Relationships between chemical changes and histological damage in the brains of scrapie affected mice

J. COMP.

PATH.

1967.

VOL.

77.

RELATIONSHIPS

393

BETWEEN

HISTOLOGICAL

CHEMICAL

DAMAGE SCRAPIE

IN

CHANGES

THE

AFFECTED

BRAINS

AND OF

MICE

BY

D. L. MOULD,

A. McL.

DAWSON,

J. S.

Moredun

Research Institute,

Gilmerton,

SLATER

and I.

ZLOTNIK*

Edinburgh

INTRODUCTION

Several workers have measured enzyme activities and levels of constituents in tissue or body fluids from animals affected with clinical scrapie, and certain differences have been observed when comparing the data with those from controls (Field, C&par-y and Windsor, 1966; Kasting and Darcel, 1963 ; Millson, 1965 ; Hunter and Millson, 1966; Slater, 1965a, b). Millson and Hunter have correlated an increase in the activities of brain &glucuronidase, deoxyribonuclease, and N-acetylglucosarninidase with development of the disease after killing groups of mice at fixed intervals following inoculation with scrapie agent. In preliminary work on cerebral proteinase activities we observed that in single mice killed six months after inoculation, not only did the enzyme activities show a wider spread than normal, but the extent of pathological damage to the brain also varied considerably. An alternative and more refined method of referring biochemical changes to progress of the disease thus became necessary. It was considered that a significant correlation of biochemical data with histological findings might lead to methods for differential diagnosis, assessmentof brain damage and some indication as to the possible mechanism of the progressive degeneration of brain tissue characteristic of scrapie. A method has therefore been devised of scoring mice for the severity of brain damage, based on the extent and intensity of brain lesions. The scores for individual mice have been set against certain physical and chemical measurements with a good measure of correlation in some cases. The type of mouse scrapie investigated was that arising in serial passagethrough mice of a strain of scrapie derived from a natural case of scrapie in a Suffolk sheep (Zlotnik and Rennie, 1962, 1963). The data reported here on brain weight, density, total dry matter, protein content, sodium and potassium levels and acid proteinase activity are concerned mainly with the possible variations in ion and water balance in scrapie-affected brains, and with the loss of material during the degenerative phase of the disease. MATERIALS

AND

METHODS

Random bred Swiss white mice from the Moredun stock colony were inoculated at 3 to 4 weeks of age with the ME7 mouse-passaged strain of scrapie (Zlotnik and Rennie, 1963). Each mouse received 0.03 ml. of a 10 per cent. (w/v) homogenate of brain tissue from scrapie affected mice. With the inoculated groups Mice.

* Present D

address:

Microbiological

Research

Establishment,

Porton,

Sal&bury,

Wilts.

