Uppsala J Med Sci 91: 245-250, 1986 Large Section Cryomicrotomy: A Basic Method for Microdissectioning and in vitro Autoradiography of Human Nervous Tissue Sten-Magnus Aquilonius Department of Neurology, University Hospital, S-75185 Uppsala, Sweden 1 . I N T R O D U C T I O N E v o l u t i o n o f t h e i m p o r t a n t t e c h n i q u e of whole body a u t o r a d i o - g r a p h y o f e x p e r i m e n t a l a n i m a l s , i n t r o d u c e d by U l l b e r g i n 1 9 5 4 , h a s been d i r e c t l y l i n k e d t o t h e development of heavy-duty c r y o - microtomes ( 1 6 ) . S i n c e t h e n , l a r g e - s e c t i o n cryomicrotomy h a s found s e v e r a l new areas of a p p l i c a t i o n . The p r e s e n t p a p e r w i l l i l l u - s t r a t e t h e u s e f u l n e s s of t h i s b a s i c methodology as e x e m p l i f i e d by s t u d i e s i n o u r own r e s e a r c h group. R e p o r t s i n c l u d i n g t e c h n i c a l d e t a i l s have r e c e n t l y been pub- l i s h e d ( 5 , 1 0 ) . 2 . T O P O G R A P H I C A L ANATOMY O F HUMAN B R A I N AND MUSCLE 2 . 1 B r a i n Computed tomography ( C T ) i s e s t a b l i s h e d a s a most i m p o r t a n t t e c h n i q u e i n n e u r o r a d i o l o g y . CT o f t h e b r a i n i s m a i n l y r e s t r i c t e d t o n e a r h o r i z o n t a l p l a n e s , a f a c t which c r e a t e s a need f o r ana- t o m i c a l knowledge of b r a i n s e c t i o n s which were n o t commonly s t u d i e d p r e v i o u s l y . T h e r e f o r e , w e a p p l i e d l a r g e s e c t i o n c r y o - microtomy o f whole human b r a i n and produced a c o l o u r a t l a s o f h o r i z o n t a l c r y o s e c t i o n s ( F i g . 1 A ) which where p h o t o g r a p h e d j u s t thawed a g a i n s t a b l a c k background ( 3 ) . However, t h i s a t l a s d o e s n o t i l l u s t r a t e t h e r e l a t i o n s h i p b e t - ween b r a i n and s u r r o u n d i n g t i s s u e s . A h e a d - b r a i n a t l a s b a s e d on t h e t e c h n i c a l m o d i f i c a t i o n s of cryomicrotomy worked o u t by Rauschning ( t h i s volume) would be a u s e f u l complement t o t h e neuroimaging t e c h n i q u e s , C T , n u c l e a r m a g n e t i c r e s o n a n c e , and . p o s i t r o n e m i s s i o n tomography. 2 . 2 Muscle Knowledge a b o u t t h e t o p o g r a p h i c a l d i s t r i b u t i o n o f motor end- p l a t e s i n human muscle i s i m p o r t a n t b o t h f o r t h e n e u r o p h y s i o l o - 16-868573 245 B Fig. 1 Different applications of large section cryomicrotomy: A) Topographical atlas of the human brain (4) adapted to computed tomography. B) Distribution of ChE staining in whole human biceps muscle ( 5 ) . C ) Tissue punches within a section through extrapyra- midal structures 5 mm behind the anterior commisure (8). D) In vitro autoradiography in a frontal section of a human hemisphere (3) using the muscarinic ligand 3H-QNB. 246 gist carrying out electromyography and for the clinician taking a muscle biopsy. However, due to technical difficulties at sec- tioning, available information was based on cholinesterase (ChE) staining of muscles from small children only. By means of the heavy-d.uty cryomicrotome (PMV 400, LKB 2250, LKB, Sweden) it is possible to obtain horizontal cryosections of adult whole human muscle. We have studied the distribution of motor end-plates in biceps brachii (Fig. 1 B), tibialis anterior and sartorius muscle from adults by staining longitudinal cryosections of the whole muscles for ChE (5). The results from a large number of cryo- sections from biceps brachii and tibialis anterior muscles were analysed by a computer to reconstruct the topographical end-plate distribution within the whole muscle (2). 