Acta Neuropathol (1992) 83:488 - 498
Acta Neuropathologtca (~ Springer-Verlag 1992
Presynaptic terminal loss from alpha-motoneurones following the retrograde axonal transport of diphtheria toxin* A. H. Pullen Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London, WC1N 3BG, UK Received July 22, 1991/Revised, accepted December 9, 1991
Summary. Intercostal motoneurones intoxicated following intraneural injection of diphtheria toxin exhibited a progressive dilatation and fragmentation of Nissl body rough endoplasmic reticulum (rER), coupled with two different forms of presynaptic terminal response. Firstly, terminal dysjunction without prior degeneration, and secondly, Wallerian-type degeneration. Dysjunction was attributed to a toxin-related failure by the motoneurones to maintain postsynaptic site structure. Degeneration was considered to arise from toxicity in presynaptic neurones, either neighbouring motoneurones or local interneurones. Morphometry revealed that by 8 days, intoxicated motoneurones exhibited a 33 % loss in terminal frequency, a 15 % loss in residual presynaptic membrane, and a 43 % loss in overall presynaptic input. The concomitant loss of synaptic sites was greater that the overall loss of presynaptic membrane, indicating a toxin-related deficiency of the maintenance of postsynaptic sites. Analyses of the relationship between changes in terminal numbers and the development of Nissl body abnormality in the postsynaptic motoneurone identified three groups of motoneurones: (i) those with normal presynaptic input and normal neuronal Nissl body rER; (ii) those showing a dramatic loss of presynaptic input and a marked dilatation and fragmentation of Nissl bodies; and (iii) neurones exhibiting a maintained or further loss of presynaptic input coupled with extreme dilatation and fragmentation of Nissl body r E R with loss of Nissl body structure. These changes are discussed in context with the known molecular action of diphtheria toxin.
sponding intercostal motoneurones [7, 19], as a result of inhibition of protein synthesis in the cell body [3, 11, 12]. The lesion is chiefly characterised by a progressive dissolution of Nissl body ultrastructure, including Nissl bodies associated with 'C'-type axon terminals [17, 19]. The neuronal lesion is accompanied by abnormal focal synaptic cleft widening and a proliferation of astroglial processes apposing the neuronal membrane. It is postulated here that these features are a prelude to presynaptic terminal withdrawal [1, 2, 22] and a direct result of the toxin-induced inhibition of synthesis of proteins forming synaptic sites and cell adhesion molecules. This hypothesis has been examined in intoxicated intercostal motoneurones by firstly delineating the qualitative and quantitative response of the presynaptic terminals, and secondly relating the presynaptic response to the development of Nissl body abnormalities in the postsynaptic neurone.
Materials and methods
Animals Results were obtained from 40 normal intercostal motoneurones from 3 adult cats, and 21 intercostal motoneurones (3 'experimental' cats) in which a diphtheritic lesion had fully developed. Prior to experimental surgical procedures and terminal perfusion-fixation, animals were anaesthetised with sodium pentobarbitone (45 mg/kg, i.p.), or ketamine-HC1 (30 mg/kg, i.m.; supplemented by 1.5 mg/kg xylazine to ameliorate muscle rigidity).
Key words: Spinal motoneurone - Diphtheria toxin Synapses - Dysjunction - Neurodegeneration
General procedure
Intraneural injections of diphtheria toxin (DTX) into cat intercostal nerves evoke a primary lesion in the corre-
In experimental animals, intercostal nerves in segments T7 or T10 were directly injected with 1-2 ~tl of a 20 ng/~tl solution of lyophilised DTX reconstituted in sterile PBS, pH 7.5, containing 0.1% BSA (dose = 0.01-0.02 flocculation units). Postoperative recovery was unremarkable and at post-mortem examination there were no signs of systemic intoxication (i.e. endocarditis, or haemorrhagic lesions in lungs, liver and mesentery.) Experimental and normal animals were perfusion-fixed under anaesthesia with
* Supported by the Medical Research Council and the Motor Neurone Disease Association
489 2.5 % glutaratdehyde-2 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal segments T7-T10 were removed, cut into 2-ram transverse slices, osmicated, and processed for electron microscopy.
Qualitative examinations Qualitative analyses of normal and experimental material were restricted to ventral horn neurones of >35-~tm diameter possessing 'C'-type presynaptic terminals on the somal membrane, i.e. alpha-motoneurones [4]. In experimental material analyses of synaptology were confined to motoneurones exhibiting the gross cytological abnormalities associated with diphtheritic toxicity [7, 19], and their presynaptic axon terminals were classified using prescribed ultrastructural criteria based on terminal size, vesicle shape and synaptic site ultrastructure [4].