394

CHANGES

IN

BRAIN

OF

SCRAPIE

MICE

twenty uninoculated mice of the same age were set aside as controls and kept under identical conditions. During the course of the experiment each mouse was weighed weekly and the clinical condition at the time of killing by decapitation was recorded. After removal of the brain the spinal cord was trimmed back to the obex for uniformity. Half brains from every mouse were examined histopathologically and graded for severity of scrapie lesions according to the scheme described in the section on histology. Examination of inoculated mice. Four groups of mice were inoculated and their brains examined as follows. Group A (95 mice) was inoculated intracerebrally with ME7/5th passage material and killed 166 to 188 days after inoculation. Estimations were made of total brain weight, dry matter, density and protein content. Group B (69 mice) was inoculated intracerebrally with ME7/5th passage material and killed 158 days after inoculation. Estimations of sodium, potassium and proteincontent were made. Group C (52 mice) was inoculated intracerebrally with ME7/3rd passage material and killed 164 to 186 days after inoculation. Protein and a-amino-nitrogen, and acid proteinase activity were determined. Group D (35 mice) was inoculated intraperitoneally with ME7/5th passage material and killed 188 to 233 days after inoculation. Estimations of protein and a-amino-nitrogen contents, and acid proteinase activity were made. Brain weighi-, density and dry matter. The brain, suspended on a wire hook, was weighed in air and in physiological saline of known density. Excess saline was removed by draining on the convex side of a watch glass. This procedure could be performed reproducibly, as shown by repeated dipping and draining, with weight changes in air of less than 1 per cent. The brain was then bisected, one half fixed in 10 per cent. form01 saline and the other half weighed and homogenized in water in a perspex and glass homogenizer (Aldridge, Emery and Street, 1960) to provide a 10 per cent. (w/v) suspension. Weighed aliquots (about 1 g.) of the homogenate were dried to constant weight in a convection oven at 105OC. (McIlwain and Rodnight, 1962) to give a figure for dry matter content. Protein. Weighed aliquots of the brain homogenates (about 0.5 g.) were mixed with 0.5 ml. of 10 per cent. trichloroacetic acid (TCA), cooled in crushed ice for 10 min. and centrifuged. The pellet was washed twice with 5 per cent. TCA, the supernatants discarded and the final pellet dissolved in 2N NaOH. Nitrogen in this solution was determined by a micro-Kjeldahl technique and protein defined as (N x 6.25). Sodium and potassium. Half brains were homogenized in glass distilled water to provide a 10 per cent. (w/v) suspension. To 1 ml. of homogenate in a conical centrifuge tube was added 1 ml. of 20 per cent. TCA. The tubes were mixed, covered and left overnight at room temperature. The suspension was centrifuged and sodium and potassium in the protein-free supernatants determined using an EEL flame photometer. Acid proteinare activity. Proteinase activity at pH 3.8 was assayed in homogenates of half brains using bovine haemoglobin (Armour) as substrate, according to the method of Marks and Lajtha (1963). The standard amino-acid was leucine and results are given as pmoles of amino group released,/g. of protein/hr., corrected for hydrolysis of endogenous protein. Free amino groups. Aliquots from half brain homogenates were extracted with an equal volume of 10 per cent. TCA at OOC. After removal of the precipitate by centrifugation, ninhydrin positive material in the supernatants was measured according to .the method of Stein and Moore (1954), using leucine as standard. The approximate value for total free ammo groups thus obtained was used for comparative purposes within groups C and D. Histology. Half brains remained fixed in 10 per cent. formol-saline for about 2 weeks pnor to histological examination. Each half brain was then divided by

D.

L.

M~ULD

et al.

395

sagittal sections into 5 blocks at the following levels : medulla oblongata and posterior cerebellum; pons and anterior cerebellum; mesencephalon and occipital cerebral lobes; diencephalon (thalamus and hypothalamus), hippocampus, lateral and entorhinal cortex; fornix, paraterminal body, corpus striatum and the anterior cortex. After dehydration the tissue was embedded in paraffin wax and all the 5 pieces of each brain were blocked together in a single block. Serial sections 7 p in thickness were cut and stained with haematoxylin and eosin, so that each slide contained transverse sections from the five brain regions. RESULTS

