Ultrastructural studies of normal and degenerating mouse neuromuscular junctions

Summary A quantitative study of the ultrastructural changes occurring at degenerating motor axon terminals of the mouse hemidiaphragm in the first 26 h following unilateral phrenicotomy has been made. Several ultrastructural characteristics of axon terminals from normal diaphragms and phrenicotomized and control hemidiaphragms of phrenicotomized preparations were analyzed. Considerable swelling and disruption of mitochondria occurred in terminals of control hemidiaphragms at 3, 6 and 9 h post-phrenicotomy after which no more damage than that seen in normal terminals after fixation for electron microscopy was observed. At terminals of the phrenicotomized hemidiaphragm, much mitochondrial damage was observed from 3 h post-phrenicotomy onwards. In phrenicotomized hemidiaphragms all terminals had completely degenerated by 26 h post-phrenicotomy. Reduced synaptic vesicle population densities occurred during degeneration. At many axon terminals on phrenicotomized hemidiaphragms the population densities of synaptic vesicles were reduced compared with controls and vesicle aggregates were noted in many engulfed or partially engulfed nerve terminals.These results are discussed with respect to the vesicle hypothesis for nerve-muscle transmission. The mechanisms underlying Schwann cell hyperactivity are also considered.     

Sequential events of degeneration and synaptic remodelling in the viper optic tectum following retinal ablation. A degeneration, radioautographic and immunocytochemical study

The ultrastructural changes taking place in the retino-recipient layers of the viper optic tectum were examined between 5 and 122 days after retinal ablation. The initial degeneration of retinotectal terminals proceeds at widely different rates and is characterized by a marked degree of polymorphism in which a number of different patterns can be discerned. In the final stages of degeneration, either both the degenerating bouton and the distal portion of the postsynaptic element are engulfed by reactive glia, or, more frequently, only the degenerating terminal is eliminated and the postsynaptic differentiation remains. The free postsynaptic differentiations are reoccupied predominantly by boutons containing pleiomorphic vesicles and which are for the most part gamma-aminobutyric acid (GABA)ergic, thus forming heterologous synapses; less frequently these sites are occupied by boutons of the ipsilateral visual contingent to form homologous synapses. These two processes, both of which depend on terminal axonal sprouting, take place within the first 3 postoperative months. They are followed by a decrease in the number of heterologous synapses and a concurrent increase in the number of homologous synapses newly formed by optic boutons generated by collateral preterminal sprouting of ipsilateral retinotectal fibres. The data suggest that partial deafferentation of the optic tectum induces a transitory GABAergic innervation of free postsynaptic sites prior to the restoration of new retinal synaptic contacts.     

Gracile nucleus of streptozotocin-induced diabetic rats

Summary This study reports ultrastructural changes in the gracile nucleus of male Wistar rats after streptozotocin-induced diabetes. During the acute phase (3–7 days) degenerating electron-dense dendrites and axon terminals were dispersed in the neuropil. Degenerating dendrites were characterized by an electron-dense cytoplasm, swollen mitochondria, dilated endoplasmic reticulum and scattered ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals may form part of a synaptic glomerulus with a central electron-dense dendrite, or they may form the central element of a synaptic glomerulus. These degenerating profiles were absent in the gracile nucleus of the 3 and 7 days insulin-treated post-streptozotocin rats. Macrophages were present in the neuropil and were in the process of engulfing neuronal elements. During the medium phase (1–6 months), most of the degenerating dendrites and axon terminals had been engulfed or removed by macrophages. During the late phase (9–12 months) a second wave of degeneration occurred in the gracile nucleus, similar to the acute phase. During the medium and late phases, dystrophic axonal profiles were also significantly increased in the rats after streptozotocin treatment.It is concluded that the ultrastructural changes observed in the gracile nucleus in the present study were the result of streptozotocin-induced diabetes rather than a toxic effect of streptozotocin, even in the acute phase.     

