Increase in presynaptic territory of C-terminals on lumbar motoneurons of G93A SOD1 mice during disease progression.

Compensatory synaptic plasticity is reported in muscle and the central nervous system of motor neuron disease patients, and transgenic SOD1 mice, but direct ultrastructural evidence for spinal motoneurons is lacking. Prompted by our observation in spinal cords from autopsied patients suggesting selective enlargement of the ultrastructurally distinctive C-type terminal synapsing with spinal motoneurons, we examined the ultrastructural synaptology of lumbar motoneurons during disease progression in age- and sex-matched wild-type mice, transgenic G93A SOD1 mice, and mice overexpressing normal human SOD1 (Wt(SOD1)). Prescribed criteria classified presynaptic terminals of motoneurons into five ultrastructural classes (S, F, T, M, and C). Computerized morphometry on electronmicrographs was used to measure their appositional lengths, coverage of the motoneuron membrane, and sizes of postsynaptic structures. No terminal degeneration occurred in wild-type or Wt(SOD1) mice. In transgenic mice, degeneration of motoneurons and S-terminals and F-terminals commenced presymptomatically (10 weeks), and continued into the symptomatic stage (18 weeks). However, C-terminals were preserved. Morphometry confirmed significant reductions in frequency and membrane coverage for S-terminals and F-terminals between 10 and 18 weeks, but a maintained frequency of C-terminals coupled with increased appositional length and coverage of the motoneuron membrane. Increased C-terminal size was matched by growth of its characterizing postsynaptic cistern and Nissl body. The results reveal selective preservation and increased presynaptic territory of the C-type terminal. As C-terminals derive from cholinergic intrasegmental propriospinal interneurons and may modulate motoneuron excitability, their increased presynaptic territory on surviving motoneurons of transgenic SOD1 mice may represent a means of maintaining excitability, compensating for the loss of overall presynaptic input.

Serine proteases purified from sera of patients with amyotrophic lateral sclerosis (ALS) induce contrasting cytopathology in murine motoneurones to IgG.

Affinity purified IgG from sera of patients with amyotrophic lateral sclerosis (ALS) is claimed to enhance transmitter release, induce apoptotic death of cultured motoneurones, and elicit a distinctive cytopathology with raised Ca(2+) in mouse motoneurones. An alternative hypothesis attributes these events to serine proteases in ALS sera. To test this, motoneurones in BALB/c mice injected intraperitoneally with plasminogen affinity purified from sera of ALS patients and healthy controls were analysed using immunochemical and ultrastructural morphometric methods. The responses were validated in motoneurones of mice injected with commercially purified plasminogen, tissue plasminogen activator (tPA), or plasmin. Motoneurones in non-injected mice had normal morphology and ultrastructure without evidence of electron-dense degeneration. Purified plasminogen from both ALS patients and healthy controls, evoked electron-dense motoneurone degeneration, as did commercially purified plasminogen and tPA. The common cytopathology comprised disruption and distension of Nissl body rough endoplasmic reticulum, cytoplasmic polyribosomal proliferation, and significant Ca(2+) enhancement in mitochondria. By contrast, using affinity purified serum immunoglobulins, ALS-IgG but not IgG from healthy or disease controls, elicited necrosis, with 30% of ALS-IgGs tested evoking electron-dense degeneration in 40% of motoneurones. The primary cytopathology was extensive swelling of Golgi endoplasmic reticulum and mitochondria, with enhancement of Ca(2+) in Golgi endoplasmic reticulum and presynaptic boutons. We conclude that serine proteases purified from sera of ALS patients elicits a distinctive cytopathology and pattern of Ca(2+) enhancement in motoneurones different from that found on passive transfer of affinity purified ALS-IgG.

The pro and the active form of matrix metalloproteinase-9 is increased in serum of patients with amyotrophic lateral sclerosis.