Histology Evaluation of brain damage. The evaluation was based on the degree of neuronal vacuolation and spongy degeneration. It was assumed that since the vacuoles became more frequent and the spongy degeneration more widespread with the progress of the disease process, these lesions represented a true picture of the degree of brain damage which could be evaluated and classified from histological sections. In the actual estimation two factors were considered-degree of brain damage and distribution of the lesions throughout the brain. Five degrees of brain vacuolation and degeneration were distinguished by the following types of lesion : I, few vacuoles of variable size in the brain substance but not within the neurones, unevenly distributed and present both in the grey and white matter. This type of change is not characteristic of scrapie, since it can be found frequently in old mice, especially over one year of age (Fig. 1). II, few vacuoles inside the neurones and scattered small vacuolations in the cxtraneuronal grey matter (Figs. 2 and 7). III, numerous vacuoles in the grey matter, evenly distributed but not confluent (Figs. 3 and 4). IV, severe vacuolation in the grey matter but only partly confluent (Figs. 4 and 5). V, very severe vacuolation in the grey matter, resulting in a complete network (Figs. 6 and 8). Distribution of brain lesions. In order to evaluate the distribution, the type of lesion in the following nine separate regions of the brain (1) medulla and Irons, (2) cerebellum, (3) mesencephalon, (4) diencephalon (thalamus and hypothalamus), (5) corpus striatum (caudate and lentiform nuclei), (6) paraterminal body, (7) hippocampus, (8) anterior half of cerebral cortex and (9) posterior half of cerebral cortex was estimated. The damage was evaluated and summated as a definitive numerical scheme of grading according to Table 1. As type I lesions alone were not characteristic of scrapie such a distribution was graded as “0-“. The presence of type II lesions in one to four regions of the brain was considered to be the minimum for diagnosis of scrapie and graded as “1 +“. When type II lesions were found in four to nine regions the grading was increased to “2 + “. If type III lesions appeared in one single region classification depended on whether type II was present in up to four regions (“2 + “) or in five to nine regions (“3 +“). The grading was then increased numerically as the number of regions in which type III occurred increased. Similarly the grading was further extended with the appearance of types IV and V in up

396

CHANGES

IN

BRAIN

OF

TABLE GRADING

Tyjc of brain damage

Number of regions aflected

I

o-9

II

SCBAPIE

MICE

1

OF LESIONS

IN THE

Tyje of brain in remaining

BRAIN

damage regions

Number of affected regions

-

o-9 o-4

1 1

o-4 5-a o-4 5-7 o-4 5-6 o-4

3”: 4+ 4+ 5+ 5+

094 o-3

:: 7+

1:: lo+ 11+ 11+

-111

o-4 5-a l-a o-7 l-7 O-6 l-6 o-5 l-5 o-4 l-4 o-3

II-III IV II-III IV II -111 IV II -111 IV II-III IV II-IV

o-a l-a o-7 l-7 O-6 l-6 o-5 1-5 o-4 l-4 o-3

: z 4 659

II

IV :

1:: 1:: 1:: II III 1:: II

6!9 V : 2’ i 4 4 5 6:9 ranging

from

“I+”

O-

l-4 5-9

III

Results

Grading

to “20+”

are

diagnostic

TABLE DISTRIBUTION

OF HISTOLOGICAL

2+

g+

12+ 12+ 13+ 14+ 14+ 15+ 15+

16+ 16+ 17+ 17+ ia+ la+ 19+

20+

for scrapie.

2 GRADING

OF LESIONS

Group Grade 0

Histological grading o-2

A 16*

t 15

3-7 a-14 15-20

29 26 24

:z

95

69

Totals * No.

of mice

in each

group.

c ::

a

3

208

:: 14

i 0

52

35

D.

L.

MOULD

et

397

U.k

to nine regions when interpretation involved the additional presence in the remaining regions of two or three less advanced types of lesions respectively. The maximum damage found in the brain was type V in six to nine regions and types II to IV in the remainder (“20 +“). Thus each brain was examined histologically, the type and spread of lesions noted and the grade of brain damage estimated over a range from “0” to “20”. To simplify the evaluation of the data the brains from the four groups of inoculated mice were divided into sub-groups designated 0, 3, 8 and 15 as shown in Table 2. These corresponded to histological scores of 0 to 2 (minimal lesions), 3 to 7, 8 to 14, and 15 to 20 (very severe lesions). The numbers of mice falling within the sub-groups are shown in Table 2. Fig. 9.

qo-

p0-

u’

$bOz c 50I :‘I,-

B c 30: Y 2 *o-

Distribution percentages

of histological gradings at each grading.