Ultrastructural correlates of synapse withdrawal at axotomized neuromuscular junctions in mutant and transgenic mice expressing the Wld gene

We carried out an ultrastructural analysis of axotomized synaptic terminals in Wld(s) and Ube4b/Nmnat (Wld) transgenic mice, in which severed distal axons are protected from Wallerian degeneration. Previous studies have suggested that axotomy in juvenile (< 2 months) Wld mice induced a progressive nerve terminal withdrawal from motor endplates. In this study we confirm that axotomy-induced terminal withdrawal occurs in the absence of all major ultrastructural characteristics of Wallerian degeneration. Pre- and post-synaptic membranes showed no signs of disruption or fragmentation, synaptic vesicle densities remained at pre-axotomy levels, the numbers of synaptic vesicles clustered towards presynaptic active zones did not diminish, and mitochondria retained their membranes and cristae. However, motor nerve terminal ultrastructure was measurably different following axotomy in Wld transgenic 4836 line mice, which strongly express Wld protein: axotomized presynaptic terminals were retained, but many were significantly depleted of synaptic vesicles. These findings suggest that the Wld gene interacts with the mechanisms regulating transmitter release and vesicle recycling.     

Nigral and cerebellar synaptic terminals in the intermediate and deep layers of the cat superior colliculus revealed by lesioning studies

The presence of degenerating nigral and cerebellar synaptic terminals in the intermediate and deep layers of the cat superior colliculus was demonstrated by electron microscopy following lesions of the substantia nigra or brachium conjunctivum. The superior colliculus was taken for analysis 4–5 days after operation. Nigral terminals underwent a dark type of degeneration following kainic acid lesion of the pars reticulata of the substantia nigra. The majority of nigral degenerating terminals and axons were found in the stratum griseum intermediate with a few in the stratum griseum profundum. Two kinds of cerebellar terminals were distinguished by general appearances such as size, type of synaptic contact and type of synaptic vesicle and by the pattern of degenerative changes following electrical lesion of the brachium conjunctivum. Large elongated synaptic terminals 4–7 μm in diameter, were found mainly in the stratum griseum profundum. They often had double termination with conventional dendrites and with vesicles containing dendrites. This kind of terminal had a filamentous type of degeneration. A second type of degenerating cerebellar terminal, characterized by an electron-lucent type of degeneration, was predominantly located in the stratum griseum intermediale. These terminals were circular, about 4 μm in diameter, and did not have synaptic contact with vesicle-containing profiles. The finding of the two types of degenerating terminal after lesion of the brachium conjunctivum can be considered as evidence of the coexistence of at least two kinds of cerebellar terminals in the superior colliculus. The presence of nigral and cerebellar terminals in the intermediate and deep layers of the superior colliculus implicates the involvement of the substantia nigra and cerebellum in control of collicular visuomotor function.     

大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系

用切断后根方法在透射电镜下观察了大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系。切断脊神经后根24小时后,胶状质内出现明显的溃变终末。量最多又持续时间最长的溃变是电子致密型溃变,此外,也观察到了少数的电子透明型溃变和神经微丝型溃变;溃变终末大部分位于胶状质突触小球的中央。据统计,电子致密型溃变作为突触前成分与胶状质内的树突或树突棘形成轴—树突触者占98.8％,作为突触后成分与周围轴突形成轴—轴突触者占1.2％。     

Unilateral retrogasserian rhizotomy causes contralateral degeneration in spinal trigeminal nuclei of cats: an ultrastructural study

We are studying the response to injury within the brainstem trigeminal nucleus following trigeminal nerve lesions. We have previously shown with light microscopy and reduced silver stains that unilateral retrogasserian rhizotomy results not only in massive degeneration throughout the ipsilateral spinal trigeminal nucleus; in addition, degeneration is seen in a ventral position at the periobex region (involving caudal pars interpolaris and rostral pars caudalis) in the contralateral spinal trigeminal nucleus. In the present study, we have used electron microscopy to identify the source of the degenerating elements seen bilaterally after unilateral retrogasserian rhizotomy in eight adult felines with survival times ranging from 3 to 20 days. At short survival times (3–7 days) degenerating terminals with round synaptic vesicles (R terminals) and type 1, asymmetric contacts predominate bilaterally, while fewer degenerating terminals with flattened synaptic vesicles (F terminals) and type 2, symmetric contacts are seen. At longer survival times more F terminal degeneration is seen, especially on the contralateral side. Postsynaptic sites and dendrites show minimal alterations. These findings suggest that the degenerating R terminals seen on the contralateral side originate from primary afferents while the degenerating F terminals seen on the contralateral side originate from intrinsic sources involving a crossed internuclear pathway. In addition, the finding of degenerating F terminals may represent a novel form of selective transynaptic change of intrinsic neurons, associated with minimal dendritic or somatic alterations.     

