiamine deficiency. However, no information is available concerning thiamine turnover rate in different nervous regions and the data available for other organs are scanty :~1,:~6,43.This fact prompted us to carry out the present investigation. The [thiazole-2-14C]thiamine turnover was evaluated in 7 rat brain areas, the spinal cord and the sciatic nerve under a steady-state condition. The liver was also included for purposes of comparison, because of its thiamine storage function. For the calculation of turnover rates, a compartmental mathematical model was developed. The parameters in the corresponding differential equations were estimated by means of a minimization procedure. A preliminary note has been published elsewhere 30 METHODS Animals and thiamine adminbtration Labeled thiamine (30 #g, corresponding to 1.25/zCi), dissolved in 0.5 ml of saline, was injected i.p. into female albino rats (Wistar strain; 230-280 g body weight), that had been starved overnight with water ad libitum. Since the amount of injected thiamine corresponded to the rat daily requirement ~, during the subsequent 24 h the rats were fed a thiamine-deficient diet, and then again a complete diet. In this way, thiamine intake and total thiamine content of the organs were kept constant (steady-state condition). Anatomical dissection The rats were sacrificed by decapitation. The brain areas (cerebral cortex, cerebellum, midbrain including the thalamic area, hypothalamus, corpus striatum, pons and medulla) were dissected according to the Glowinski and Iversen 17 procedure. The spinal cord was isolated after opening the vertebral canal, while the sciatic nerve was dissected following its course to the Achilles' tendon, after dislocation of the coxofemural articulation and sectioning of the posterior limb muscles. The lobum quadratum of the liver was removed after the opening of the abdomen. All dissections were carried out in the cold, at given time intervals ranging from 5 min to 96 h after labeled thiamine injection. In preliminary experiments the dissected brain areas had been controlled by light microscopy. Blocks of tissue were fixed in Carnoy fixative (without acetic acid), included in paraffin and serially cut. The sections (15 #m) were coloured with a simplified Nissl method. Thiamine extraction The tissues, rapidly dissected and weighed, were homogenized with 4 ml of cold 0.5 N HCI in a precooled porcelain mortar, containing washed and limed quartz sand. After centrifugation at 0 °C for 15 min at 15,000 × g, the supernatant was separated a n d deproteinized with 0.5 ml of 40 ~ TCA, while the precipitate was once again extracted and centrifuged as above. In every case, TCA-precipitated proteins were eliminated by

371 l0 rain cold centrifugation at 15,000 × g. The final volume of the extracts was brought to 10 ml with 0.5 N HC1. The whole procedure was performed at 0-5 °C.

Chemical determination of thiamine Using 1 ml of TCA extract, free and total thiamine contents were determined by a modification of the fluorometric method of Burch et al. 3, respectively before and after overnight incubation with an acid phosphatase (Takadiastase) at pH 4.5. The bianks were aliquots of TCA extract alkalinized with NaOH, without addition of potassium ferricyanide. Thiochrome produced by ferricyanide oxidation was extracted with 3 ml of freshly distilled isobutanol. Fluorescence was measured with a spectrofluorophotometer (wave lengths: 370 nm for excitation, and 430 nm for emission). Thiamine content was calculated by means of a standard curve, set up for each set of measurements using I ml of thiamine pure solutions, containing 3-13 ng of thiamine chloride hydrochloride, and treated as reported above. Phosphorylated thiamine was calculated as the difference between total and free thiamine. The reliability of the entire analytical procedure had been assayed in preliminary experiments by adding known amounts of thiamine chloride to cerebellar tissue. The mean recovery of thiamine was 98 ~ (range 91.5-106.3 ~). Separation and determination of labeled free and phosphorylated thiamine The chromatographic method of Sharma and Quaste138 was employed for the separation of labeled free and phosphorylated thiamine, using 3 ml of TCA extract brought to pH 6.5 with a 15 %oNaOH solution. Phosphorylated thiamine passed into the percolate, while free thiamine was fixed on the resin and then eluted with 0.5 N HC1. Radioactivity measurements of thiamine samples were made on 0.5 ml of percolate or eluate, placed on aluminum planchets and desiccated during 3 h exposition to an infrared lamp. The standard error of the measurements was less than 2 %: efficiency was 81 o~. From the radioactivity values, labeled thiamine contents were calculated by means of a standard curve prepared with known amounts of labeled thiamine. In this case also, the reliability of the analytical procedure had been checked in 10 preliminary experiments, by adding known amounts of labeled thiamine to cerebellar tissue which was then carried through the entire procedure. The mean recovery was 97 ~ (range 89.5 108 %). Water content This was determined by placing the tissues in an oven at 55-65 °C and then in a desiccator till constant weight was attained. Reagents and diets' All the reagents were of analytical grade and supplied by : Merck, Darmstadt, G.F.R. (quartz sand, TCA, sodium acetate, sodium hydroxide, potassium chloride, perhydrol, isobutanol) ; British Drug House Ltd., Poole, U.K. (CG-50 Amberlite resin, acetic acid, potassium ferricyanide). Takadiastase was a product of Parke Davis, Detroit, Mich., U.S.A. Unlabeled thiamine chloride hydrochloride was supplied by Pro-