within

Groups

A and B, expressed

as cumulative

All inoculated groups showed variations in the extent to which mice were affected by scrapie. Groups A and B received the same inoculum, but were killed at periods after inoculation of 166 to 188 days and 158 days respectively. The distribution of histopathological grading for the two groups plotted as a cumulative percentage is shown in Fig. 9. The progressive development of the lesions with time is clearly shown by the displacement towards a higher grading of the curve A relative to the B curve. Curve B also illustrates the extent of variation of tissue damage present in the brains of a group of mice all killed at the same time after inoculation. Both curves show a development of damage in five steps. The histological grading scheme was based on five degrees of observable damage and the appearance of the curves may be a reflection of the grading technique. Alternatively, since both groups can be regarded as a randomised distribution

398

CHANGES

IN

BRAIN

OF

SCRAPIE

MICE

of affected mice, there is a strong suggestion that the spread of tissue damage proceeded by successive outbursts of activity. In observations of the clinical condition of mice at an early stage before the onset of severe clinical signs (150 days) it is not uncommon for apparent signs to regress over a period of 10 to 14 days before a further deterioration takes place. Group C, killed at 164 to 186 days after inoculation, received an earlier passage of the ME7 strain of scrapie and appeared to have progressed more rapidly than groups A and B. Group D, which was inoculated intraperitoneally, had only just entered the degenerative phase of the disease as signified by the low histological scores. Mice inoculated by this route usually exhibit lesions comparable to those in intracerebrally inoculated mice about 60 days later. The animals in group D should, therefore, have been kept rather longer before killing. Body Weight The clinical signs in mouse scrapie follow a descriptive pattern (Dickinson and Mackay, 1964), but are subjective and individual observers tend to place significance on different signs. No attention was paid here to clinical signs except in cases where the animal was obviously sick. As an objective measurement alI the mice were weighed once a week and the rate of change in body weight used as a criterion of condition. The various quantitative estimations involving group A were such that only six mice could be killed each day. The mice were, therefore, removed over a period of 166 to 188 days. This should not affect the assessment of the results since the histological grading was taken throughout as the fixed parameter. Mean changes in body weight are listed in Table 3. The changes are expressed both as weight gain (g./wk.) averaged over the last three weeks before slaughter and also as a percentage of the mean body weight during this period. The mice taken from the random-bred stock colony varied considerably in size as shown by the large standard deviations of the mean. Some of the affected mice deteriorated rapidly whilst others exhibited little or no change (Dickinson and Mackay, 1964). N evertheless mean changes in growth rate over the groups of approximately 20 animals showed significant differences. There was a high significance (P < O-01) in growth rates between grade 0 and all the others in both groups A and B. In group A differences were significant (P = O-05) between grades 3 and 8 and (P < 0.0 1) between grades 8 and 15. In group B differences were not significant between grades 3 and 8, but significant (P < O-01) between 3 and 15, and (P = 0.05) between grades 8 and 15. There is, therefore, some correlation between the histological grading and clinical condition of the animals. The reduced response in group B is presumably related to the shorter period of the experiment. It is of interest to mention that in group A no definite clinical signs were evident in any mice with a histological grading between 0 and 3. Brain Weight The wet weights of brains removed from mice in groups A and B are combined together in Table 4. A progressive decrease in brain weight with increase in histological grading is apparent. The significance of the difference between grades

grading

24

:z 24

16

No. of mice

+0.50

-2.84

* Groups

A and B combined.

8-14 3-7 15-20

ii 15

Inoculated

o-2

Histological grading

0 Uninoculated

Grade

Brain wt. * (w.)

IN CONDITION

400 ki 40 420 31 (47) 395 + 30 (32) (46)

432 +f 28 (40) 434 36(37)

VARIATION

+

zk 1.13

k 0.52 1.78 + 1.70 i zt 1.24

g./wk.