Convergence of hippocampal and dopaminergic input onto identified neurons in the nucleus accumbens of the rat

The hippocampal input to the nucleus accumbens was interrupted by an electrolytic lesion of the fimbria-fornix. Boutons degenerating as a result of this lesion were found in asymmetric synaptic contact with dendritic spines and shafts in the medial part of the nucleus accumbens. Dopaminergic fibres and terminals in this area, identified using an antibody to tyrosine hydroxylase, established symmetrical synaptic contacts with dendritic shafts, spines and somata. In material where neurons in the nucleus accumbens had been Golgi-impregnated, it was found that the hippocampal and dopaminergic inputs converge onto the same neurons, and that the post-synaptic targets could be either spiny or aspiny neurons. It has been suggested that hippocampal dysfunction is involved in schizophrenia and this convergence of input from the hippocampus onto the same neurons that are post-synaptic to the dopaminergic input, which presumably originates from neurons in the ventral tegmental area, may provide an anatomical basis for the therapeutic effects of neuroleptic drugs which are dopamine antagonists.     

大鼠前庭脊髓束终末与脊髓小脑束起源细胞间的突触联系

目的　探讨前庭脊髓束终末与脊髓小脑束起源细胞间的突触联系。方法　采用HRP逆行追踪和顺行溃变结合电镜技术 ,对大鼠的前庭脊髓束终末与脊髓小脑束起源细胞之间的突触联系进行了研究。结果　发现溃变的前庭脊髓束轴突终末与HRP标记的脊髓小脑束神经元胞体之间形成的突触数量较少 ,其中轴 -树型突触数量多于轴 -体型突触。突触前终末含有丰富的清亮型扁平突触小泡 ,其次为清亮型圆形小泡。前庭脊髓束轴突溃变终末与脊髓边缘细胞形成的突触数量多于Clark s柱细胞形成的突触 ,并形成一些以标记树突为中心的突触复合体。结论　本研究从形态上证实前庭脊髓束终末直接与脊髓小脑束起源细胞发生突触联系 ,也表明前庭脊髓束对脊髓小脑束起源细胞向小脑的信息传递具有直接调控使用     

Increase in synaptic sites in goldfish tectum after partial tectal ablation

In young goldfish after partial ablation of the tectum, the number of synaptic contacts and terminals in a column through the stratum fibrosum et griseum superficiale of the tectum was increased to an extent corresponding to the greater input of optic axons projecting to the remaining tectum. This finding is consistent with previous results based on counting degenerating optic terminals, and therefore establishes that the optic axons make approximately normal numbers of terminals in the remaining tectum. The conflict between this finding and results obtained on older fish suggests that tectal cells of younger fish can more readily accept an increased number of optic terminals.     

Structural features of synaptic connections between descending systems and spinal neurons in the cat

The degeneration of axon terminals of the lateral (cortico and rubrospinal) and ventral (vestibulo- and reticulospinal) descending and propriospinal (lateral and ventral funiculus) systems was investigated by electron microscopy in the cat spinal cord. We studied the mean diameters of axon terminals, their arrangement on the neuronal structures in the grey matter, as well as the variety of the destructive changes in synaptic terminals. The average size of the ‘dark’ type degenerating axon terminals of fibers in the lateral descending systems was smaller than that of the ‘light’ type degenerating axon terminals in the medial descending systems. The average size of the both ‘dark’ and ‘light’ type degenerating propriospinal terminals of the ventral funiculus differed from those of the lateral funiculus.The findings concerning the sizes of synaptic terminals of the different descending and propriospinal systems correlate to a certain extent with the electrophysiological data about the nature of their synaptic action upon the spinal neurons.     

Observations on the process of degeneration of the afferent connections to the sensori-motor cortex of the monkey.