372

E2 a23

a133

E3 a43

a53

a1~3

i E~ ~_ E5 I. . . . . ~E'I_~

a123

E12

Fig. I. Compartmental model quantitatively describing the distribution and disaptcearance of tabe~ed thiamine from rat tissues. El, peritoneum; E2, liver; E3, plasma and residual tissues; E ~-E12.different regions of the nervous system; Et3. accumulator; ajk, fractional rate constants.

dotti Roche, Milan, Italy, while [thJazole-2-14C]thiamine was prepared by the Radiochemical Centre, Amersham, U.K. (specific activity, 14 mCi/mmol). Both the complete and the thiamine-deficient diets were prepared by Piceioni and Co., Brescia, Italy. Instruments The fluorometric measurements were carried out with an Aminco-Bowman 4-8202 SPF spectrophotofluorometer (Silver Spring, Md., U.S.A. ). Radioactivity was measured with a Geiger-MiJller low-background gas-flow counter (Nuclear Chicago, mod. 512). The refrigerated centrifuge was an International FR-2 model (Boston, Mass.. U.S.A.) and the p H meter a Beckman, Zeromatic SS-3 (Fullerton, Calif.. U.S.A.). Mathematical model The distribution and disappearance of labeled thiamine from rat tissues were quantitatively described by means of the compartmental model represented in Fig. 1. This model included 13 compartments, 10 ot which (the liver and 9 different nervous structures) were experimentally determined, while two (Ea and Eta) were unknown, and one (El, i.e. peritoneum) was known only at the beginning of the experiment, when it contained 1250 nCi of injected labeled thiamine. The introduction of two completely unknown compartments was justified by the following considerations: (a) the Eta compartment allowed us to describe a closed system so that at each instant the sum of the activities of all the compartments was constant (equal to 1250 nCi); (b) the Ea compartment made up for the lack of experimental data on plasma thiamine. Actually. it derives from the association of two possible compartments: "plasma thiamine', a compartment which here is experimentally unknown, but is identifiable, and 'residual body thiamine'. which is unknown and unidentifiable. Assuming linear kinetics, the model corresponds to the following system of differential equations: 2)t (t) (a31 - a21) yl 3?2 (t) --: a21 yl - a23 y3 a32 y2

[1 ]

373

~?z (t) =

12

13

E azjyj--

Z airy3

j--!

j--2

(t) -- ajz yz -- a3j yj ~13 (t) =-- a 1 3 3 y3 yt (0) = 1250; yj(0) = 0 3',i

. . . . . . . . . . .

12

j -: 2, . . . . . . . . . . .

13

j =

4,5

where the parameters ajk indicate the fractional rate constants.

Estimation of model parameters In order to estimate the numerical value of parameters a = (ajk), the following cost function was minimized

NeNj[ 1

Z F (a) -= Z j=l i:l

Yji (a)

