An efficient method for estimating the total number of neurons in rat brain cortex

An efficient method for estimating the total number of neurons in rat brain cortex

Journal o[ Neurowtence Methods, 31 (1990) 93-100 9~ Elsewer NSM 01029 An efficient method for estimating the total number of neurons in rat brain ...

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Journal o[ Neurowtence Methods, 31 (1990) 93-100

9~

Elsewer

NSM 01029

An efficient method for estimating the total number of neurons in rat brain cortex L~se K o r b o ~, Bente P a k k e n b e r g ~, Ole L a d e f o g e d 2 H a n s J o r g e n G. G u n d e r s e n 3 Peter A r l i e n - S o b o r g 1 a n d H e n n x n g P a k k e n b e r g I Neurologtcal Research Laboratory, Unwersl O' Hospttal of Hvtdo~,re. HeMoere (DenmarL). : I n s n tute of Toxtcologo', Nattonal Food A gem ~. Copenhagen ( Denmarl, ). and ~¢Stereologwal Research Laboratory, A arhu~ Unn,er~tt~, 4 arhu~ (Denmarl,) ( Recewed 24 Aprd 1989) (Revised 23 August 1989) (Accepted 28 August 1989)

Key words Dlsector: Neurotoxicology; Paraffin embedding, Rat brain cortex, Stereology: Toluene: Total neuron number An efficient method for the unbmsed est,mauon of the total number of neurons m rat brain cortex ~s presented The method i~ reasonable fast, the counting procedure takes 2 - 3 h per rat, and gwes an esumate of the total neuron number m cortex cerebn wtth an CE of 0 10 The rat cerebral cortex is found to have a mean volume of 253 mm ~ and to contain a mean of 21 mdhon neurons The method has been used m a neurotoxlcologlc study, m which rats were gwen toluene perorally m dose~ of 200, 400 and 800 m g / k g / d a y , respecuvely, for 12 weeks, followed by an exposure-free period of 4 weeks The total number of neurons in rat brain cortex cerebn showed no stgmficant difference between exposed and non-exposed animals The apphcabdlt) of the method ~s dl~cussed

Introduction Different counting methods have been apphed m mammalian brain cortex for estimating the total cell number or, more often, the cell density in a given area of the cortex, as described by Konlgsmark (1970). These methods were both time consuming a n d / o r biased by histological procedures such as shrinkage of the tissue during preparation, staining etc. In 1984 a new method was described (Sterlo 1984) by which the total number of arbitrary particles can be unbtasedly estimated, mdependent of size and shape m a given structure

Correspondence

Llse Korbo, M D., Neurological Research Laboratory, Pav 4, Hvtdovre Hospital, DK-2650 Hvldovre, Denmark

of known volume. The present method, in which the total number of neurons m rat brain cortex is estimated, is a modification and marked slmphfication of the procedure described by Pakkenberg and Gundersen (1988) for esumatmg total neuron number m human brain nuclei Toluene is a commonly used organtc solvent parUcularly in the graphic industry Additionally, tt is often abused by sniffers m the pure form, or as a component of glue (see Lazar 1983) Chnlcal observauons of sniffers and occupationally exposed workers indxcate that long-term exposure to toluene may cause a chronic toxic encephalopathy characterized by chronic organic psychosyndrome and often dyscoordlnation, as described by Gregersen and Hansen (1986) Fornazzari et al. (1983) have reported atrophy of the cerebral cortex and especially of the cerebellar cortex esUmated

0165-0270/90/$03 50 ~c 1990 Elsevier Science Publishers B V (Biomedical Division)

from CT scans. Only a few neurotoxlcologmal long term studies have been made on animals, most of them being behavmral studies, see W H O (1985) Neuronal degenerative changes following toluene inhalation have been reported at the ultrastructural and at the lightmlcroscoplc level by Vasques-Nln et al (1980) and Naalsund (1987) Honma et al. (1983) have shown reducnon of the content of acetylcholine m rat brains after exposure to toluene, indicating a disturbance of several brain functions