The ultrastructural features of degeneration of the afferent connections to the sensorimotor cortex of the primate have been studied with the electron microscope. Thalamo-cortical terminals show cytoplasmic darkening and swelling of vesicles, and neurofilaments and glycogen are often prominent in the larger terminals, although true neurofilamentous hypertrophy is rare. The terminals of the commissural and association projections similarly show darkening and some swelling of vesicles but are usually smaller and neurofilaments and glycogen are rarely seen. Ethanolic-phosphotungstic acid specifically stains degenerating axons and axon terminals. Degeneration occurs more rapidly in the terminals of the association projections than in those of the longer commissural projection although terminals at different stages of degeneration are present simultaneously in both instances. Micropinocytosis of degenerating terminals has been found and both glial and neuronal profiles are involved in this. Exposed post-synaptic thickenings have been seen and these induce subsurface cisternae in the neuronal structures to which they become apposed, including axon initial segments and unmyelinated axons.Damage to a part of the central nervous system thus causes consequential changes in other regions, and these changes are relevant to the interpretation of experimental studies and to the possibility of reorganization of connections following injury.     

The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. I. General description.

Lesions were made in the lateral geniculate nucleus of the rat and the consequent degeneration in area 17 of the cerebral cortex was studied by light and electron microscopy. These lesions produced prominent degeneration of axon terminals in layer IV extending into layer III and a much lesser amount in layers I and VI. The darkened degenerating axon terminals forming asymmetric synaptic junctions and were frequently surrounded by hypertrophied astrocytic processes. These terminals appeared to be disposed randomly, forming no discernible patterns. In layer IV 83% of the synapsing, degenerating terminals formed junctions with dendritic spines, 15% with dendritic shafts, and 2% with neuronal perikarya. The dendritic shafts and neuronal perikarya appeared to belong to spine-free stellate cells. The dendrites giving rise to the spines receiving degenerating axon terminals could not be identified, for most of the spines appeared as isolated profiles that could not be traced back to their dendritic shafts. One example of a degenerating axon terminal synapsing with an axon initial segment was encountered. Small, degenerating myelinated axons were prevalent in layers VI, V and IV, but were only infrequent in the supragranular layers. These results are compared with those obtained in other studies of thalamocortical projections.     

Metamorphic changes within the lateral-line system of Anura

Summary The metamorphic loss of lateral-line organs, lateral-line nerves and second order lateral-line neurons was examined in two Anuran species. At the onset of metamorphic climax, terminals within the lateral-line neuropil showed accumulation of glycogen-like granules. Neither the lateral-line nerve nor the organs or the nerve terminals inside the organs displayed any sign of degeneration at this stage. A few second order neurons exhibited accumulations of chromatin into conspicuous masses. These cells were partially or completely engulfed by phagocytes. At mid-metamorphosis all lateral-line organs were lost. The proximal parts of the lateral-line nerve fibers entering the rhombencephalic alar plate showed signs of degeneration. Within the lateral-line neuropil, pre- and some postsynaptic elements exhibited the flocculent type of degeneration or, to a lesser extent, the dark type of degeneration. Second order lateral-line neurons underwent an electron-dense or electron-lucent type of degeneration and were taken up by phagocytes. At the end of metamorphic climax the distal parts of the lateral-line nerves showed numerous dark degenerating fibers inside an intact myelin sheath. Within the lateral-line neuropil, numerous dark degenerating presynaptic elements were found next to some elements showing flocculent degeneration. Fewer degenerating second order neurons were found in the alar plate. They showed predominantly the dark type of degeneration. In contrast to earlier reports, our data suggest that the degenerative metamorphic changes observed in the present study are initiated in all parts of the lateral-line system simultaneously, and lead to the complete loss of all lateral-line organs and nerves and presumably all second order lateral-line neurons as well.     

An ultrastructural study of nerve terminal degeneration in muscle spindles of the tenuissimus muscle of the cat

Summary Muscle spindles in the tenuissimus muscle of the cat were studied between 12 and 168 h after cutting or freezing the nerve to this muscle. Degenerative changes in sensory and motor nerve terminals on intrafusal muscle fibres were observed using the electron microscope. Comparisons were made with spindles from unoperated or sham-operated cats.The earliest degenerative changes were seen in sensory and motor terminals at 20–24 h after the lesion. No nerve endings were seen by 114 h after denervation. The most consistent initial signs of degeneration were: (1) the presence of abnormal mitochondria and dense bodies in sensory terminals, and (2) a decrease in the number and clumping of synaptic vesicles combined with an increase in glycogen and neurofilaments in motor endings. Intrafusal fibres participate in the removal of degenerating sensory endings. Schwann cells phagocytose degenerating motor terminals. The disappearance of nerve terminals precedes the complete degeneration of preterminal myelinated fibres within the muscle spindle.     