yjt

~rji

where N e is the number of experimental curves, Nj is the number of experimental data (yji) for the jtn curve, yji (a) are the solutions of equations [1] for a fixed parameter vector a, and aj~ are the standard deviations related to the experimental data (yj~). For the minimization of F(a) a restricted step hybrid algorithm combining the LevenbergMarquardt and steepest descent methods was chosen TM. The correction of the parameters on each iteration was restricted by requiring it to be the Levenberg-Marquardt step, the descent step or a step interpolating both. The system [1] for a fixed set of parameters (a) was numerically solved by means of a variable order-variable step formulation of the Adams predictor-corrector method (see ref. 37). The dynamic change of the order and step allowed a remarkable gain in computation time. In order to evaluate the accuracy of the parameters identified, which are the components of the vector a* corresponding to the minimum of F(a), and approximation of the variance-covariance matrix Va of the estimates was computed through the solution of the sensitivity system obtained by linearization and related to the system [1 ]2,33. Given the Va matrix of the estimates, we were able to determine which parameters, or linear combination of parameters, were well determined (small variance) and which were poorly determined (large variance). The standard deviation of a single component of the vector parameters a* was given by the square root of the corresponding main diagonal element of V~*. Table II reports the estimated parameters aij and the corresponding standard deviations. More complete statistical information can be obtained by carrying out the eigenvalue decomposition of Va*. Of this analysis, we report here only the square root of the largest eigenvalue of the variance-covariance matrix V,~*, which gives the largest elongation of the hyperellipsoid related to the matrix. This value was found to be 0.253, which was also the standard deviation of the parameter azl (the estimated value of this parameter was 4.913). However, we observed the same situation for the other eigen-

374 values, e a c h o f t h e m b e i n g in p r a c t i c e t h e s t a n d a r d d e v i a t i o n o f a g i v e n p a r a m e t e r : T h e s e results i n d i c a t e t h a t a* = (ajk*) was well d e t e r m i n e d in all d i r e c t i o n s . T h e p a r a meters a*

(a.ik*) c a n be i n t e r p r e t e d as f r a c t i o n a l t u r n o v e r c o n s t a n t s a n d can be

t r a n s f o r m e d i n t o t u r n o v e r rates by m u l t i p l y i n g t h e m by t h e t h i a m i n e c o n c e n t r a t i o n o f t h e r e g i o n 28. RESULTS Normal thiamine content

T o t a l (free -

p h o s p h o r y l a t e d ) t h i a m i n e c o n t e n t s o f tissues f r o m rats r e a r e d on a

c o m p l e t e diet are g i v e n in T a b l e I. T o t a l t h i a m i n e m a i n l y reflected p h o s p h o r y l a t e d t h i a m i n e c o n t e n t , since free t h i a m i n e was o n l y a s m a l l p e r c e n t ( 5 - 7 ~ ) o f t o t a l t h i a m i n e (see ref. 29). A m o n g t h e n e r v o u s structures, t h e c e r e b e l l u m h a d the h i g h e s t t o t a l thiam i n e c o n t e n t a n d t h e sciatic n e r v e t h e lowest. Histological evaluation

T h e light m i c r o s c o p i c e x a m i n a t i o n o f t h e d i s s e c t e d brain r e g i o n s s h o w e d the t y p i c a l tissue o r g a n i z a t i o n o f t h e r e s p e c t i v e areas. Radioactivity distribution and time course

T h e t i m e c o u r s e s o f l a b e l e d t o t a l t h i a m i n e c o n t e n t in t h e b o d y r e g i o n s s t u d i e d a r e r e p o r t e d in Fig. 2. T h e t h e o r e t i c a l curves, p r e d i c t e d by t h e m a t h e m a t i c a l m o d e l , v e r y closely f o l l o w e d t h e e x p e r i m e n t a l curves, t h u s d e m o n s t r a t i n g t h e reliability o f o u r m o d e l . T h e a m p l i t u d e of t h e m a x i m a l p e a k o f t o t a l r a d i o a c t i v i t y was d i f f e r e n t f o r t h e single b r a i n areas, b e i n g g r e a t e s t f o r t h e c e r e b e l l u m . I n s o m e n e r v o u s s t r u c t u r e s (sciatic n e r v e , m e d u l l a a n d spinal c o r d ) t h e p e a k was r e a c h e d in a r e l a t i v e l y s h o r t t i m e ( 9 - t 6 h ) ; TABLE l Weight, water and free, phosphorylated and total thiamine contents o f liver and different region,sol rat nervous system. Means o f at least 25 samples ~: S.E.