Materials and methods

Exposure Forty-two 3-month-old male Wlstar rats were divided into 4 groups. Three groups of 10 animals each were exposed to toluene in doses of 200, 400, and 800 m g / k g / d a y , respectively. The toluene mixed with soya oil was introduced daily by a feeding tube for 5 days a week over a 12-week period. The last group of 12 ammals served as controls, and these were given soya oil by a feeding tube. The exposure period was followed by an exposure-free period of 4 weeks. All ammals were weighed once a week and allowed free access to food and drinking water during the entire test period. Two of the animals m the high dosage group died during the exposure period, probably due to aspiration of the solvent, leaving groups of 12, 10, 10, and 8 rats for the study. At the time of death the rats were anesthetized with ether and perfused through the left ventricle with formalin (4%, pH 7.4). After removal the brains were fixed in Lillie's liquid (4% formalin, phosphate-buffered) for 3 months,

Preparation of the brains The cerebellum, the brainstem and the olfactory bulb were removed and the cerebrum embedded in toto m paraffin. Each paraffin block was marked on the upper face with an interval of 1.5 mm using parallel razor blades, and cut serially providing 7 - 8 1.5-mm slabs (see also Fig. 7 in Gundersen et al., 1988). The first cut was placed at a random position to the frontal pole of the brain with the subsequent cuts in known, fixed

distances from the first t, ut. ~e.. a ,~stetnat~c random sectlomng procedure, as described b~ Gundersen and Jensen (1987) Before cuttuag, the embedded brain was heated at 60°(_ m an oven lor 15 mm, to make it possible to cut the block without damaging the tasbue. Each slab was mounted on the top of a paraffin block w~th the cut face down, and 40 serial sections ol 4 >m wele cut from the block. Sections 39 and 40 were stored and stained w~th gallocyanln-chromalum, and used for the estimation of total neuron number Gallocyanln-chromalum IS a specific Nlssl qmn described by Emarson (1951). ('ells were ~dentlfied as nerve cells, when the nucleus was ~urrounded by Nlssl stained cytoplasm If no nssue was present m section 40 no section pair was obtained from this slab. The average sectmn thickness was found by measuring the height of the block with a micrometer, 100 sections were cut and the thickness of the block measured again.

Estimates The total number of nerve cells in the cortex was estimated with the d~sector method, for further information see Sterlo (1984) and Gundersen et al. (1988). Total number of neurons IS estimated as a product of volume and numerical density.

Cortex volume One of the sections in each of the 7-8 pairs of sections from each brain was used for estimating the volume of cortex and of the whole cerebrum by the Cavahen principle, described by Gundersen and Jensen (1987). The area of the cortex in each section was estimated unbiasedly by systematic point counting at a magnification of × 17.5 using a projection microscope. At this magnification &fferentlation between cortex and white matter can easily be performed. The counting grid had a point distance of 30 ram, wtuch gave on the average 60 hits per cortex and 165 hits per cerebrum. The border of the cortex was defined using a stereotaxlc atlas of the rat brain by Kt~mg and Khppel (1963), and both the neo- and rhino-cortex were included. The total volume (V) of each fixed, dehydrated and paraf-

95

Numert~al den~ttv E a c h s e c t i o n p a i r w a s s t u d i e d a t × 700 m a g m f i cation using 2 adjacent microscopes projecting the

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images onto a table. Two counting frames of area a ( f r a m e ) = 71 × 80 m m w e r e p l a c e d o n t o t h e p r o jected images, since neurons were counted both f r o m o n e s e c t i o n to t h e o t h e r a n d v l c a v e r s a , see G u n d e r s e n ( 1 9 8 6 ) a n d G u n d e r s e n et al ( 1 9 8 8 ) for further details Four to 5 fields were counted from e a c h s e c t i o n w~th a k n o w n , f i x e d i n t e r v a l , c o u n t l n g o n l y t h o s e n e r v e cell n u c l e i , t h e t r a n s e c t s o f which were sampled with the unbiased counting f r a m e ( G u n d e r s e n 1977), p r o v t d e d t h e n u c l e i w e r e s e e n in o n e s e c t i o n o n l y . T h e n u m e r i c a l d e n s i t y , t h e t o t a l n u m b e r o f n e r v e cells p e r t o t a l c o r t e x v o l u m e ( N ~ ) , ts