Pro and active-matrix metalloproteinase-9 (MMP-9) was measured in sera from patients with amyotrophic lateral sclerosis (ALS), Guillain-Barre syndome (GBS), and healthy subjects. Both forms of MMP-9 were elevated in sera of ALS and GBS patients, compared with healthy controls. It has been postulated that elevated MMP-9 reflects damage to peripheral nerve and muscle. This possibility was investigated in sera, and tissue extracts of sciatic nerves and muscle from mice 5 and 12 days after axotomy of the sciatic nerve. Pro-MMP-9 was elevated in sera and extracts of damaged nerve and muscle, suggesting such damage may be followed by elevated pro-MM9-9 in sera. Active MMP-9 was only elevated in the sera. However, in situ activation of MMP-9 is tightly regulated and localised, and probably difficult to demonstrate by ELISA, resulting in a short half-life active MMP-9, implying any active MMP-9 in the serum may have a more immediate origin than injured muscle or nerve, for example circulating blood cells.

Passive transfer of purified IgG from patients with amyotrophic lateral sclerosis to mice results in degeneration of motor neurons accompanied by Ca2+ enhancement.

It has been reported that immunoglobulins (IgG) in sera of patients with amyotrophic lateral sclerosis (ALS) kill cultured motoneurones (MN), but whether they also cause MN degeneration in vivo is unclear. To test this, protein-A affinity purified and dialysed IgGs were prepared from sera of 44 ALS patients without paraproteinemias, 20 healthy controls and 15 disease controls. Control and ALS-IgGs were injected intraperitoneally into groups of mice for 5 consecutive days and examined at day 8. IgG was localised immunocytochemically and spinal MN were characterised histologically and ultrastructurally and by comparative counts of Ca(2+) containing organelles revealed with oxylate-pyroantimonate histochemistry. ELISA revealed no differences in IgG concentration between ALS patients and control subjects. Immunocytochemistry showed IgG was present in MN of mice injected with control or ALS-IgG, but densitometry showed immunostaining in MN was stronger in mice injected with ALS-IgG. Compared to MN of non-injected mice, control-IgG-treated mice showed near normal MN morphology and numbers of Ca(2+)-containing organelles. Disease control IgGs evoked negligible or minor morphological changes according to disease, but normal numbers of Ca(2+) containing organelles. Ultrastructurally, about 70% of ALS-derived IgGs induced a population of MN with electron lucent cytoplasm, distended Golgi, disrupted Nissl and mitochondria (i.e., necrosis). However 30% of ALS-IgGs additionally induced electron-dense degeneration in 40% of the MN. These MN exhibited shrinkage, condensed nuclear chromatin and ill-defined nuclear membranes and resembled preliminary stages of apoptosis. We conclude that passive transfer of ALS-derived, but not control IgGs, does result in MN degeneration in the recipient mice. This appears to be associated with abnormal calcium homeostasis, but the exact target of ALS-IgG remains conjectural, and the possibilities are discussed.

Ultrastructural analysis of spinal motoneurones from mice treated with IgG from ALS patients, healthy individuals, or disease controls.

Reports that ALS-IgG injected into mice results in ultrastructural abnormalities and enhanced deposition of Ca(2+) in their spinal motoneurones are unverified. To obtain verification, affinity purified IgG's from ten healthy subjects, seventeen ALS patients and eight disease controls (e.g. cases of LEMS, MS,) were injected into groups of mice in 4 daily doses by either i/m injection, or i/p (Total doses, 2-4 mg i/p, 1 mg i/m). Immunocytochemistry identified human IgG in lumbar motoneurones 48 h after the final dose. Their morphology was examined by EM and intraneuronal Ca2+ was revealed by oxylate-pyroantimonate histochemistry and its identity verified by 2 degrees emission spectroscopy. In the EM, motoneurones of non-injected mice, and mice receiving healthy IgGs had a lucent cytoplasm, intact mitochondria, and Golgi complexes comprising stacks of narrow ER. About 40% of Nissl bodies comprised alternate rER lamellae and polyribosome arrays (Type 1 structure): 10% formed polyribosome clusters (Type 3). Mitochondria, Golgi ER and presynaptic terminals contained Ca(2+) associated pyroantimonate. I/m and i/p ALS-IgG produced similar results. Some ALS IgGs (i.e. patients) produced electron dense degenerative cytology: all promoted fragmented and distended Golgi ER, polyribosmal hyperplasia, reduced numbers of Type I but raised numbers of Types 2 and 3 Nissl bodies, and a greater proportion of Golgi ER and presynaptic terminals containing Ca(2+)-antimonate. With 4/8 disease control IgGs motoneurones had normal Golgi and Nissl body organisation but dilated rER. Ca2+ content was normal. Remaining IgGs produced normal ultrastructure. Results support claims that ALS-IgG may be cytotoxic, and that it enhances the Ca(2+) content of motoneurones and synaptic terminals.