All the differences in total thiamine content between the cerebellum and the other nervous structures are statistically significant (P _< 0.001 ,Student's t-test). Region

Cerebellum Corpus striatum Pons Midbrain (plus thalamicarea) Medulla Hypothalamus Cerebral cortex Spinal cord Sciatic nerve Liver

Weight, g

Water content, Content ( Itglg w.t.) o f thiamine Free

Phosphorylated Total

0.23 t 0.011 0.096 ± 0.004 0.070 ± 0.003

79.58 ::: 0.41 75.74 T 0.52 72.55 _~_0.59

0.14 :r_ 0.005 0.12 • 0.007 0.12 • 0.008

4.24 ~ 0.07 3.20 ~ 0.15 3.15 ~ 0.1]

4.38 z 0.07 3.32 i: 0.16 3.27 F 0.11

0.150 0.178 0.075 0.76 0.20 0.11 9.07

75.84 :z 72.59 z: 78.12 ± 79.14 :z 68.73:1_ 67.51 ± 69.65 ±

0.12 0.12 0.12 0.12 0.11 0.06 0.29

2.98 2.78 2.74 2.50 1.96 1.39 8.78

3.10 2.90 2.86 2.62 2.07 1.41 9.07

~: 0.006 z: 0.010 :i: 0.003 ~ 0.07 z: 0.014 ~ 0.006 ~ 0.36

0.18 0.25 0.99 0.17 0.19 0.46 0.22

~: 0.006 ~_ 0.007 ~_ 0.008 z 0.006 -3:0.007 ± 0.004 _~ 0.04

i 0.11 :z: 0.15 ~ 0.12 ~ 0.14 ± 0.10 5_ 0.05 ~ 0.74

i 0.11 ~: 0.15 r_ 0.12 ....... ± 0.10 _+ 0.02 :-.- 1376

375

in others (pons, cerebellum, hypothalamus, midbrain plus thalamic area, corpus striaturn and cerebellar cortex), it was reached much more slowly (20-37 h). In the liver the radioactivity peak was much higher (44 nCi/g) and much more rapidly attained (30 min) than in the nervous structures (Fig. 2). F r a c t i o n a l rate c o n s t a n t s

The mathematical model enabled us to calculate two fractional rate constants for each region: one for labeled thiamine entry (influx), and the other for thianaine disnCi/g

nCL/g tO

~

~o~ -~ ....

LEGEND EXPERIMENTAL 1 CALCULATED J

15"

0

'=()

20

10

48 96 0

L~

48 96

h

CORPUS STRIATUM

CEREBRAL CORTEX

10.

/ ~

0

~o

MIOBRAIN

~

~96h

10

20

't20

2,~0 h

CEREBELLUM

HYPOTHALAMUS

40 L

3O .,I---,

o

b

2b

4896 o

PONS

•~

~

4896 h

MEDULLA

'°l 10 20 SPINAL CORD

48 96

10 20 SCIATIC NERVE

4 8 9(5 h

L

0

-i6

2o

1~ ~4o h

LIVER

Fig. 2. Time course of total thiamine radioactivity in different brain regions and liver. Experimental values (open circles) and calculated values (filled circles) are reported. Total thiamine radioactivity, expressed in nCi/g of wet tissue (ordinate), and time in hours (abscissa) are plotted in each diagram. Every experimental value is the average of 4 determinations, each carried out on a pool of one (cerebral cortex) up to 10 (pons, hypothalamus and corpus striatum) samples of tissue.

376 TABLE 11 E s t i m a t e d i n f l u x a n d e f f l u x f r a c t i o n a l ra ~.onstants ( F R C ) ~ S . D . . turnover times " K) and turnover rates o f total t h i a m i n e in different regions o / ' r a t nervous s y s t e m a n d liver

K*

1 iinflux FRC). For parameters see differential equations system [1] and Fig. I

Model eompartments

Regions

Ea

Peritoneum

E2 E4 E5 E6 E7 E8 E,~ Elo EH

Liver Cerebellum Pons Cerebral cortex Medulla Hypothalamus Corpus striatum Spinal cord Midbrain (plus thalamic areal Sciatic nerve Accumulator

E ~2 E~:~

F r a c t i o n a l rate constants Turnover rates . . . . . . . . . . . . . . . . . . . . . . . . Parain[lux.h: l ' l O -a Para- e f f t u x , h ~ K*,h ,tg/g'h meters meters

a2~ a ,3 a~3 a63 a73 a83 a~a alo:~

1.07 J:O.15 1.79=L0.03 0.91 +0.05 0.36~0.02 0.90:z0.04 1.07-I_0.03 0.68 ~ 0.03 0.51 ~r0.03

all3 a~2:~ al~a

0.49 ~-0.02 0.48!0.05 t0.84-f 0.32

a,,i a:~1 aa2 a:34 a:~ aa,~ a:~7 aa8 a3.~ a:~lo

0.172LO.O07 4.914r0.253 0.105i0.010 0.126mO.O05 0.139±0.013 0.061-2:0.006 0.187m0.013 0.125=0.006 0.081~-0.005 0,188~0.015