F~g 1 Samphng of the counting fields m the cortex cerehrl The m~croscope xs moved step-w~se through the cortex from a field of wslon to the adjacent one. and the fields m which more than half ~s ms.de the cortex ~s counted A random number between 1 and 16 ~s looked up m a random number table, and indicates the number of the first counting field Hereafter every 16th field ~s a counting field In this example the fields marked w~th dots are the counting fields

fin-embedded Cavahen

cortex

was

estimated

usmg

N~-

where SQ ~s t h e t o t a l n u m b e r o f n e r v e cells counted in a total dtsector volume of S~,(dts), t,(dls) = t . a ( f r a m e ) . T h e a v e r a g e s e c t i o n t h i c k n e s s w a s 4 / ~ m , a ( f r a m e ) = 11,600 ~ m 2, c o r r e c t e d for m a g n i f i c a t i o n . A n e x a m p l e o f s a m p h n g t h e field~ m o n e s e c t i o n is s h o w n m F~g 1.

the

formula'

V(cortex) = T. a(p).

SQSt,

NP

Total number o f neurons w h e r e T is t h e f i x e d t h i c k n e s s o f t h e s l a b s o f t h e b r a i n (1.5 m m ) , a l p ) ~s t h e a r e a a s s o c i a t e d w i t h each point of the grid, corrected for magnification, a n d 2 P is t h e t o t a l n u m b e r o f p o i n t s t h a t h i t t h e cortex.

T h e t o t a l n u m b e r o f n e r v e cells ( N ) cortex can now be determined: NQ--

N -

V_NQ

.Np.

St'

m the rat

T-alp)

t . -X'a ( f r a m e )

TABLF I MEAN VALUES OF THE PARAMETERS FOR THE DIFFERENT GROUPS ('oefflc~ent of variation (CV) = SD/mean ts shown m parentheses. Toluene dosage (mg/kg/day)

Body weight after 12 weeks exposure (gl Body weight after 16 weeks (g) Brain weight (g) Cerebrum volume (mm 3 ) Cortex volume (mm3) Total neuron number (106) * *

Control

200

467

475

(0 072)

494 (0.078) 1 56 (0 032) 726 (0 069) 250 (0 116) 21 0 (0 179)

400 (0 065)

516 (0 073) 1 55 (0 041 ) 722 (0 088) 251 (0071) 20 9 (0 172)

2P < 0 0l, * 2P < 0 05, Student's t-test, deviation from control values

430

800 (0 069) * *

485 (0 060) 1 51 (0 046) 728 (0 081 ) 249 (0 126) 19 4 (0 213)

407

II) 0741 * *

466 (0 068) 1 48 t0 047) * 699 (0 074) 260 (00651 23 8 (0 150I

V6

Results

The weights of the animals after the 12 weeks exposure and after the total experimental period. the brain weights, the volumes of cortex and whole cerebrum and the total neuron number for the different groups are shown in Table I. The animals exposed to the medium and the highest dose had a significant lower body weight after 12 weeks exposure compared to the control group (2P < 0.01 and 2 P < 0 . 0 0 1 , respectively). In the following exposure-free period the exposed animals gained m weight, and at decapitation no significant difference was found between the mean body weight of the groups. A significant difference in brain weight was found between the control group and the group given the highest dose (2P = 0.015). Regarding the volume of cortex and cerebrum and the total number of neurons no significant differences were found between the groups As shown m Figs. 2 and 3 there was a clear-cut toluene dosage dependence of brain weight (slope" - 3 4 mg per doubling, P = 0.019) and of body weight after the total experimental period (slope: - 25 g per doubhng, P = 0.0015). The total number of neurons in rat brain cortex was estimated to be on the average 21 millions (Table I)

Discussion

In the present study no significant difference was found between the exposed and non-exposed animals with respect to the total neuron number.