Protracted elevation of neuronal nitric oxide synthase immunoreactivity in axotomised adult pudendal motor neurons.

Neuronal nitric oxide synthase immunoreactivity (NOS1-ir) in sacral motor neurons of normal adult cats was compared with that in cats surviving 1-10 wk after unilateral transection and ligation of the pudendal nerve. Levels of immunostaining were measured by microdensitometry. In nonoperated cats 60% of motor neurons in the ventrolateral nucleus (VL) and Onuf's nucleus (ON) showed high levels of NOS1-ir with lower NOS1-ir in 40%. Following axotomy, motor neurons in ON on both sides of the cord showed an acute rise in mean level of NOS1-ir at 1 wk, with a further increase at 2 wk. Mean levels of NOS1-ir in the ipsilateral and contralateral ON remained elevated at 10 wk after axotomy. Elevation of NOS1-ir occurred in the VL with a similar time-course to that in ON, implying a wider response in motor nuclei synaptically coupled to ON. Measurements of neuronal size in ON and VL revealed an increase in neuronal size in ON but not VL, indicating increased NOSI-ir in ON was not an artifact of neuronal atrophy. The proportion of motor neurons in ON and VL possessing higher levels of NOS1-ir increased from 60% in controls to 100% at 2-3 wk postaxotomy. The proportion slightly declined by 8 wk due to re-emergence of motor neurons exhibiting low NOS1-ir, but remained greater than normal at 10 wk in both nuclei. Based on evidence from related analyses of synaptology, we argue that acute axotomy induced alterations in presynaptic complement which increased overall Ca2+ influx and thereby stimulated NOS1-ir.

Time-dependent alterations in NOS1 immunoreactivity in feline pudendal motoneurons following retrograde axonal transport of diphtheria toxin.

Neuronal nitric oxide synthase immunoreactivity (NOS1-ir) in sacral somatic motor neurons of normal adult cats was compared with NOS1-ir in cats surviving 1 to 10 weeks after injection of the ADP-ribosylating protein diphtheria toxin (DTX) into one-half of the external anal sphincter. Levels of immunostaining were measured by microdensitometry. In non-operated cats, 60% of motor neurons in the ventrolateral (VL) and Onuf's nucleus (ON) showed high levels of NOS1-ir with lower NOS1-ir in 40%. Intramuscular injection of DTX caused cytopathology in motoneurons in ON, but not in VL with onset at 1 week, and regression by 10 weeks. Immunocytochemistry and microdensitometry disclosed an associated rise in levels of NOS1-ir in both the ipsilateral and contralateral ON at 1 week, which persisted up to 4 weeks, but reduced to normality by 10 weeks. Simultaneous neuronal swelling in ON precluded raised staining intensity being an artifact of neuronal atrophy. Despite restriction of cytopathology to ON, motoneurons in VL also exhibited acute elevation with subsequent normalisation of NOS1-ir over an identical time-course. Conclusions. Since DTX inhibits protein synthesis, (i) activation of NOS1 in acute toxicity probably reflects raised intracellular calcium due to loss of calcium homeostasis; (ii) the bilateral response in ON may indicate uptake of DTX by contralateral pudendal axons crossing the sphincter midline; and (iii) raised NOS1-ir in VL indicates a wider response in nuclei synaptically coupled to ON. Recovery of neuronal morphology and normalisation of NOS1-ir in sublethal toxicity contrast with the protracted elevation of NOS1-ir reported by others following axonal lesions associated with neuronal death and muscle target deprivation.