Mo~hometry As in previous analyses of cat motoneurone synaptology [15, 18] morphometric analyses were undertaken on random profiles of normal and intoxicated motoneurones sectioned through the near midnuclear plane. Advantages of this method over methods based on serial- and semi-serial sections are discussed elsewhere [15]. Previous analyses of synaptology showed that estimates of relative standard error (RSE) for calculations of presynaptic terminal frequency and cover based on samples of either 10 or 15 neurones provided values of 14% (n = 10) and 9% (n = 15), where RSE = 100 (SE/mean) [15]. To reduce RSE further, sample sizes were increased to a minimum of 20 neurones. Electron micrograph photo montages (magnification x 25,000) were constructed for each motonenrone and computer-assisted techniques applied to obtain the following data [14]: the length of the neuronal membrane (~m); the numbers of presynaptic terminals (total, and for each terminal category); the appositional length of each presynaptic terminal (gm); and the numbers of synaptic specialisations (total, and for each terminal. From these were derived: (1) axon terminal frequencies (total, and for each category; no./100gm neuronal membrane); (2) axon terminal cover (total, and for each category; terminal appositional length (~tm)/100-~tmneuronal membrane); and (3) synaptic specialisation fiequency; expressed as (i) no./terminal, (ii) no./100-~tm terminal apposition, and (iii) No./100%tm neuronal membrane. Differences between normal, and experimental data were tested with Student's t-test. Distributions of size or frequency were tested with a Kolmgorov-Smirnow procedure.
Results
Motoneuronal ultrastructure Normal m o t o n e u r o n e s viewed in 0.5-~m 'plastic' sections stained with toludine blue (Fig. la) possessed a smooth-contoured neuronal m e m b r a n e and contained and a regularly shaped nucleus.The unstained cytoplasm contained a mixture of punctate dense-staining bodies (lysosomes) and larger basophilic regions representing Nissl bodies. Additional features of normal motoneurones seen in electron micrographs (Fig. lb) were intact mitochondria with prominent cristae, mulltilamellate Golgi cisterns, and highly structured Nissl bodies (Fig. 2a). Nissl bodies consisted of 10- to 40-nm wide cisterns of rough endoplasmic reticulum (rER), alter-
nating with linear arrays of lamellae-associated polyribosomes. No cytoplasmic vacuolation occurred in normal motoneurones. In confirmation of previous data [19], different motoneurones within the same spinal segment 5 - 8 days after injection of toxin exhibited a characteristic range of morphological abnormalities. In 0.5-gm 'plastic' sections, these ranged from normal cell shape and basophilia but slight cytoplasmic vacuolation and nuclear crenellation (Fig. lc), to extreme vacuolation. While some motoneurones showed reduced basophilia, others were hyperbasophilic. Ultrastructurally, motoneurones exhibited a corresponding wide range of abnormalities which ranged from slight vacuolation coupled with normal mitochondria and normal spatial relationships between Nissl body r E R , r E R - b o u n d ribosomes and lamellae-associated polyribosomes, to extreme cytoplasmic vacuolation, irregular cell shape, eccentric nuclei, and mitochondrial swelling with loss of cristae (Fig. ld). In these neurones, Nissl bodies displayed extreme dilatation of r E R cisterns (Fig. 2c-d), fragmentation of r E R lamellae, and loss of spatial relationships between r E R and polyribosomes. De-granulation occurred in only the severest stages of Nissl body dissolution. O t h e r neurones showed intermediate stages of Nissl body abnormality (Fig. 2c), suggesting that Nissl body dissolution was progressive. Motoneurones were uniform in their response. None showed a different form of pathology: for example, the acute loss of multilamellated Nissl body structure, loss of r E R , and formation of polyribosomal aggregates associated with intercostal nerve transection [8], which in these experiments would have indicated a neuronal response to axonal damage induced by intraneural injection.