TABLE WITH

3

OF BRAIN

+1.52

$0.67 -4.34 -8.87 -9.53 i

+ -1 + i

DEGREE

4

zt zk 0.0016 i 0.0019 0.0020 i 0.0019 k + 0.0018

1.0455 1.0431 I.0443 1.0440 1.0444 1.0440

(92)

(29) (16) (27) (23)

(20)

OF DAMAGE

3.11

1.38 7.91 6.30 5.76

Density of brain (6 ml.)

WITH

TABLE

OF DAMAGE

Per cent. t

DEGREE

over same period.

GroujJ -4

OF GROWTH

+0.34 -2.59 -1.43

IN RATE

* change in body wt. averaged over last 3 weeks before slaughter. t rate of change in body wt. as percentage of average body weight

15-20

15

Uninoculated

o-2 3-7 8-14

Histological

0 ii

Grade

VARIATION

TISSUE

23.48

23.51 23.45 23.43

23.31 23.44

(92)

0.41 (28) (16) 0.63 (24) 0.50 (23)

0.52 0.46 (20)

i 0.50

i zk h + +

Total dry matter (per cd.)

T O BRAIN

22

+0.25 -0.60 -0.99 -2.01

TISSUE

flo. of mice

T O BRAIN

+ + zt zt

0.60 0.76 1.08 1.25

g.lwk.

Groq

B

rt i f rt

1.73 240 2.95 3.94

124.0

121.8 124.3 124.0 123.7 123.5

6.0 9.7 7.5 7.1 7.7

(35) (46) (39) (31)

(40)

+ 7.8 (155)

f + ii f

Brain protein (mg./g. wet time)

+0.69 -1.82 -2.84 -6.82

Per cent.

R ?

ch

400

CHANGES

IN

BRAIN

OF

SCRAPIE

MICE

0 and 3 is slight (P = O*l), but high between grades 0 and 8, and 0 and 15 (P < 0.01). The data confirmed previous observations where the mean brain weight of normal uninoculated mice was found to be 433 + 33 mg. (87) and the corresponding value from mice exhibiting very definite clinical signs of scrapie was 401 ?I 40 mg. (85) (Mould and Slater, 1964). Brain Tissue Density. There is a very slight decrease (P < 0.01) in the mean value of density for all inoculated animals compared with the mean value for uninoculated controls (Table 4). The decrease is more pronounced in the groups of low histological grading, with significant differences (P < O-01) between the uninoculated group and grade 0 and (P = 0.01) between the uninoculated group and grade 3. Total dry matter. No alteration other than a barely significant (0.2 > P>O*l) slight increase in total dry matter was observed (Table 4). Protein content. The protein content throughout all the histological grades remained constant with a weakly significant (P = 0.1) tendency towards a 2 per cent. increase over the content of brains from uninoculated animals (Table 4). TABLE 5 VARIATION

OF SODIUM

Grade

AND

POTASSIUM

Histological grading

Uninoculated

CONTENT

OF BRAINS

lWTH

Potassium (mg./g. wet tissue)

DEGREE

OF DAMAGE

(mg./g.

T O BRAIN

TISSUE

Sodium wet time)

4.37 ztO.16

(20)

1.38IkO.11

(19)

0

o-2

4.10 kO.34

(22)

1.36kO.19

(22)

3

3-7

4.21 ztO.39

(19)

1.35zto.10

(18)

8

8-14

3.94 kO.23

(20)

1.37 r0.16

(20)

15

15-20

4.12 ztO.12

(8)

1.36ztO.11

(8)

Sodium and potassium content. The sodium content of the scrapie affected brains remained constant and unchanged from the value in the uninoculated group (Table 5). There was a 5 per cent. decrease in potassium content significant (P
D.

L.

MOULD

et

401

d.

grades previously considered are listed in Table 6. The high standard error of the mean value in grades 0 and 3 was caused by the change in activity over these regions. The standard error of the mean value for the uninoculated group was lower than that for the scrapie affected brains. With an increasing extent and degree of damage (10 to 20) in the brain, erratic values are probably to be expected. Fig.

10.