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Fig 2 Relatlonshap between brain weight for the exposed groups and the logarithm of the exposure dose A clear-cut toluene dosage dependence is found (slope - 3 4 m g per doubling, P = 0 019)

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Fig 3 Relauonsbxp between body weight after the total experimental period for the exposed groups and the logarithm of the exposure dose The regression hne drawn has a slope of - 25 g per doubhng, P = 0 0015

However, the rats seem to have been affected by the toluene in the exposure-period at least in the 2 highest dosed groups, leading to a lower body weight of the rats. Possible biases may be: the exposure period may have been too short, and the rats more resistant than expected to toluene, so the neurons did not show evidence of cell death w~thin the time limits of the test period. Different discrete brain areas might not be equally sensitive to toluene (Haghd and Kjellstrand, 1985). Cell loss m distinct areas may be overlooked, when the analysis does not distinguish between different areas. Any method providing solely information about the total cortex is unspeofic in the sense that It requires a general cell loss in the whole cortex, before a difference between exposed and non-exposed animals will appear. If a certain chemical is expected to give cell loss in only a specific part of the brain, the total number of neurons in this area may be estimated, if indeed the volume of this area can be defined. Toluene was given orally. It is completely absorbed from the gastrointestinal tract and has been shown to have sirmlar tissue distribution in acute intoxication by this type of administration as after inhalation exposure ( W H O 1985). Whether an induction of liver enzymes appears when rats are exposed for a longer period is not known, but an enhanced metabolism of toluene in rats has been reported after prior administration of phenobarbital (WHO, 1985).

97

A significant decrease in brain weight was f o u n d in the exposed rats, while the volume of the cerebrum in the 4 groups were found to be almost identical. This was surprising. Since b o t h variables are estimated on brains which had been in fixative for months, this m a y be due to a different degree of swelling in the fixation period. Since b o t h brain weight and brain volume m a y be biased, results based only on these parameters may be biased as well. The relation between the cell density and the cortex volume for the different groups of rats in this study can be seen in Fig 4. The regression analysis of the groups shows, that there is no

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VOLUME

F i g 5 R e l a t i o n s h i p b e t w e e n total n u m b e r o f n e u r o n s a n d v o l u m e of c o r t e x for all r a t s T h e d i f f e r e n t g r o u p s a r e p l o t t e d w~th the s a m e s y m b o l s as m F i g 4 A s c a n b e seen m the figure a r a t h e r s t r o n g c o r r e l a t i o n ~s f o u n d b e t w e e n the 2 v a r i a b l e s ( r = 0 52, 2 P < 0 0 0 1 )

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CORTICAL VOLUME Fig 4 density control toluene

Relationship between the cortex volume and the cell The 4 groups are plotted with different symbols • D, A, a groups receiving 200, 400 and 800 m g / k g / d a y Regression analysis for each group shows no relauonship between the 2 variables m any group

relation between these 2 variables This observation makes it unlikely, that s w e l h n g / s h r m k a g e i~ a major factor for the variation in cortex volume. As shown in Fig. 5, there is a rather strong correlation between the total cortex volume and the total n u m b e r of neurons. Since p r o n o u n c e d variation of shrinkage is an unlikely explanation for this correlation (see also Appendix), it is possible to postulate that there ts a direct p r o p o r t l o n a h t y between the volume of the brain and the total n u m b e r of neurons, even though these 2 variables are coupled (cf. the formula for estimating the total n u m b e r of neurons). The m e t h o d is not only usable in neurotoxlcology, but is applicable on all kinds of tissue, where a total n u m b e r of well-defined objects or particles is wanted In a well-defined region

¢)8

Appendix

unbiased countmg principle of the dlsecto) ~ used lor esttmatmg N~ (neurons/cortex), alld (2) the reference volume v(dis) = t a(frame) m the dlsectot refers to a volume with precisely the same defimtJon as that estimated by Cavalier/s prmc~ple Specifically, any shrmkage or deformamm of the corttcal tissue during fixation, deh'~drauon. paraffin embedding, sectLomng and section stretching cannot influence the estimate of total number The ~solated estimates of ,,V, (neurons/ cortex) and V(cortex) refer on the contrar) to the fixed, dehydrated, shrunken nssue m paraffin, and the relationship of these values to the hvmg animal is not well-defined Moreover, the combined procedure J., both rea-