Presynaptic terminal ultrastructure Classified on the basis of size, vesicle shape and synaptic site ultrastructure [4], axon terminals presynaptic to normal cat thoracic m o t o n e u r o n e s exhibited five morphological classes. Using nomenclature introduced by Conradi [4], these were classified as; S, F, T, M, and C. S-terminals exhibited spherical synaptic vesicles and several focal synaptic sites. F-terminals possessed pleomorphic or 'flattened' vesicles and focal synaptic sites. T-terminals had spherical vesicles, and focal synaptic sites which typically showed a row of two to five electron-dense particles associated with the postsynaptic m e m b r a n e 'density'. These particles were Taxi bodies. M-type terminals were large relative to the other classes, contained spherical synaptic vesicles and possessed several long focal synaptic sites, some of which were associated with a row of sub-synaptic Taxi bodies. The main characterising feature, however, was a small terminal presynaptic to the axo-somatic M-terminal, classified as the P-terminal. C-type terminals typically showed a high packing density of 50 nm spherical vesicles, an apparent lack of focal synaptic sites, and a narrow 15-nm-wide sub-synaptic cistern with an associated Nissl
490
491
Fig. 2. a Nissl body ultrastructure in normal cat thoracic motoneurone. Lumen width of rER cisterns = 20 nm. b-d Different thoracic motoneurones 8 days after an ipsilateral intraneural injection of diphtheria toxin, b Normal multilamellated Nissl structure and composition. Lumen width of rER cisterns = 40 nm. e Fragmentation and distension of Nissl body rER lamellae with
some loss of spatial relationship between rER and polyribosomal arrays. Mean lumen width = 180 nm. d Extreme dilatation and dissolution of residual Nissl body lamellae postsynaptic to a C-type presynaptic terminal, with degranulation of ER and loss of associated polyribosomal arrays. Mean lumen width >300 nm. Scale bars = 1 ~tm
body. T h e presence of C-type terminals on neurones ensured quantitative examinations were confined to a l p h a - m o t o n e u r o n e s [4]. All five classes apposed intoxicated m o t o n e u r o n e s (Figs. 3, 4), indicating that no class was selectively vulnerable to postsynaptic toxicity. Irrespective of morphological class, axon terminals presynaptic to normal m o t o n e u r o n e s exhibited an elec-
tron-lucent non-vacuolated cytoplasm, intact mitochondria with p r o m i n e n t cristae, and occassional short profiles of neurotubules. Synaptic clefts a p p r o x i m a t e d 20 n m in width and extended the complete length of terminals without interruption by glial cell processes or localities of extreme cleft widening. N o r m a l terminals did not possess p r o m i n e n t neurofilaments, E R , or contain m e m b r a n e u s inclusion bodies. In contrast, qualitative evidence suggested terminals were 'lost' f r o m the surface of intoxicated m o t o n e u r o n e s by two different methods: d e t a c h m e n t f r o m the neuronal surface without degenerative changes in the terminal (Fig. 3 a - f ) , and terminal degeneration with out prior d e t a c h m e n t (Fig. 4 a - d ) . Evidence of d e t a c h m e n t without degenerative changes included focal widenings of the synaptic cleft (Fig. 3a,b), separation of pre- and post-synaptic m e m b r a n e s along the m a j o r length of the synaptic apposition, penetration of astroglial processes into the widened cleft especially at the periphery of
Fig. 1. a The normal motoneurone viewed in a toluidine bluestained 0.5-~m 'plastic' section exhibits a smooth contour and posseses a central round nucleus. The cytoplasm contains several basophilic Nissl bodies and is unvacuolated, b Normal ultrastructure is characterised by multilamellated rough endoplasmic reticulum (rER) (arrow) and intact organelles, c,d Intoxicated motoneurones show characteristic crenellated nuclei, cytoplasmic vaculation, mitochondria swelling and disrupted rER. Scale bars = 10 ~m
492
Fig. 3a-f. Detachment without degeneration of axon terminals from intercostal motoneurones 8 days after an ipsilateral injection of diphtheria toxin showing: focal cleft widening (partial detachment; arrowed) in an S-type (a) and F-type terminal (b); insertion
of astroglial processes (arrowed) at the periphery of a T-type terminal (c); insinuation of astroglial processes (arrowed) along the entire synaptic cleft (d,e); total enclosure of terminals by astroglial processes (arrowed; f). Scale bars = 1 ~m
terminals (Fig. 3c-e), and enclosure of some terminals within a 'shell' of astroglial processes (Fig. 3d-f). Cleft widening (partial dysjunction) was seen in all terminals but total dysjunction was mainly apparent in S,T,F, and M-terminals. Only one example of C-type terminal detachement was encountered. Since C-type terminals comprise only 1% of the normal synaptic complement of cat thoracic motoneurones [18], a considerably larger number of C-type terminals than that analysed would be
required to obtain multiple examples of C-type terminal detachment (cf. quantitative data). The majority of terminals exhibiting cleft widening had a normal electron-lucent cytoplasm with normal or pleomorphic synaptic vesicles, mitochondria, and one or two small profiles of ER. In contrast, some S,T, F and M-terminals showed frank degenerative changes not necessarily accompanied by cleft widening (Fig. 4). Degenerative changes included two or more abnormal multisized
493
Fig. 4a-f. Wallerian-type degeneration. Degenerative changes include vacuolar inclusions and vesicle swelling (terminal in a), vesicular bodies with membraneous inclusions (b), increased neurofilament prominence (c), and abnormal cytoplasmic density
with accumulation of mitochondria (d). C-type terminals invaginate into the soma (e) and become totally absorbed into the postsynaptic motoneurone together with their subsynaptic cistern and associated Nissl body rER (f). Scale bars = 1 ~m
vacuoles (Fig. 4a), multivesicular bodies containing membrane fragments (Figs. 4a,b), and clumped synaptic vesicles (Fig. 4 b - d). Some terminals exhibited prominent neurofilaments (Fig. 4c) or an abnormal 'grey' cytosol and numerous mitochondria (Figs. 4d). Electron-dense degeneration of C-type terminals was not found. Instead, examples of resorption of the C-type presynaptic terminal into the soma (Fig. 4e,f) indicated
that some were lost from the motoneurone surface in the manner found after partial central deafferentation by spinal hemisection [18]. A small proportion of terminals with an electronlucent cytoplasm contained a mixture of spherical and polymophic synaptic vesicles. On the basis of the predominant form of vesicle shape, such terminals were assigned to one of two supplementary classes; S' (pre-
494
Morphometry - synaptic sites
dominantly spherical), and F' (predominantly flattened).