Comparative analysis of nitric oxide synthase immunoreactivity in the sacral spinal cord of the cat, macaque and human.

Nitric oxide synthase immunoreactivity (bNOS-ir) was examined in the sacral spinal cord of the cat, macaque monkey and human using an antibody to the c-terminal region of neuronal NOS. In S2 of all 3 species NOS-ir was identified in both dorsal and ventral horns. In cat, monkey and human, bNOS-r occurred in sensory neurons of superficial laminae and the base of the dorsal horn, in autonomic neurons around the central canal and in the intermediolateral sacral spinal nucleus. In all 3 species, a large proportion of somatic motor nuclei in the ventromedial (VM), ventrolateral (VL) nuclei, and Onuf's nucleus (ON) showed high bNOS-ir, while others exhibited markedly lower immunoreactivity. Validatory experiments showed separate cellular localisation for bNOS, inducible NOS (iNOS), and endothelial NOS (eNOS) with only bNOS being localised to neuronal perikarya. Comparative morphometric analyses of the relative proportions and diameters of motor neurons in the VL, VM and ON exhibiting high and low levels of bNOS-ir revealed statistically significant differences in proportions in individual nuclei, and differences in size were generally not statistically significant. Finally, a comparison between cat sacral and thoracic spinal cord showed bNOS-ir in motor neurons of S2 was subject to less animal and rostrocaudal segment variation than in T10.

Morphological and morphometric characterisation of Onuf's nucleus in the spinal cord in man.

In the absence of a systematic morphometric study of Onuf's nucleus in man, this investigation defines the limits of variation of segmental position and the range of length and volume of Onuf's nucleus in 6 normal humans displaying no neurological disease (2 males, 4 females). Serial section reconstruction methods in conjunction with the disector method provided information on the numbers, sizes and shapes of the constituent motor neurons of Onuf's nucleus. In contrast to previous descriptions, the cranial origin of Onuf's nucleus occurred in rostral S1 in 50% of subjects, and midcaudal S1 in the remaining subjects. Onuf's nucleus varied in length between 4 and 7 mm, and was 0.2-0.37 mm3 in volume. Differences in length or volume between males or females, or between the left and right side of the cord were not statistically significant. Neurons in Onuf's nucleus varied in diameter between 10 microns and 60 microns (mean 26 microns) and their mean number was 625 +/- 137. A higher density of neurons occurred at the cranial and caudal ends of the nucleus relative to the middle. While 37% of neurons were approximately spherical (shape index approximately 1), 44% were ellipsoid and 19% fusiform (shape indices varying between 0.26 and 0.8). These findings are compared with previous studies of Onuf's nucleus in man and animals. The results form a basis for further studies on Onuf's nucleus in normality and neurodegenerative diseases.

Diversity in localisation of nitric oxide synthase antigen and NADPH-diaphorase histochemical staining in sacral somatic motor neurones of the cat.

Nitric oxide synthase (NOS) immunoreactivity occurred in about 60% of ventromedial, ventrolateral, and sphincteric motoneurones in cat sacral spinal cord. Proportions of sacral motoneurones histochemically stained for NADPH-diaphorase, were similar, and equivalent in size to those immunoreactive for NOS, suggesting co-localisation of diaphorase and NOS. Double staining techniques revealed that NOS co-localised with NADPH-diaphorase in approximately 60% of sacral motoneurones. Some remaining motoneurones exhibited neither NOS nor NADPH-diaphorase, while others exhibited only NOS or NADPH-diaphorase, indicating phenotypic differences among sacral motoneurones.