,4nalyzlng and opttmtzmg the estimation o f rat total neuron number

The estimator of total neuron number N(neurons) = N) (neurons/cortex). V(cortex) developed in this study has several attracttve statistical properties and is less laborious than its predecessor described for human brain nuclei by Pakkenberg and Gundersen (1988). It is foremost unbiased because it solves 2 major problems m number estimatmn (1) the

0

5

25

50

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PREDICTED

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I ANIMALS

l 25 T.,CKNESSES

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FOR N V (NEURONS/CORTEXI

25 FIELDS

7 SECTIONS FOR V(CORTEX) E~

60 POINTS

Fig. 6 Dlstnbutlon of samphng vanances m the estimates of total number of neurons in cerebral cortex m rats The horizontal columns show estimated variances m percent of total observed vanance among animals within a group, the bars at the end indicate the vanablhty, SD, of the estimated variances among the 4 groups of 8-12 rats each The uppermost bar indicates the variance predicted by the analysis described in the text The bar marked 'ammals' shows the varaance which remains when all variances contributed from the samphng steps in the stereologlcal analysis has been accounted for. It is therefore the natural vanatlon of neuron number among such rats. The following bars show how much of the total vanance wtuch could be attributed to the vanablhty of mean values of a finite number of samphng items as indicated when only 200 neurons are counted in only 25 fields (each with 2 disectors on only 7 section pairs) for the estimate of Nv(neurons/cortex ) m a rat the bruited sample sizes gave nse to some additional variance As also indicated the low number of not very precise estimates of the section thackness is the major methodological source of variation m the original sampling design

9q

s o n a b l y fast (it takes 2 3 h per rat, everything m c l u d e d ) a n d q m t e precise. T h e analysis of the overall variance of the e s t i m a t e a n d its division into c o m p o n e n t s c o n t r i b u t e d from all s a m p h n g steps is described m detail in P a k k e n b e r g and G u n d e r s e n (1988) and is b a s e d m a i n l y on recent d e v e l o p m e n t s in variance est~manon in s y s t e m a n c ,~amphng designs ( G u n d e r s e n a n d Jensen, 1987) The analysis of overall variance is p e r f o r m e d in several steps T h e p r e d i c t e d c o n t r i b u t i o n s from the various s a m p l i n g steps is first c a l c u l a t e d using formulae m G u n d e r s e n a n d Jensen (1987). This p r e & c n o n of total variance ~s then c o m p a r e d to the actual, o b s e r v e d variance between a m m a l s within the groups F o r the 4 groups in this study, the o b s e r v e d relative variation or the c o e f h c l e n t of variation C V = S D / m e a n ranged from 0.15 to 0 21 w~th a mean of 0 18 Since the p r e & c t e d values were on the average 98 7 _+ 5.8% of the o b s e r v e d (see F~g. 6), the m o d e l s u n d e r l y i n g the analys~s seem to be robust enough for calculating the c o n t r i b u t i o n s from the s a m p h n g steps to the total varmnce T h e result of th~s analys~s ~s also shown m F~g. 6 The s~mple biological fact that different rats have different total n u m b e r s of neurons in thmr cerebral cortices a c c o u n t e d for the largebt fraction of the variance, = 41 ,% The corres p o n d i n g estimate of the true b~olog~cal variation of the total n u m b e r of neurons of the cerebral cortex a m o n g the rats is ~,'(0.41 - 0.18: ) = 0 12. This figure ~s therefore the a b s o l u t e m i n i m a l v a n a N l l t y of the final results, which can be achieved ~f the s a m p h n g effort was increased without any hm~t at all s a m p h n g levels. A n a l y z i n g the steps m the stereologlcal estHnat~on, it seems that the b a l a n c e b e t w e e n effort a n d resulting c o n t r i b u t i o n to the overall variance ~s not far from the o p t i m u m . (In analysis of hierarchical s a m p h n g schemes the o p t i m a l &strlb u t l o n of effort between two levels ~s the one p r o p o m o n a l to the variances of the 2 levels the larger the variation between s a m p l i n g items, the more of them should be analyzed, as ~s wellknow n ) F o r example, the effort of cutting 7 slabs from the cortex is of the same o r d e r of m a g m t u d e as that of c o u n t i n g a total of 60 test p o i n t s on the slabs, n a m e l y a few minutes, and this ratio m a t c h e s the roughly equal c o n t r i b u t i o n s from the 2 sam-

