Large Section Cryomicrotomy: A Basic Method for Microdissectioning and in vitro Autoradiography of Human Nervous Tissue

1 . INTRODUCTION Evolut ion of t h e important technique of whole body au to rad io graphy o f experimental animals , introduced by Ullberg i n 1954 , has been d i r e c t l y l i n k e d t o t h e development of heavy-duty cryomicrotomes ( 1 6 ) . Since t h e n , l a rge sec t ion cryomicrotomy has 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 paper w i l l i l l u s t ra te 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 exemplif ied by s t u d i e s i n o u r own r e s e a r c h group. Reports i nc lud ing 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 publ i s h e d ( 5 , 1 0 ) .

gist carrying out electromyography and for the clinician taking a muscle biopsy. However, due to technical difficulties at sectioning, 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 cryosections from biceps brachii and tibialis anterior muscles were analysed by a computer to reconstruct the topographical end-plate distribution within the whole muscle (2).

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 cholinergic 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 reductions 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 localization of neurotransmitter systems tissue sampling by methods 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.

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).

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 3Halpha-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.

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 receptor 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 possibility to perform in vitro large section autoradiography on post-mortem brain sections in parallel to positron emission tomography using the same 11C-labelled ligand. Such studies have recently been carried out in our laboratories using a bensodiazepine receptor ligand (d'Argy et a1 to be published).

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, 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.
C h i c h e s t e r , 1 9 8 3 .