°o o f the cerebellum value

9.52 7.93 7.19 16.39 5.32 8.00 12.34 5.32

0.952 0.55l 0.454 0.159 0.543 0.357 0.268 0.389

172 100 82 29 98 65 48 70

a:m 0.695=i=0.005 10.52 a:~12 0.413= 0.048 2 . 4 2

0.294 0.582

53 t05

appearance (efflux) (Table I1). These constants have the physical dimensions of h -1 a n d are related to the probability that one particle of labeled t h i a m i n e has o n e n t e r i n g or leaving a given structure in a u n i t of time. The rate (/~g/g.h) of t h i a m i n e influx or efflux was o b t a i n e d by multiplying the fractional rate constants by the t h i a m i n e c o n c e n t r a t i o n of a given region. U n f o r t u n a t e l y the influx rate could n o t be calculated, since the t h i a m i n e c o n t e n t of the E a c o m p a r t m e n t (Fig. 1) was u n k n o w n . However. a c o m p a r i s o n of the influx rate constants of the different regions can be made. T h u s the c e r e b e l l u m e x h i b i t e d the highest fractional influx rate constant, similar to that of the liver, while the cerebral cortex showed the lowest rate (Table I11. Efflux l ates, however, could be calculated, since both fractional rate constants a n d t h i a m i n e regional c o n c e n t r a t i o n s were k n o w n . The efflux rates are true turnover rates of thiamine, expressing the a m o u n t ¢,/zg/g.h) of t h i a m i n e which disappeared (either metabolized or intact) from a given region. The cerebellum was the brain area with the highest t h i a m i n e t u r n o v e r rate, followed by the m e d u l l a a n d the pons (respectively 98 a n d 82 ~ of the rate of the cerebellum). The cerebral cortex h a d the lowest t h i a m i n e t u r n o v e r rate, while the liver a n d sciatic nerve had a higher value t h a n the cerebellum (Table IlL Finally, the t u r n o v e r times, representing the time required for the complete renewal of t h i a m i n e in a given region zT, ranged from 2.4 to 16.4 h, b u t u s u a l l y falling between 5 a n d 10 h (Table 1I).

377 DISCUSSION The total thiamine, or thiamine pyrophosphate (TPP), contents we found in nervous structures were similar to those reported by previous authors 11,'~a,aS,4°. The small differences derived from the different dissection procedures used. We found the cerebellum to be the brain area richest in thiamine, the differences with the othel regions being statistically significant and independent of their water content (Table I). Labeled thiamine entered most easily into the cerebellum and least easily into the cerebral cortex. The fractional influx rate constants can be ordered in the following sequence: cerebellum > liver, hypothalamus, pons and medulla > corpus striatum > spinal cord, midbrain (plus thalamic area) and sciatic nerve > cerebral cortex. No apparent relationship can be found between fractional rate constants and regional hematic flows a4 or glucose a9 and 0215 utilization. Rather, influx constants appear to be related to number of cells to be found in the region. The cerebellum has the highest cellular density 21 and accordingly, the highest influx constant. The radioactivity peak amplitudes are apparently unrelated to total thiamine content (Table 1 and Fig. 2). This could mean that the total thiamine content of a given nervous structure does not depend only on thiamine efflux and influx rates, but also on some other factors such as different ratios of neuronal to glial cells, differing metabolic activity or ability to phosphorylate thiamine, etc. Histochemical studies have shown that thiamine tends to localize in neuronal cells, especially Purkinje cells tg, in higher concentrations than in glial cells or fibers 6. Orally administered labeled thiamine is taken up more quickly by neurons than by the glia or fibers, while in the cerebellum, Purkinje cells apparently accumulate much more thiamine than do granular cellsa. Liver apart, the investigated structures may be ordered with respect to their thiamine turnover rates in the following manner: cerebellum and sciatic nerve > medulla and p o n s > spinal cord and hypothalamus > midbrain (plus thalamic area) and corpus striatum > cerebral cortex. Thus the cerebellum was the cerebral region having the highest thiamine turnover rate and the cerebral cortex the lowest; the medulla and pons did not differ greatly from the cerebellum. These findings are in good agreement with the sensitivity of the different cerebral areas to thiamine-deficiency. In the cerebellum 11,e3 and ports 2a total thiamine depletion, caused by dietary thiamine deficiency, is more marked than in the cerebral cortexll, 2a and midbrain 2a. In rodents, the residual content of total thiamine~*, za or TPP a5 is higher in the cerebral cortex than in other structures during avitaminosis. Brain areas had turnover times ranging mainly from 2 to 12 h, but that of the cerebral cortex was the highest (16.4 h). Considering that total thiamine is the sum of free thiamine and thiamine mono- and pyrophosphate, which have very different halflives al (i.e. free thiamine 20 min; thiamine monophosphate 160 min, and thiamine pyrophosphate 8 h), the present value of liver total thiamine lurnover time (9.5 h) can be considered to confirm the value found previously by Rindi et al. a'. The sciatic nerve, with the lowest total thiamine content, had a very high thiamine turnover rate, comparable to that of the cerebellum, and a very low turnover time. This might be related to the rapid neural transport of thiamine in the rat sciatic nerve2a,40, 41.