3. THE "PUNCH-TECHNIQUE" OF TISSUE SAMPLING FROM CRYOSECTIONS OF HUMAN BRAIN AND SPINAL CORD A defined and reproducable method of tissue dissectioning is a prerequisite for regional neurochemical studies in post-mortem nervous tissue. For this purpose the "punch-technique" of tissue sampling has been rather extensively used in animal (see 14 for review) and human studies (see 5 for review). We introduced this technique (Fig. 1 C) in an investigation on the regional distribution of choline acetyltransferase (ChAT) within the brain of controls and Huntington chorea cases (6). Thereby, it was possible to obtain the most detailed localization of human brain ChAT available at that time and to demonstrate the marked reduction of neostriatal ChAT in Huntington's disease, probably corresponding to a pronounced degeneration of choliner- gic interneurons. More recently the "punch-technique" of microdissectioning has been used in investigations on the topical localization of ChAT within the human spinal cord (4). In addition to an area of high activity in the ventrolateral part of the ventral horn traced into the ventral root region and probably corresponding to motor neurons, another area of high ChAT activity was found in the apical part of the dorsal horn. In motor neuron disease the re- ductions in spinal ChAT are not restricted to the motor neuron areas (9) indicating a more general involvement of cholinergic structures. In view of the rapid development and application in human post-mortem tissue of immunohistochemical methods for the locali- zation of neurotransmitter systems tissue sampling by methods 241 like the "punch-technique" might be of less importance in the future. However, biochemical analysis of enzyme activities within well defined tissue specimens will still represent an important complementary technique especially in quantitative investigations. 4. LARGE SECTION CRYOMICROTOMY FOR IN VIVO RECEPTOR AUTORADIOGRAPHY In recent years in vitro autoradiographic techniques (17) have been extensively employed for localization of receptor sites within different tissues. So far, the methods have mainly been applied to rather small sections, like in our own studies, the spinal cord (8, 11). However, technical modifications have now been worked out permitting in vitro autoradiographic studies in very large tissue sections (10). 4. 1 Muscle After freezing whole human biceps brachii muscles between metal plates and horizontal cryosectioning, in vitro receptor autoradiography using 3H-alpha-bungartoxin (3H-alpha-Btx) as ligand for the nicotinic cholinergic receptors has been performed (7). It could be demonstrated that in motor neuron disease 3H- alpha-Btx binding occurred over the entire muscle, indicating uncovering of extrajunctional sites while in the control the binding was restricted to the motor end-plate region. 4. 2 Brain In the last years the development of large section in vitro receptor autoradiography (Fig. 1 D) has enabled studies on regional distribution as well as quantitation of different re- ceptor sites within the whole human brain (12, 15, see 13 for review). Such studies represent important complements to in vivo receptor autoradiographic studies in man by positron emission tomography. Of special interest in this connection is the possi- bility to perform in vitro large section autoradiography on post-mortem brain sections in parallel to positron emission tomo- graphy using the same 11C-labelled ligand. Such studies have re- cently been carried out in our laboratories using a bensodiaze- pine receptor ligand (d'Argy et a1 to be published). 5. CONCLUSIONS In clinical neuroscience large section cryomicrotomy has found valuable applications in topographical neuroanatomy and as a tool in microdissection of tissue for neurochemical analysis. Recently, 248 cryomicrotomy-in vitro-autoradiography has been introduced as a most important technique for the localization of neuroreceptors and provides a useful complement to human in vivo studies by positron emission tomography. REFERENCES 1. Aquilonius, S.