Ultrastructural abnormalities with inclusions in Onuf's nucleus in motor neuron disease (amyotrophic lateral sclerosis).

This study describes an ultrastructural examination focused on motor neurons in Onuf's nucleus in the spinal cord of four control patients without neurological disease (45-70 years) and six motor neuron disease (MND) patients (38-79 years; duration 8 months-19 years) who showed no somato-vesical dysfunction. Prompted by recent studies suggesting some sphincteric motor neurons may succumb to MND, this study sought to determine whether the wider population of neurons in Onuf's nucleus display ultrastructural cytopathology which is normally undetectable in histological preparations. Spinal cords were removed 3-20 h after death, and 1 mm slices of cord rapidly fixed in modified Karnovsky medium were processed for both light- and electronmicroscopy. 'Control motor neurons' had intact neuronal and nuclear membranes. Nissl bodies chiefly comprised ordered structures of alternate lamellae of rough endoplasmic reticulum and arrays of polyribosomes. The Golgi complexes consisted of multilamellated curvilinear stacks of ER. No intraneuronal filamentous or Bunina body inclusions were observed, but occasional axonal spheroids were seen in the neuropil. In MND, histological evidence of sparing in Onuf's nucleus was associated with abnormal ultrastructure of the motor neurons. Some sphincteric neurons were atrophic, whereas in the others, Nissl bodies were reduced in number, showed loss of structural organization or comprised polyribosomal aggregates. Golgi complexes had disrupted lamellated organization or consisted solely of distended ER. Intraneuronal filamentous Lewy-body or skein-like inclusions and Bunina bodies were identified in Onuf's nucleus of three subjects (duration of MND 8 months-2 years). The results of the present study indicate that Onuf's nucleus is vulnerable in MND, and preservation of sphincter function with qualitative histological evidence of 'sparing' does not necessarily imply a corresponding lack of ultrastructural cytopathology in this nucleus.

Phosphorylated neurofilament antigen redistribution in intercostal nerve subsequent to retrograde axonal transport of diphtheria toxin.

A novel enzyme-linked immunosorbent assay technique using specific monoclonal antibodies has been used to examine the proximal-distal distribution of phosphorylated neurofilament proteins (pNF) in normal feline intercostal nerve, and to compare it with that following retrograde axonal transport of the ADP-ribosylating protein diphtheria toxin (DTX) to thoracic motoneurones. The molecular target of DTX is elongation factor 2 which resides solely in the cell body. Normal intercostal nerves exhibited significantly higher amounts of the 200-kDa pNF-H, 160-kDA pNF-M, and 68-kDA pNF-L in proximal nerve than in the distal nerve. The overall content of all three triplet pNF proteins decreased 3 days after injection of DTX, but the normal proximal-distal gradient was retained. By 8 days post DTX injection, the proximal-distal gradient had reversed, with proximal nerve starved of pNF-H and pNF-M and distal nerve showing abnormally high pNF-L content. Correlative immunocytochemistry of spinal cords from normal animals verified that pNF-H and pNF-M are confined to efferent axons in the spinal grey matter, and that motoneurones are only reactive for pNF-L. At 8 days following toxin treatment, motoneurones in the ipsilateral ventral horn were strongly immunoreactive for all pNF. Contralateral motoneurones were non-reactive. Onset of abnormal perikaryal pNF immunoreactivity at 3 days precedes onset of ultrastructural cytopathology. Together these results indicate an early deficit in transference to the axon of NF proteins synthesised prior to full toxicity, probably because of a toxin-induced failure in regulation of phosphorylation-dependent NF assembly and turnover immediately prior to entry into the proximal axon. Results are discussed in relation to diphtheritic motoneuronopathy.

Neurofilament reorganisation and neurofilament antigen redistribution in spinal motoneurones following retrograde axonal transport of diphtheria toxin.