p i i n g steps, n a m e l y 5.7% a n d 5.5% of total ~anance, respectively (cf. Fig. 6) T h e r e are onl?r two reservations against the s a m p l i n g scheme raised b'v the analys~s of v a r i a n c e distributions. The first is the rather large fraction of the total varmtlon which is due to the relatively Imprecise estimation of section thickness T h e CV of the readings of section thickness with a c a h p e r was 0 15, xvhich for an average n u m b e r of estimates of 2.5 per a n i m a l gave a C E of 0 10, which In turn c o n t r i b uted 31.6~ to the total ob,~erved variance In the o p p o s i t e &rect~on one may note, that a large fraction of the effort was spent on c o u n t i n g = 200 n e u r o n s in = 50 & s e c t o r s per animal, the o n l \ step in the s a m p h n g scheme which takes hours rather than minutes. Since these 2 latter steps c o m b i n e d c o n t r i b u t e only 14 8% to the total v a n a n t e , this is u n h k e l y to be an o p u m a l d i s t r i b u t i o n of effort. In future s t u & e s of similar rats one should therefore i m p r o v e the s a m p l i n g design b \ e s t i m a t i n g the >ectlon thickness m all 7 blocks, but then use a s o m e w h a t greater & s t a n c e between the held,, used for counting, a i m i n g at a total count ol no more than 100 neuron~ per rat in ~ 25 dlsectors. (Until n o w ' , It has not been necessar,e to c o u n t m o r e than = 100 1terns per m & v l d u a l in any of the m a n y studms w,here the dlsector prlnc~pie has been used for n u m b e r est~matum In somc cases c o n s i d e r a b l y less were sufficient (see e g G u n d e r s e n , 1986)) T h e expected total variation a m o n g rats after such a redistributIon and reduction of the resources spent on a stereologlcal >tudx is 0 1 8 - l ( 1 + 0 . 1 4 8 + ( 2 . 5 / 7 - 1 ) > 4 0 3 1 6 ) = 017. It ~s noteworthly, that coronal sections through the whole cortex d i d not show' any c o n t r i b u t i o n to the total variance f r o m any l n h o m o g e n e l t y with respect to N~ ( n e u r o n s / c o r t e x ) In all 4 groups, the o b s e r v e d v a r m n c e a m o n g sections w~thln a m m a l s c o u l d be e x p l a i n e d by the c o n t r i b u t i o n from the s a m p l e v a r i a n c e of the 3.7 fields studied p e r section W~th respect to s a m p l e variance we ma~ therefore c o n c l u d e that sectmns do not shou a n y a p p r e c m b l e v a r i a t i o n a b o x e that c o n t r i b u t e d from the h e l d - t o - h e l d variation. This doe,,, not exclude, however, some s y s t e m a t i c v a r l a n o n m the numerical d e n s i t y of n e u r o n s from the frontal to the occipital lobe As illustrated in Fig 7. a closer

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50. FRONTAL MIDBRAIN OCCIPITAL CORTEX Fig 7 Systemauc v a n a t m n m numerical density N v ( n e u r o n s / cortex) m rats Mean values for all 40 rats are shown The bars on the mean value for the frontal regmn are SD a m o n g ammals, those on mean n u m e n c a l densities m the rmdbram and occipital regmns are SEM of the dewauons from the prewous regmns (paired observatmns)

analysts of arty systematic variaUon revealed a higher numerical density m the occipital region. The increase by 30% from 8 1 . 1 0 3 - m m -~ neurons in the frontal region to 106 • 103 • m m 3 neurons m the occipital region was statistically sigmficant (Student's paired t-test = 3.81, 2 P < 0.001).

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