378 On the whole, our results point out the peculiarity o f the cerebell am in corn parison to other cerebral areas as far as thiamine metabolism is concerned. This region exhibited the highest total thiamine content, influx fractional rate constant and turnover rate. although it did not have the highest hematic flow or glucose and Oz consumptions. Further, on a weight basis, the cerebellum showed a higher thiamine ~avidit~" (influx rate constant) than the liver. On the contrarv, the cerebral cortex has a thiamine content which is 60 ~/o o f that o f the cerebellum and a thiamine influx rate constant and turnover rate respectively corresponding to 20 and 29 o / o f the cerebellar ones. while its hematic flow, glucose and Oz consumptions are all higher than those of the cerebellum Ls. 34,z9. Moreover. the total thiamine or T P P content o f the cerebral cortex is lowered less by thiamine deficiency than that of the cerebellum 1 t,.,3,~. These features of the cerebellum as regards thiamine metabolism could be merely a consequence o f its very high cellular density (3-5 times higher than that of the other regions studiedl zl However. there are other considerations. As can be inferred from histological findingsS,~, 19 and f r o m the relative paucity o f glial cells 16, most cerebellar thiamine is probably located in the neurons. It is well k n o w n that glial cells have a low metabolic activity, respiring 2-10 times less than the mean neuron10, t5 and therefore probably a low thiamine content. Actually the total thiamine content of the rat corpus catlosum, a structure colaraining mainly glial cells, was 2 :rz 0.24 #g/g (mean of 4 determinations, each on a pool o f 10 samples) (Patrini and Rindi, unpublished data ~. Thus the cerebellar neuron, especially the Purkinje cell. should be particularly rich in total thiamine. Unfortunately no data is available in the literature on this subject. Since the mean cerebellar neuron has a metabolic activity much lower than the mean cerebral cortex neuron~4, tS. while it admittedly has a high total thiamine content. one could speculate that in the cerebellum thiamine function might not be limited to tissue metabolism only. Recently, neurochemical studies have shown that thiamine deficiency selectively impairs serotonin uptake by cerebellar synaptosoma[ preparations 24. and that the seroto,ainergic mossy fibers are particularly affected 4. Interestingly thiamine deficiency produces specific histologic lesions in the rat cerebellum z6. and the cerebellar glial cells have been considered by Collins and Converse :~ to be the site of primary avitaminic alterations in the nervous system. In general, the values of the turnover rates f o u n d in this study reflected the sensitivity o f brain areas to thiamine deficiency. The pons and medulla, which have high total thiamine contents and turnover rates, not far f r o m those of the cerebellum, are particularly vulnerable to thiamine deficiency. Thiamine depletion causes the greatest decrease o f total thiamine or T P P content in the pons zz. in the vestibular nuclei;~ as well as in the cerebellum ~ Thiamine deficiency in the rat induces its earliest and most characteristic histologic lesions in the vestibular nuclei and in the brain stem structures. These lesions are variously described in the literature as affecting the glial cells7, s.z°.3z, neuronal processes 42 or the presynaptic knobs and axons"L ACKNOWLEDGEMENTS The present research was partially supported by a contribution (no. 7200863.04) of C.N.R., Rome.

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