Single unilateral injections of diphtheria toxin (DTX) into the external anal sphincter muscle or internal intercostal nerve of cat induced characteristic ultrastructural lesions in corresponding ipsilateral spinal motoneurones 6-8 days later. The chief neuronal lesion was a progressive disruption of Nissl body composition and organisation, which between days 8-19 post injection was accompanied by a progressive accumulation of neurofilaments in motoneuronal perikarya and dendrites. Some axons in the ipsilateral ventral horn became hypertrophied due to neurofilamentous accumulation. Related immunocytochemical investigations 6-35 days after injection of DTX revealed abnormal immunoreactivity intoxicated motoneurones for 200-kDa and 160-kDa phosphorylated neurofilament proteins, but not in contralateral motoneurones. By day 35 abnormal neurofilament immunostaining also occurred in ipsilateral and some contralateral interneurones but not contralateral motoneurones. Abnormalities of Nissl body endoplasmic reticulum, neurofilament organisation, and neurofilament protein immunostaining were identical after either intraneural and intramuscular injections of DTX, indicating abnormalities were attributable to toxicity and not injection-related axonal damage. Since DTX acts specifically in the soma to inhibit protein synthesis, neurofilament abnormalities are secondary to cytotoxicity and probably result from deficits in transference of existing partially phosphorylated neurofilaments to the axonal transport system, or axonal transport per se.

Ultrastructure of pre-synaptic input to motor neurons in Onuf's nucleus: controls and motor neuron disease.

Ultrastructural analyses of sphincteric motoneurons in Onuf's nucleus at S2 were undertaken in human spinal cord obtained 3-6 h post-mortem from three subjects with no neurological disease ('controls') and five in which death was due to motor neuron disease (MND). Neurons in specified locations within Onuf's nucleus of control subjects ranged between 17.8 and 71.7 microns diameter (mean 38.6 microns). Analyses of synaptology revealed five ultrastructural classes of presynaptic terminal synapsing with the neuronal surface membrane. When classified by size, vesicle morphology, and synaptic site structure these conformed to the S, F, T, M and C-terminals defining somatic motoneurons. No terminals characteristic of autonomic motoneurons were found. In MND subjects, neurons in Onuf's nucleus at S2 were preserved despite a paucity of neurons in medial and lateral motor nuclei and were of similar size range to those in control subjects. The morphological classes of pre-synaptic terminal found in controls, also characterized sphincteric motoneurons in MND subjects, including the C-type terminal. The presence of C-terminals indicates (i) that sphincteric motoneurons are somatic alpha-motoneurons, and (ii) that hypotheses explaining the survival of sphincteric motoneurons in MND on the basis of Onuf's nucleus being an extension of the pre-ganglionic parasympathetic nucleus, or having intrinsic autonomic properties are incorrect.

Presynaptic terminal loss from alpha-motoneurones following the retrograde axonal transport of diphtheria toxin.

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 rER with loss of Nissl body structure. These changes are discussed in context with the known molecular action of diphtheria toxin.

An in vitro brainstem preparation preserving peripheral auditory function.

We describe an in vitro preparation of the brainstem and auditory periphery of the turtle. Properties of cochlear nucleus units recorded from this preparation are consistent with studies of both behavioural responses and auditory nerve fibre activity. A modified preparation for in vitro HRP tracing is also described.

Morphometric evidence from C-synapses for phased Nissl body response in alpha-motoneurones retrogradely intoxicated with diphtheria toxin.