900cwArc4 wbx 800

-

700

-

000

-

500

-

0

I

inoculated

mice

z i

0

0

I

2

3

4

S

6

7

8

HISTOLoGIC*L

Acid proteinase activity expressed as pmoles tein, using bovine haemoglobin as substate. n - control group TABLE VARIATION

OF ACID

PROTEINASE

Grade

Histological grading

Uninoculated

a-Amino

ACTIVITY

AND

a-AMINO T O BRAIN

9

IO

II

12

14

IS

lb

17

amino-group released/hr./g. 0 - group C

I8

I9

20

brain pro0 - group D

6 ACID CONTENT TISSUE

Proteinase activity (pmoles c( -NH,/hr./g. protein)

313*59

I3

GRADING

(15)

OF BRAINS

WITH

DEGREE

OF DAMAGE

Total 0: amino groups pmoles leuck/g. wet wt.

53.1 ~~5.2

(24)

0

o-2

317 f 140 (22)

47.0 G.2

(26)

3

3-7

504 zk 127 (18)

50.2 +I.3

(18)

8

8-14

468 f 114 (24)

52.0 + 3.5 (24)

15

15-20

463*107

53.3 zk4.6

(14)

(14)

Nitrogen

The method used provided only an approximate guide to the quantities of free amino-acids present in brain. The results were intended to be comparative only and show no gross differences in the amounts of total free amino-acids between brains from inoculated and control mice (Table 6).

402

CHANGES

IN

BRAIN

OF

SCRAPIE

MICE

DISCUSSION

In general the physical and chemical data correlate well with the histological observations. The degenerative effect of scrapie is perhaps best shown by the simple measurement of brain weight, with losses approaching 10 per cent. as the animals become very severely affected. Some sort of balance is however being continually applied, since the total dry matter and protein content, expressed as a percentage of the wet weight, alter very little during the course of the disease. Acid proteinase activity is greatly increased and this presumably suggests a mechanism whereby the endogenous protein is destroyed. Since both protein content and total amino-acids are virtually unchanged, products of protein hydrolysis must be removed rapidly from the brain. There is certainly an outflow of water, producing excess cerebrospinal fluid, and it is surprising that more profound changes do not occur in the cerebrospinal fluid (Millson, West and Dew, 1960). However, the time scale of the incubation period is so long as to allow for continual removal of breakdown products from the cerebrospinal fluid. Alternatively, the release of waste products may occur in sudden localised outbursts, with which sampling would have to coincide for increased concentrations to be detected. Kasting and Darcel (1963) have reported increased amino-acid concentrations in the brain of a scrapie-affected Suffolk sheep. Proliferation of astroglia, because of the importance now attached to these cells in maintaining proper water and ion equilibrium in the organ (de Robertis, 1963), has suggested to some workers that ion balance in the brain may be altered (Pattison, 1965 ; Field et al., 1966). The last mentioned demonstrated an elevated sodium ion concentration and reduced potassium ion concentration in scrapie mouse brains, but in the present work only the decrease in potassium levels was observed. Field et aL (1966) also found a higher water content in scrapie affected brains, an effect we could not detect even in the most severely affected animals. The discrepancies between these results could be explained by differences in the character and distribution of histopathological lesions arising with the “Suffolk strain” of mouse scrapie (Zlotnik and Rennie, 1962, 1963) compared with other strains. Millson (1965) has shown that some L‘lysosomal” enzymes show an increased activity in scrapie brain. The acid proteinases also fall into this category, but whereas Millson demonstrated an apparent progressive increase in activity throughout the incubation period, the indications for proteinase are an explosive rise in activity while the histological score is low. Millson in fact determined activities on bulked homogenates of twenty brains from mice killed at monthly intervals after inoculation. If the other lysosomal enzymes behave as proteinase and if the range of histological score is as great as we found, the number of brains in each group with high enzyme activity would increase monthly and give the impression of a smooth upward trend during the incubation period. These two possible interpretations merit further study in view of their implications on the causes and effects of scrapie. Some of the quantitative changes presented here are significant when averaged over a group of animals, but none is striking enough to have a direct application in assisting diagnosis or assessing cerebral damage in an individual animal.