Pooled data also revealed a concomitant loss of synaptic sites from intoxicated motoneurones (Table 1). Numerically, synaptic sites expressed in relation to mean terminal apposition were 34 % of normal, and 25 % of normal when expressed per 100-~m neuronal membrane. While for normal motoneurones 40 % of terminals possessed > 1 synaptic site, in intoxicated motoneurones only 22 % exhibited >1 site, with 78 % displaying 1 or no sites.
Glia In this material glia associated with terminals exhibiting cleft widening and total detachment were invariably astrocytes: microglia rarely occurred in the immediate neuropil surrounding presynaptic terminals on either normal or intoxicated cat motoneurones. Isolated instances of microglia near to, but not engulfing degenerating terminals were encountered. One rare feature encountered in intoxicated motoneurones, but not normal neurones, is illustrated in Fig. 3f. Namely, a focal desmosome-like junction between an astroglial process displacing a presynaptic terminal from the postsynaptic membrane. These have previously been seen in association with motoneurones subjected to partial 'central' deafferentation by a rostral spinal hemisection. This lesion also induces terminal displacement and degeneration and is followed by glial cell occupancy of vacated synaptic territory [18].
Relative changes in terminal classes Proportional counts of the different morphological classes of presynaptic terminal apposed to the membranes of normal and intoxicated motoneurones confirmed the qualitative impression that alteration in terminal numbers involved all classes with no evidence of class selectivity. This conclusion remained valid following aggregation of counts for S and F terminals with the supplementary classes associated with intoxicated motoneurones, i.e. (S + S') and (F + F').
Morphometry - presynaptic terminals Relationship between presynaptic changes and development of neuronal abnormality
Morphometric data (Table 1) revealed that relative to normals, intoxicated motoneurones showed a 33 % reduction in mean terminal frequency, a 15 % reduction in mean length of presynaptic terminal apposition, and a 43 % reduction in overall presynaptic cover. Reductions in presynaptic cover were not attributable to differences in size between normal and intoxicated motoneurones since their relative diameters were similar (normals: range 26.5-58.9 ~tm, mean 40.7 + 7.8 ~m; intoxicated: range 3 3 . 5 - 56.8 9m, mean 39.5 + 5.8 9m; P >0.1). Values of R S E for terminal frequency and cover for normal motoneurones were 6.7 %, and 7 . 1 % , respectively (n = 30neurones). The higher RSE values obtained for intoxicated motoneurones (frequency: 10 %, cover: 11% ; n = 21 neurones) was more an effect of the wide spectrum of neuronal pathology within the pooled data, than lower sample n u m b e r (n = 21).
The relationships between altered presynaptic cover, including numbers of active synaptic sites, and the development of the neuronal lesion was examined using a two-step procedure first devised to analyse the progression of pathology in the Nissl body elements [17]. Nissl body r E R lumen width in normal motoneurones ranges from 11- to 40 nm (mean 21.2 + 5.2 nm; [17]), but post-intoxication r E R cisterns undergo distension, thereby extending lumen width to over 200 nm in intoxicated motoneurones (range 10-210 nm, mean 56 + 7.1 nm). While r E R distension occurs in other cytotoxic neuronopathies and is, therefore, probably a non-specific response to diphtheritic toxicity, lumen width is both measureable and variable. On this basis it provides a convenient morphometric index for catego-
Table 1. Pooled data from morphometric analyses of presynaptic terminal frequency, terminal apposition, overall presynaptic cover and synaptic site frequencies for normal and intoxicated cat thoracic motoneurones
Terminal frequency (no./100 ~tm neuronal membrane) Mean terminal apposition (~tm) Terminal coverage (Total terminal apposition/1004tm neuronal membrane) Mean no. Synaptic sites/terminal No. Synaptic sites/100-Bm terminal apposition No. Synaptic sites/100-p,m neuronal membrane
Intoxicated motoneurones n Range Mean sd
Normal motoneurones n Range Mean sd
se
30 11.6- 38.6
23.9
1.54 21 2.5-31.8
16.2
30 0.8- 2.8 30 8.2- 83.2
2.0 48.9
0.4 0.07 21 1.1- 2.6 19.1 3.49 21 6.6-63.1
1.7 28.2
30 0.9- 2.2 30 41.8-190
1.5 77.6
0.3 27.7
0.9 0.3 0.06 0.01
Acta Neuropathologtca (~ Springer-Verlag 1992
Presynaptic terminal loss from alpha-motoneurones following the retrograde axonal transport of diphtheria toxin* A. H. Pullen Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London, WC1N 3BG, UK Received July 22, 1991/Revised, accepted December 9, 1991