Diphtheria toxin (DTX) kills cells by inactivating ribosomal translocation and when used to retrogradely intoxicate cat intercostal motoneurones produces marked morphological alterations in Nissl bodies, including those specifically sited postsynaptic to C-type axon terminals. Here, qualitative examinations of 'intoxicated' postsynaptic Nissl bodies reveal a progressive structural alteration marked by rER dilatation, rER lamellae fragmentation but retention of both the highly ordered multilamellate organization and ribosomal attachment until final stages of Nissl body dissolution. Morphometric results identified 3 broad phases to the postintoxication response which differed in the degree of rER cisternal dilation, and the numerical and spatial relationships between rER-lamellae, rER-bound ribosomes and rER-associated polyribosomes. These phases reflect the known molecular basis of diphtheritic toxicity and contrast with the fast developing Nissl body reaction associated with the neurotoxin ricin which also invokes ribosomal dysfunction and has been used to mimic certain features of motor neurone disease. The cytopathology of DTX and ricin are compared in the Discussion.

Quantitative synaptology of feline motoneurones to external anal sphincter muscle.

Motoneurones innervating the cat external anal sphincter muscle were labelled retrogradely following intramuscular injections with horseradish peroxidase (HRP). Labelled motoneurones were examined by correlative light and electron microscopy (LM and EM) with special regard to a qualitative and morphometric analysis of the axon terminals resident on the neuronal membrane. By LM, labelled motoneurones were (1) ipsilateral to the injections; (2) all in S1-S2; (3) found only in the superior dorsomedial region of Onuf's nucleus; and (4) exhibited a broad spectrum of diameters (25-72 micron, mean 47.4 +/- 11.3 micron). By EM, axon terminals on the neuronal membrane when classified according to size, vesicle shape, and synaptic complex ultrastructure conformed to the S-, F-, T-, M-, and C-type terminals previously described for cat lumbosacral motoneurones. C-terminals confirmed these sphincteric motoneurones to be skeletomotor. Pooled data from midnuclear sections through 15 random labelled motoneurones (20-64-micron diameter) revealed that S- and F-type terminals predominated, with numerically few M and C types. Notwithstanding their low frequency (0.3/100 micron membrane) C-terminals contributed 1% of the mean areal coverage by terminals, which implies a potentially larger synaptic influence relative to other terminal types. Linear relationships occurred between terminal frequency (or cover) and motoneurone diameter. While motoneurones greater than 40 micron in diameter exhibited all five terminal types, labelled motoneurones less than or equal to 30 micron generally possessed only S-, F-, and occasional T-type terminals, and in this respect resembled gamma motoneurones.

Feline 'C'-type terminals possess synaptic sites associated with a hypolemmal cistern and Nissl body.

Previous findings suggest that axosomatic 'C'-type terminals on rat spinal motoneurones possess 'active' synaptic sites associated with the characteristic subsynaptic cistern and postsynaptic Nissl body, but 'C'-terminals in cat do not. A re-examination of feline 'C'-terminals undertook to compare the synaptic ultrastructure in aldehyde-fixed material stained by either osmium or ethanolic phosphotungstic acid (E-PTA). In agreement with previous findings osmicated cat 'C'-terminals failed to reveal synaptic complexes in regions possessing the subsynaptic cistern ('cisternal regions'). In contrast, cisternal regions of E-PTA stained 'C'-terminals exhibited a linear array of presynaptic dense projection all opposed by a single extended postsynaptic density which abutted directly onto both postsynaptic membrane and the cistern. This close topographical relationship is suggested to be functionally significant in the trans-synaptic trophism previously demonstrated for feline 'C'-terminals.

Target dependence of Nissl body ultrastructure in cat thoracic motoneurones.

The effects on Nissl body (NB) ultrastructure of muscle reinnervation or neuroma formation were determined in cytochemically identified cat thoracic motoneurones subjected to axotomy by either nerve crush or nerve section with proximal ligation. Normal NB ultrastructure comprised highly ordered lamellae of rough endoplasmic reticulum (RER) with associated linear arrays of unbound polyribosomes. This ultrastructural orderliness was lost following axotomy, with or without light microscopic chromatolysis. While NBs were seen in the light microscope at late stages following both nerve crush or ligation, normal NB ultrastructure was only observed following nerve crush. An inductive effect of the periphery on NB ultrastructure is proposed and the implication of NB ultrastructure discussed in relation to protein synthesis.