-M., Arvidson, B., Askmark, H. & Gillberg, P.-G.: Topographical localization of end-plates in cryosections of whole human biceps muscle. Muscle Nerve 5:418, 7982. 2. Aquilonius, S.-M., Askmark, H., Gillberg, P.-G., Nandedkar, S., O l s s o n , Y, & Stblberg, E.: Topographical localization of motor endplates in cryosections of whole human muscles. Muscle Nerve 7:287-293, 1984. 3. Aquilonius, S.-M. & Eckernas, S.-8.: A colour atlas of the human brain adapted to computed tomography. Esselte Studium, Stockholm, 1980. graphical localization of choline acetyltransferase within the human spinal cord and a comparison with some other species. Brain R e s 211:329-340, 1981. section cryomicrotomy in human neuroanatomy and neurochemistry. In: Brain Microdissection Techniques (ed. A.C. Cuello), pp. 165- 170. John Wiley & Sons, Chichester, 1983. 6. Aquilonius, S.-M., Eckernas, S . - 8 . & Sundwall, A.: Regional distribution of choline acetyltransferase in the human brain: Changes in Huntington's chorea. J Neurol Neurosurg Psychiat 4. Aquilonius, S.-M., Eckernas, S.-A. & Gillberg, P.-G.: Topo- 5. Aquilonius, S.-M., Eckernas, S . - 8 . & Gillberg, P.-G.: Large 38:669-677, 1975. 7. Askmark, H., Gillberg, P.-G. & Aquilonius, S.-M.: Autoradio- graphic visualization of extrajunctional acetylcholine re- ceptors in whole human biceps brachii muscle. Changes in amyo- trophic lateral sclerosis. Acta Neurol Scand 72:344-347, 1985. 8. Gillberg, P.-G. & Aquilonius, S.-M.: Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography. Acta Neurol Scand 72:299-306, 1985. 9. Gillberg, P.-G., Aquilonius, S.-M., Eckernas, S.-A., Lundqvist, G. & Winblad, B.: Choline acetyltransferase and substance P- like immunoreactivity in the human spinal cord: changes in amyotrophic lateral sclerosis. Brain Res 250:394-397, 1982. IO.Gillberg, P.-G., Jossan, S.S., Askmark, H. & Aquilonius, S.-M.: Large-section cryomicrotomy for in vitro receptor autoradio- graphy. J Pharmacol Methods 15:169-180, 1986. Il.Gillberg, P.-G., Nordberg, A. & Aquilonius, S.-M.: Muscarinic binding sites in small homogenates and in autoradiographic sections from rat and human spinal cord. Brain Res 300:327-333, 1984. 12.JohnstonI M.V., Silverstein, F . S . , Reindel, F.O., Penney, J.B. Jr. & Young, A.B.: Muscarinic cholinergic receptors in human infant forebrain: 3H-quinuclidinyl benzilate binding in homo- genates and quantitative autoradiography in sections. Dev Brain Res 19:195-203. 1985. the human brain. TINS 6:284-289, 1986. areas by the punch technique. In: Brain Microdissection Tech- niques (ed. A.C. Cuellof, pp. 1-36. John Wiley & Sons, 13.Palacios, J.M., Probst, A. & Cortgs, R.: Mapping receptors in 14.Palkovits, M. & Brownstein, M.J.: Microdissection of brain 249 C h i c h e s t e r , 1 9 8 3 . n e u r o t r a n s m i t t e r r e c e p t o r s i n H u n t o n g t o n ’ s disease. N e u r o l o g y , 1 5 . P e n n e y , J . B . & Young, A . B . : Q u a n t i t a t i v e a u t o r a d i o g r a p h y of 3 2 : 1 3 9 1 - 1 3 9 5 , 1 9 8 2 . 1 6 . U l l b e r g , S . : The t e c h n i q u e of w h o l e b o d y a u t o r a d i o g r a p h y . C r y o s e c t i o n i n g of l a r g e s p e c i m e n s . S c i e n c e T o o l s , The LKB I n s t r u m e n t a l J o u r n a l , S p e c i a l I s s u e 2 - 2 3 , 1 9 7 7 . r a d i o g r a p h y : 3 H - o p i o i d r e c e p t o r s i n r a t b r a i n . B r a i n R e s 1 7 9 : 1 7 . Young, W.S. & K u h a r , M . J . : A new m e t h o d f o r r e c e p t o r a u t o - 2 5 5 - 2 7 0 , 1 9 7 9 . Address f o r r e p r i n t s : S t e n - M a g n u s A q u i l o n i u s D e p a r t m e n t of N e u r o l o g y U n i v e r s i t y H o s p i t a l S-751 8 5 U p p s a l a SWEDEN 250