Summary. Intercostal motoneurones intoxicated following intraneural injection of diphtheria toxin exhibited a progressive dilatation and fragmentation of Nissl body rough endoplasmic reticulum (rER), coupled with two different forms of presynaptic terminal response. Firstly, terminal dysjunction without prior degeneration, and secondly, Wallerian-type degeneration. Dysjunction was attributed to a toxin-related failure by the motoneurones to maintain postsynaptic site structure. Degeneration was considered to arise from toxicity in presynaptic neurones, either neighbouring motoneurones or local interneurones. Morphometry revealed that by 8 days, intoxicated motoneurones exhibited a 33 % loss in terminal frequency, a 15 % loss in residual presynaptic membrane, and a 43 % loss in overall presynaptic input. The concomitant loss of synaptic sites was greater that the overall loss of presynaptic membrane, indicating a toxin-related deficiency of the maintenance of postsynaptic sites. Analyses of the relationship between changes in terminal numbers and the development of Nissl body abnormality in the postsynaptic motoneurone identified three groups of motoneurones: (i) those with normal presynaptic input and normal neuronal Nissl body rER; (ii) those showing a dramatic loss of presynaptic input and a marked dilatation and fragmentation of Nissl bodies; and (iii) neurones exhibiting a maintained or further loss of presynaptic input coupled with extreme dilatation and fragmentation of Nissl body r E R with loss of Nissl body structure. These changes are discussed in context with the known molecular action of diphtheria toxin.
sponding intercostal motoneurones [7, 19], as a result of inhibition of protein synthesis in the cell body [3, 11, 12]. The lesion is chiefly characterised by a progressive dissolution of Nissl body ultrastructure, including Nissl bodies associated with 'C'-type axon terminals [17, 19]. The neuronal lesion is accompanied by abnormal focal synaptic cleft widening and a proliferation of astroglial processes apposing the neuronal membrane. It is postulated here that these features are a prelude to presynaptic terminal withdrawal [1, 2, 22] and a direct result of the toxin-induced inhibition of synthesis of proteins forming synaptic sites and cell adhesion molecules. This hypothesis has been examined in intoxicated intercostal motoneurones by firstly delineating the qualitative and quantitative response of the presynaptic terminals, and secondly relating the presynaptic response to the development of Nissl body abnormalities in the postsynaptic neurone.
Materials and methods
Animals Results were obtained from 40 normal intercostal motoneurones from 3 adult cats, and 21 intercostal motoneurones (3 'experimental' cats) in which a diphtheritic lesion had fully developed. Prior to experimental surgical procedures and terminal perfusion-fixation, animals were anaesthetised with sodium pentobarbitone (45 mg/kg, i.p.), or ketamine-HC1 (30 mg/kg, i.m.; supplemented by 1.5 mg/kg xylazine to ameliorate muscle rigidity).
Key words: Spinal motoneurone - Diphtheria toxin Synapses - Dysjunction - Neurodegeneration
General procedure
Intraneural injections of diphtheria toxin (DTX) into cat intercostal nerves evoke a primary lesion in the corre-
In experimental animals, intercostal nerves in segments T7 or T10 were directly injected with 1-2 ~tl of a 20 ng/~tl solution of lyophilised DTX reconstituted in sterile PBS, pH 7.5, containing 0.1% BSA (dose = 0.01-0.02 flocculation units). Postoperative recovery was unremarkable and at post-mortem examination there were no signs of systemic intoxication (i.e. endocarditis, or haemorrhagic lesions in lungs, liver and mesentery.) Experimental and normal animals were perfusion-fixed under anaesthesia with
* Supported by the Medical Research Council and the Motor Neurone Disease Association
489 2.5 % glutaratdehyde-2 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal segments T7-T10 were removed, cut into 2-ram transverse slices, osmicated, and processed for electron microscopy.
Qualitative examinations Qualitative analyses of normal and experimental material were restricted to ventral horn neurones of >35-~tm diameter possessing 'C'-type presynaptic terminals on the somal membrane, i.e. alpha-motoneurones [4]. In experimental material analyses of synaptology were confined to motoneurones exhibiting the gross cytological abnormalities associated with diphtheritic toxicity [7, 19], and their presynaptic axon terminals were classified using prescribed ultrastructural criteria based on terminal size, vesicle shape and synaptic site ultrastructure [4].