D.

Fig. 1. Fig. 2. Fig. 3. Fig. 4.

Medulla-type I. Cerebral cortex-type Cerebral cortex-type Cerebral cortex-type

L.

MOULD

et

d.

H&E x 140 II. H&E x 140 III. H & E x 140 IV, and hippocampus type

III.

H & E x 90

CHANGES

Fig. Fig. Fig. Fig.

5. 6. 7. 8.

Paraterminal body-type Paraterminal body-type Hippocampus-type II. Hippocampus-type V.

IN

BRAIN

IV. V. H & H &

H H E E

OF

SCRAPIE

& E x 140 & E x 140 x 140 x 140

MICE

D.

L.

MOULD

403

et al.

SUMMARY

An attempt has been made to correlate chemical changes with the degree and extent of histological damage in mouse brains infected with the “Suffolk strain” of scrapie. A scheme is described for the combined grading of degree of brain damage and distribution of lesionsbased on neuronal vacuolation and spongy degeneration. Considerable variation was found in the extent to which the mice were affected with scrapie. There was good correlation between change in body weight and histological grading and also a progressive decrease in brain weight with increase in extent of lesions. A slight decrease in mean density of brain tissue was observed. Total dry matter and protein concentrations remained constant. The sodium concentration remained constant, but there was 5 per cent. decrease in potassium concentration. A rise in acid proteinase activity appeared at the same time as minimal lesions, but reached a maximum before widespread lesions were present. There were no gross changes in the concentration of total free amino-acids. Since the total dry matter and protein concentrations alter very little, excess products of protein hydrolysis arising from increased acid proteinase activity must be removed rapidly from the brain. It is possible that the release of waste products may occur in sudden localised outbursts. ACKNOWLEDGMENTS

We are grateful to Mr. W. Smith for supplying infected brain tissue, to Mr. J. C. Rennie for assistancein histological preparation and examination, and to Mr. J. Sommerville, Mr. J. T. Williams and Miss M. Young for technical assistance. REFERENCES

Aldridge. W. N., Emery, R. C., and Street, B. W. (1960). B&hem. J., 77, 326. Dickinson, A. G., and Mackay, J, M. K. (1964). Heredity, 19, 279. Field, E. J., Caspary, E. A., and Windsor, G. D. (1966). Res. vet. Sci., 7, 72. Hunter, G. I)., and Millson, G. C. (1966). J. Neurochem., 13, 375. Kasting. R., and Darcel, C. le Q. (1963). Res. vet. Sci., 4, 518. Marks, N., and Lajtha, A. (1963). Biochem. J., 89, 438. McIlwain, H., and Rodnight, R. (1962). Practical Neurochemistry (1st Edit.), p. 17. J. & A. Churchill Ltd.; London. Millson, (:. C. (1965). J. Neurochem., 12, 461. Millson, G. C., West, L. C., and Dew, S. M. (1960). J, camp. Path., 70, 194. Mould, D. I,., and Slater, J. S. (1964). Scrapie Seminar (ARS 91-.53), p. 270. U.S. Dept. Agriculture; Washington D.C. Pattison, J. H. (1965). Nat. Inst. Nervous Diseases & Blindness, Monograph No. 2, p. 249. de Robertis,, E. (1963). Wld. Neurol., 3, 98. Slater, J. S. (1965a). Res. vet. Sci., 6, 92; (196513).Ibid., 155. Stein, W. H., and Moore, S. (1954). J. biol. Chem., 211, 915. Zlotnik, I., and Rennie, J. C. (1962). 1. camp. Path., 72, 360; (1963). Ibid., 73, 150. [Received

for publication,

]anuary

16th, 19671