Spinal projections to the lateral reticular nucleus in the rat

Summary The synaptic relationships and the distribution of the afferent terminals of the spinal pathway to the lateral reticular nucleus (LRN) of the rat were examined following induced degeneration. After high cervical hemisections, the spino-LRN projection was first examined with the Fink-Heimer silver impregnation method. Degeneration was confined primarily to the ipsilateral LRN and all three divisions of the nucleus were involved. Maximum degeneration was observed in the caudal regions of the parvocellular division. The magnocellular division, except for the extreme dorsomedial area, showed substantial degeneration as well. The subtrigeminal division throughout its entire length contained only sparse degeneration.Electron microscopic examination following spinal cord lesions revealed both round and pleomorphic-vesicle terminals in various stages of electron dense degeneration. The majority of the degenerating terminals were of the round-vesicle variety. Both types of terminals contacting somata were also observed to degenerate but their number was small in comparison to those on dendritic profiles. Terminals in synaptic contact with two dendritic profiles were also observed to degenerate. Some of the large terminals belonging to synaptic configurations (glomeruli) underwent degeneration and were therefore of spinal origin as well.     

皮质—脑桥核—小脑通路的HRP结合溃变电镜法研究

目的：观察大鼠脑桥核内皮质纤维终末的溃疡变型及其与脑桥核小脑投射神经元的突触联系方式,方法：采用HRP逆行标记结合溃变电镜技术。结果：（1）皮质纤维终末出现电子致密和微丝增生两种溃变类型。以电子致密型为主。后者终末又有两种不同形态,即含圆形清亮型小泡和多形清亮型小泡者,以圆形清亮型小泡终末占优势。（2）皮质纤维终末与脑桥核小脑投射神经元形成轴－树和少量轴－体突触,部分溃变终末与HRP标记的投射神经元形成单突触联系,结论：皮质纤维与脑桥核小脑投射神经元间存在的单突触联系构成了皮持－脑桥核－小脑通路。     

Ultrastructure of supraspinal dorsal root projections in the toad. II. The cerebellar granular layer

Summary Following section of the left dorsal roots, degenerating fibres and boutons were observed in the granular layer of the ipsilateral cerebellum. The degenerating terminals were identified as largeen passant varicosities of mossy fibres contacting the dendrites of presumptive granule cells. They contained round synaptic vesicles and neurofilaments and established Gray type I contacts. The terminals initially underwent filamentous degeneration with neurofilamentous hypertrophy, swollen mitochondria and loss of synaptic vesicles. At later survival times (6–30 days) they acquired an electron-dense appearance due to an increase and clumping of the filamentous component.After injection of horseradish peroxidase into the left cerebellum, all ipsilateral spinal ganglia showed a few (2–3%) labelled cells, indicating that a primary afferent contribution to this pathway originated from each segment of the spinal cord.     

Electron microscopy of degenerating axons and terminals in spinal trigeminal nucleus after tooth pulp extirpations

Following multiple tooth pulp extirpations, electron microscopic preparations show degenerating axons and synaptic terminals in the same region of the brain stem trigeminal nucleus previously demonstrating degeneration by light-optical methods. The observations confirm the phenomenon of transganglionic degeneration in this system and identify the class of central nervous system axons and synapses specifically related to innervation of the teeth.     

Direct synaptic connections between corticothalamic axon terminals and interneurons in the cat ventrobasal complex

Lesion-induced degeneration was combined with immunocytochemistry to study, with electron microscopy, the synaptic connectivity between corticothalamic axon terminals from the first and second somatosensory areas and local circuit neurons of the ipsilateral ventrobasal complex (VB), selectively labelled with an antibody raised against gamma-aminobutyric acid (GABA). Four days from the cortical ablation many degenerating axon terminals, forming asymmetric synapses, were found on dendritic trees of both labelled and unlabelled neurons of VB and occasionally on presynaptic dendrites. The main finding of the present paper is that 64.01% of degenerating axon terminals synapsed with GABA-immunopositive dendrites, suggesting that the principal target of the cortical projection to VB are local circuit neurons.