Mo~hometry As in previous analyses of cat motoneurone synaptology [15, 18] morphometric analyses were undertaken on random profiles of normal and intoxicated motoneurones sectioned through the near midnuclear plane. Advantages of this method over methods based on serial- and semi-serial sections are discussed elsewhere [15]. Previous analyses of synaptology showed that estimates of relative standard error (RSE) for calculations of presynaptic terminal frequency and cover based on samples of either 10 or 15 neurones provided values of 14% (n = 10) and 9% (n = 15), where RSE = 100 (SE/mean) [15]. To reduce RSE further, sample sizes were increased to a minimum of 20 neurones. Electron micrograph photo montages (magnification x 25,000) were constructed for each motonenrone and computer-assisted techniques applied to obtain the following data [14]: the length of the neuronal membrane (~m); the numbers of presynaptic terminals (total, and for each terminal category); the appositional length of each presynaptic terminal (gm); and the numbers of synaptic specialisations (total, and for each terminal. From these were derived: (1) axon terminal frequencies (total, and for each category; no./100gm neuronal membrane); (2) axon terminal cover (total, and for each category; terminal appositional length (~tm)/100-~tmneuronal membrane); and (3) synaptic specialisation fiequency; expressed as (i) no./terminal, (ii) no./100-~tm terminal apposition, and (iii) No./100%tm neuronal membrane. Differences between normal, and experimental data were tested with Student's t-test. Distributions of size or frequency were tested with a Kolmgorov-Smirnow procedure.
Results
Motoneuronal ultrastructure Normal m o t o n e u r o n e s viewed in 0.5-~m 'plastic' sections stained with toludine blue (Fig. la) possessed a smooth-contoured neuronal m e m b r a n e and contained and a regularly shaped nucleus.The unstained cytoplasm contained a mixture of punctate dense-staining bodies (lysosomes) and larger basophilic regions representing Nissl bodies. Additional features of normal motoneurones seen in electron micrographs (Fig. lb) were intact mitochondria with prominent cristae, mulltilamellate Golgi cisterns, and highly structured Nissl bodies (Fig. 2a). Nissl bodies consisted of 10- to 40-nm wide cisterns of rough endoplasmic reticulum (rER), alter-
nating with linear arrays of lamellae-associated polyribosomes. No cytoplasmic vacuolation occurred in normal motoneurones. In confirmation of previous data [19], different motoneurones within the same spinal segment 5 - 8 days after injection of toxin exhibited a characteristic range of morphological abnormalities. In 0.5-gm 'plastic' sections, these ranged from normal cell shape and basophilia but slight cytoplasmic vacuolation and nuclear crenellation (Fig. lc), to extreme vacuolation. While some motoneurones showed reduced basophilia, others were hyperbasophilic. Ultrastructurally, motoneurones exhibited a corresponding wide range of abnormalities which ranged from slight vacuolation coupled with normal mitochondria and normal spatial relationships between Nissl body r E R , r E R - b o u n d ribosomes and lamellae-associated polyribosomes, to extreme cytoplasmic vacuolation, irregular cell shape, eccentric nuclei, and mitochondrial swelling with loss of cristae (Fig. ld). In these neurones, Nissl bodies displayed extreme dilatation of r E R cisterns (Fig. 2c-d), fragmentation of r E R lamellae, and loss of spatial relationships between r E R and polyribosomes. De-granulation occurred in only the severest stages of Nissl body dissolution. O t h e r neurones showed intermediate stages of Nissl body abnormality (Fig. 2c), suggesting that Nissl body dissolution was progressive. Motoneurones were uniform in their response. None showed a different form of pathology: for example, the acute loss of multilamellated Nissl body structure, loss of r E R , and formation of polyribosomal aggregates associated with intercostal nerve transection [8], which in these experiments would have indicated a neuronal response to axonal damage induced by intraneural injection.
Presynaptic terminal ultrastructure Classified on the basis of size, vesicle shape and synaptic site ultrastructure [4], axon terminals presynaptic to normal cat thoracic m o t o n e u r o n e s exhibited five morphological classes. Using nomenclature introduced by Conradi [4], these were classified as; S, F, T, M, and C. S-terminals exhibited spherical synaptic vesicles and several focal synaptic sites. F-terminals possessed pleomorphic or 'flattened' vesicles and focal synaptic sites. T-terminals had spherical vesicles, and focal synaptic sites which typically showed a row of two to five electron-dense particles associated with the postsynaptic m e m b r a n e 'density'. These particles were Taxi bodies. M-type terminals were large relative to the other classes, contained spherical synaptic vesicles and possessed several long focal synaptic sites, some of which were associated with a row of sub-synaptic Taxi bodies. The main characterising feature, however, was a small terminal presynaptic to the axo-somatic M-terminal, classified as the P-terminal. C-type terminals typically showed a high packing density of 50 nm spherical vesicles, an apparent lack of focal synaptic sites, and a narrow 15-nm-wide sub-synaptic cistern with an associated Nissl
490
491
Fig. 2. a Nissl body ultrastructure in normal cat thoracic motoneurone. Lumen width of rER cisterns = 20 nm. b-d Different thoracic motoneurones 8 days after an ipsilateral intraneural injection of diphtheria toxin, b Normal multilamellated Nissl structure and composition. Lumen width of rER cisterns = 40 nm. e Fragmentation and distension of Nissl body rER lamellae with
some loss of spatial relationship between rER and polyribosomal arrays. Mean lumen width = 180 nm. d Extreme dilatation and dissolution of residual Nissl body lamellae postsynaptic to a C-type presynaptic terminal, with degranulation of ER and loss of associated polyribosomal arrays. Mean lumen width >300 nm. Scale bars = 1 ~tm
body. T h e presence of C-type terminals on neurones ensured quantitative examinations were confined to a l p h a - m o t o n e u r o n e s [4]. All five classes apposed intoxicated m o t o n e u r o n e s (Figs. 3, 4), indicating that no class was selectively vulnerable to postsynaptic toxicity. Irrespective of morphological class, axon terminals presynaptic to normal m o t o n e u r o n e s exhibited an elec-
tron-lucent non-vacuolated cytoplasm, intact mitochondria with p r o m i n e n t cristae, and occassional short profiles of neurotubules. Synaptic clefts a p p r o x i m a t e d 20 n m in width and extended the complete length of terminals without interruption by glial cell processes or localities of extreme cleft widening. N o r m a l terminals did not possess p r o m i n e n t neurofilaments, E R , or contain m e m b r a n e u s inclusion bodies. In contrast, qualitative evidence suggested terminals were 'lost' f r o m the surface of intoxicated m o t o n e u r o n e s by two different methods: d e t a c h m e n t f r o m the neuronal surface without degenerative changes in the terminal (Fig. 3 a - f ) , and terminal degeneration with out prior d e t a c h m e n t (Fig. 4 a - d ) . Evidence of d e t a c h m e n t without degenerative changes included focal widenings of the synaptic cleft (Fig. 3a,b), separation of pre- and post-synaptic m e m b r a n e s along the m a j o r length of the synaptic apposition, penetration of astroglial processes into the widened cleft especially at the periphery of
Fig. 1. a The normal motoneurone viewed in a toluidine bluestained 0.5-~m 'plastic' section exhibits a smooth contour and posseses a central round nucleus. The cytoplasm contains several basophilic Nissl bodies and is unvacuolated, b Normal ultrastructure is characterised by multilamellated rough endoplasmic reticulum (rER) (arrow) and intact organelles, c,d Intoxicated motoneurones show characteristic crenellated nuclei, cytoplasmic vaculation, mitochondria swelling and disrupted rER. Scale bars = 10 ~m
492
Fig. 3a-f. Detachment without degeneration of axon terminals from intercostal motoneurones 8 days after an ipsilateral injection of diphtheria toxin showing: focal cleft widening (partial detachment; arrowed) in an S-type (a) and F-type terminal (b); insertion
of astroglial processes (arrowed) at the periphery of a T-type terminal (c); insinuation of astroglial processes (arrowed) along the entire synaptic cleft (d,e); total enclosure of terminals by astroglial processes (arrowed; f). Scale bars = 1 ~m
terminals (Fig. 3c-e), and enclosure of some terminals within a 'shell' of astroglial processes (Fig. 3d-f). Cleft widening (partial dysjunction) was seen in all terminals but total dysjunction was mainly apparent in S,T,F, and M-terminals. Only one example of C-type terminal detachement was encountered. Since C-type terminals comprise only 1% of the normal synaptic complement of cat thoracic motoneurones [18], a considerably larger number of C-type terminals than that analysed would be
required to obtain multiple examples of C-type terminal detachment (cf. quantitative data). The majority of terminals exhibiting cleft widening had a normal electron-lucent cytoplasm with normal or pleomorphic synaptic vesicles, mitochondria, and one or two small profiles of ER. In contrast, some S,T, F and M-terminals showed frank degenerative changes not necessarily accompanied by cleft widening (Fig. 4). Degenerative changes included two or more abnormal multisized
493
Fig. 4a-f. Wallerian-type degeneration. Degenerative changes include vacuolar inclusions and vesicle swelling (terminal in a), vesicular bodies with membraneous inclusions (b), increased neurofilament prominence (c), and abnormal cytoplasmic density
with accumulation of mitochondria (d). C-type terminals invaginate into the soma (e) and become totally absorbed into the postsynaptic motoneurone together with their subsynaptic cistern and associated Nissl body rER (f). Scale bars = 1 ~m
vacuoles (Fig. 4a), multivesicular bodies containing membrane fragments (Figs. 4a,b), and clumped synaptic vesicles (Fig. 4 b - d). Some terminals exhibited prominent neurofilaments (Fig. 4c) or an abnormal 'grey' cytosol and numerous mitochondria (Figs. 4d). Electron-dense degeneration of C-type terminals was not found. Instead, examples of resorption of the C-type presynaptic terminal into the soma (Fig. 4e,f) indicated
that some were lost from the motoneurone surface in the manner found after partial central deafferentation by spinal hemisection [18]. A small proportion of terminals with an electronlucent cytoplasm contained a mixture of spherical and polymophic synaptic vesicles. On the basis of the predominant form of vesicle shape, such terminals were assigned to one of two supplementary classes; S' (pre-
494
Morphometry - synaptic sites
dominantly spherical), and F' (predominantly flattened).
Pooled data also revealed a concomitant loss of synaptic sites from intoxicated motoneurones (Table 1). Numerically, synaptic sites expressed in relation to mean terminal apposition were 34 % of normal, and 25 % of normal when expressed per 100-~m neuronal membrane. While for normal motoneurones 40 % of terminals possessed > 1 synaptic site, in intoxicated motoneurones only 22 % exhibited >1 site, with 78 % displaying 1 or no sites.
Glia In this material glia associated with terminals exhibiting cleft widening and total detachment were invariably astrocytes: microglia rarely occurred in the immediate neuropil surrounding presynaptic terminals on either normal or intoxicated cat motoneurones. Isolated instances of microglia near to, but not engulfing degenerating terminals were encountered. One rare feature encountered in intoxicated motoneurones, but not normal neurones, is illustrated in Fig. 3f. Namely, a focal desmosome-like junction between an astroglial process displacing a presynaptic terminal from the postsynaptic membrane. These have previously been seen in association with motoneurones subjected to partial 'central' deafferentation by a rostral spinal hemisection. This lesion also induces terminal displacement and degeneration and is followed by glial cell occupancy of vacated synaptic territory [18].
Relative changes in terminal classes Proportional counts of the different morphological classes of presynaptic terminal apposed to the membranes of normal and intoxicated motoneurones confirmed the qualitative impression that alteration in terminal numbers involved all classes with no evidence of class selectivity. This conclusion remained valid following aggregation of counts for S and F terminals with the supplementary classes associated with intoxicated motoneurones, i.e. (S + S') and (F + F').
Morphometry - presynaptic terminals Relationship between presynaptic changes and development of neuronal abnormality
Morphometric data (Table 1) revealed that relative to normals, intoxicated motoneurones showed a 33 % reduction in mean terminal frequency, a 15 % reduction in mean length of presynaptic terminal apposition, and a 43 % reduction in overall presynaptic cover. Reductions in presynaptic cover were not attributable to differences in size between normal and intoxicated motoneurones since their relative diameters were similar (normals: range 26.5-58.9 ~tm, mean 40.7 + 7.8 ~m; intoxicated: range 3 3 . 5 - 56.8 9m, mean 39.5 + 5.8 9m; P >0.1). Values of R S E for terminal frequency and cover for normal motoneurones were 6.7 %, and 7 . 1 % , respectively (n = 30neurones). The higher RSE values obtained for intoxicated motoneurones (frequency: 10 %, cover: 11% ; n = 21 neurones) was more an effect of the wide spectrum of neuronal pathology within the pooled data, than lower sample n u m b e r (n = 21).
The relationships between altered presynaptic cover, including numbers of active synaptic sites, and the development of the neuronal lesion was examined using a two-step procedure first devised to analyse the progression of pathology in the Nissl body elements [17]. Nissl body r E R lumen width in normal motoneurones ranges from 11- to 40 nm (mean 21.2 + 5.2 nm; [17]), but post-intoxication r E R cisterns undergo distension, thereby extending lumen width to over 200 nm in intoxicated motoneurones (range 10-210 nm, mean 56 + 7.1 nm). While r E R distension occurs in other cytotoxic neuronopathies and is, therefore, probably a non-specific response to diphtheritic toxicity, lumen width is both measureable and variable. On this basis it provides a convenient morphometric index for catego-
Table 1. Pooled data from morphometric analyses of presynaptic terminal frequency, terminal apposition, overall presynaptic cover and synaptic site frequencies for normal and intoxicated cat thoracic motoneurones
Terminal frequency (no./100 ~tm neuronal membrane) Mean terminal apposition (~tm) Terminal coverage (Total terminal apposition/1004tm neuronal membrane) Mean no. Synaptic sites/terminal No. Synaptic sites/100-Bm terminal apposition No. Synaptic sites/100-p,m neuronal membrane
Intoxicated motoneurones n Range Mean sd
Normal motoneurones n Range Mean sd
se
30 11.6- 38.6
23.9
1.54 21 2.5-31.8
16.2
30 0.8- 2.8 30 8.2- 83.2
2.0 48.9
0.4 0.07 21 1.1- 2.6 19.1 3.49 21 6.6-63.1
1.7 28.2
30 0.9- 2.2 30 41.8-190
1.5 77.6
0.3 27.7
0.9 0.3 0.06 0.01
