Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells

Summary With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5–9.8 m) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4–1.8 m long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 m, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.     

Recurrent dorsal root potentials and motoneuron morphology in the frog spinal cord

About one third of motoneurons stimulated intracellularly evoked dorsal root potentials (DRP) in the lumbar segments of the isolated and perfused frog spinal cord. Axon collaterals were found in one of the 22 motoneurons filled with HRP (horseradish peroxidase) through the stimulating electrode. In further experiments injecting individual motoneurons with cobalt, and filling the ventral roots with HRP or cobalt, the frequency of occurrence of axon collaterals was about 2% of the number of labelled motor cells. It is suggested that the presence of motor axon collaterals is not indispensable in the generation of the DRP evoked by ventral root or motor cell stimulation.     

小鼠臂丛离断对脊髓运动神经元树突结构与形态退变的影响

目的 研究臂丛离断后脊髓运动神经元树突退变与时间和损伤距离的相关性。 方法 在距离椎间孔3 mm或10 mm处处离断小鼠臂丛,术后7、14、28、56 d取材,采用MAP2免疫荧光染色和体视学分析、Golgi-Cox染色和Sholl分析观测颈膨大处脊髓前角运动神经元的树突结构和形态变化;术后28 d比较距离椎间孔3 mm和10 mm臂丛离断对脊髓运动神经元树突的影响。 结果 MAP2免疫荧光显示臂丛离断导致脊髓前角内树突的密度和完整性随时间延长逐渐下降;Golgi-Cox染色和Sholl分析显示运动神经元最长树突、总树突长度、树突最大跨度、树突3级分支的数量均呈时间依赖性下降。与距离椎间孔10 mm处离断组相比,3 mm处离断引起的树突长度退变更为明显。 结论 脊髓运动神经元树突在周围神经损伤后会发生退变,随时间延长其退变程度加重,随损伤部位与脊髓的距离延长树突长度退变程度减轻。     

The relationship of dorsal root afferents to motoneuron somata and dendrites in the adult bullfrog: a light and electron microscopic study using horseradish peroxidase

The relationship of lumbar dorsal root afferents to lateral motor column motoneurons was studied using anterograde injury filling of dorsal roots and retrograde injury filling of ventral roots with horseradish peroxidase. At the light microscopic level, horseradish peroxidase labelled dorsal root axons were observed to separate into a medial division of large diameter axons which enter the dorsal funiculus and a lateral division of small diameter axons which form a compact bundle in the dorsolateral funiculus which may be homologous to the mammalian tract of Lissauer. Within the spinal gray, primary afferents terminate in two distinct regions. The more ventral of these terminal fields, which receives collaterals of primary afferent axons in the dorsal funiculus, overlaps the dendritic arborizations of the lateral motor column motoneurons. Some axons leave the ventral terminal field to enter the dorsal lateral motor column. Here they terminate on the primary dendrites and somata of lateral motor column motoneurons. At the electron microscopic level, labelled primary afferent terminals were seen to synapse upon lateral motor column motoneuron dendrites as well as upon the somata of dorsally positioned lateral motor column motoneurons. These terminals contain small spherical vesicles and occasional dense-cored vesicles. The synaptic specializations are characterized by a small amount of postsynaptic material. The lateral motor column may be divided into dorsal and ventral portions on the basis of the primary afferent distribution and this is in accord with functional, physiological and developmental data.     

Nerve regeneration in the dorsal funiculus of the rat spinal cord: a light and electron microscopic study

In an attempt to identify regenerating axons in the central nervous system, a partial transection of the dorsal funiculus in the rat spinal cord was carried out with a pair of microdissection scissors, and a nylon thread loop was inserted into the lesion to demarcate the severed tissue. Nerve regeneration through the demarcated lesion was observed 4-20 days after the operation by light and electron microscopy. In the early stage, many naked axons appeared from the caudal part of the lesion, and some of these further extended into the demarcated space. They contained an accumulation of mitochondria, smooth-surfaced endoplasmic reticulum and vesicles in the axoplasm; this axoplasmic feature indicated that they were regenerating axons. They gradually increased in number, and took highly irregular courses exhibiting various fluctuations in diameter throughout their lengths. Immature Schwann cells as well as glial cells including oligodendrocytes and astrocytes appeared in close association with these regenerating axons. Oligodendrocytes eventually formed thin myelin sheaths. On the other hand, naked axons were present deflecting outside the thread loop; they showed no axoplasmic characteristics as described above. These axons could be regarded as uninjured ones merely undergoing demyelination due to the surgery. Thus, regenerating axons were clearly distinguished from merely demyelinated ones, and some of them were shown to grow through the traumatic lesion in the dorsal funiculus of the rat spinal cord.     

Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study

Cholecystokinin-like immunoreactivity was investigated with an indirect immunoperoxidase technique in the whole spinal cord with the light microscope and in the dorsal horn with the electron microscope. Intraparenchymal injections of colchicine were performed to allow the detection of cholecystokinin-like immunoreactive cell bodies. Rats treated at birth with capsaicin were also studied at the light microscope. Numerous cholecystokinin-like immunoreactive fibres and varicosities were found in the two superficial layers of the dorsal horn and in the intermedio-medial nucleus; cholecystokinin-like immunoreactive cell bodies were also present in these two regions. After neonatal capsaicin treatment, the number of cholecystokinin-like immunoreactive fibres and varicosities was strongly reduced in the dorsal horn. At the electron microscope level, cholecystokinin-like immunoreactivity was localized in numerous neurites often filled with vesicles (axon terminals and dendrites containing vesicles) and in few cell bodies and dendrites. The immunoreaction was found mainly associated with ribosomes, granular reticulum, neurotubules and vesicles. Large granular vesicles were filled with the reaction product whereas small and medium-sized vesicles showed a varying degree of immunoprecipitate around their membrane. In addition dense "granules" of precipitate were observed in numerous presynaptic neurites. Cholecystokinin-like immunoreactive axons were of small calibre and mostly unmyelinated. Cholecystokinin-like immunoreactive axon terminals made asymmetric synaptic contacts with generally unlabelled dendrites or dendritic spines. A single labelled nerve terminal could contact several different dendrites in structures resembling glomeruli. Few axo-somatic synapses but a relatively high number of axo-axonic contacts were seen. About half of these axo-axonic contacts involved pre- and postsynaptic profiles. Both light and electron microscopic observations led us to the conclusion that some of the cholecystokinin-like immunoreactive fibres of the dorsal horn originate in the spinal ganglia via capsaicin-sensitive C afferents; and some from intrinsic neurons, particularly islet cells. Other fibres may come from supraspinal centres, other local neurons or capsaicin-insensitive afferents from the spinal ganglia. The results are discussed with regard to data in the literature, particularly those concerned with the specificity of the cholecystokinin antibodies; it is hypothesized that several types of cholecystokinin-like immunoreactive peptides may be present in the dorsal horn, depending on their origin (supraspinal, intrinsic or peripheral).(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonergic innervation of the dorsal horn of rat spinal cord: light and electron microscopic immunocytochemical study

Summary The ultrastructure of serotonergic projections to the dorsal horn of the rat spinal cord has been investigated, using a highly specific polyclonal antiserum. The highest concentrations of immunoreactive profiles were found in lamina I and the outer part of lamina II (IIo). Intermediate concentrations were found in laminae III and IV, while the inner part of lamina II (IIi) was almost devoid of immunoreactivity. Whereas 60% of the profiles show at least one varicosity studded with synaptic vesicles, only one-fifth of the latter contributes to classical synapses, the remaining profiles being devoid of a facing postsynaptic density. The results are compared with those in the literature and our own results relative to other regions of the cord. It is concluded that the pauci-synaptic projections to the dorsal horn could correspond to a diffuse influence of serotonin, the targets for which are determined by the corresponding serotonergic receptors.     

Vesicle-containing dendrites in the nucleus raphe dorsalis of the cat. A serial section electron microscopic analysis

Summary The nucleus raphe dorsalis of the cat was examined by serial section electron microscopy and the presence of vesicle-containing dendrites is reported. Such dendrites were divided into two classes according to their synaptic contact. Dendrites containing round and/or pleomorphic vesicles associated with a clear synaptic specialization which was generally intermediate between a Gray's type I or II were classified as presynaptic dendrites. These presynaptic dendrites were presynaptic to conventional dendrites and dendritic spines. In addition, some profiles containing a sparse population of vesicles which may be dendritic in nature were observed involved in serial synaptic arrangements. A second class of dendrites were characterized by the presence of vesicles which were never found associated with any synaptic membrane specialization. Commonly, the vesicles were densely packed and associated with unusual densities. Serial section analysis of these densities showed that they were not presynaptic dense projections. We suggest that the existence of vesicle-containing dendrites in the nucleus raphe dorsalis of the cat constitute the morphological support for the dendritic release of neurotransmitters.     

A light and electron microscopic study of the CB1 cannabinoid receptor in the primate spinal cord

The distribution of cannabinoid receptors was studied in the monkey spinal cord by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in all portions of the grey matter of the spinal cord. These included small diameter 9–16µm neurons in the dorsal horn, larger (40–60µm) neurons in the intermediate grey, and very large (60–100µm), motor neurons in the ventral horn. Reaction product was observed in dendrites postsynaptic to unlabelled axon terminals. Since cannabinoid receptor activation decreases neuronal excitability by several mechanisms, including inhibition of voltage dependent calcium channels, the dense staining of CB1 in dorsal horn neurons suggests that CB1 could reduce calcium influx through such channels in these neurons. This, in turn, could decrease calcium-dependent changes in synaptic transmission and decrease sensitisation to nociceptive stimuli in these neurons. Similarly, the dense staining of CB1 in ventral horn cells suggests that cannabinoid receptors could limit calcium influx through voltage dependent calcium channels in these neurons, and could be significant in terms of neuroprotection to these neurons.     

A study of motoneuron groups and motor columns of the human spinal cord

Eight normal human spinal cords were studied for motoneuron (Mn) groups and columns. Spinal segments (C1 to Coc.) were identified and embedded in paraffin wax. Serial cross sections were cut at 25 μm and stained by cresyl violet. Cross-sectional profiles of the spinal cord were traced for each segmental level and the outlines of the various Mn groups superimposed. These charts (maps) were used to examine intra and intersegmental changes in the relative positions of the columns. An attempt was made to provide topographical picture of Mn groups of individual segments. In the cervical region neuronal groups were more numerous but smaller and less distinct, while in the lumbosacral region they were fewer, larger and at many levels better circumscribed. The average number of Mn groups at any segmental level was 3–4 and never exceeded 5. C4, C5, C6, C7, L4, L5 and S1 contained numerous Mn groups. Maximum intrasegmental changes were noted at C3, C4, C7, T1, and S2, while at C5, C6, all thoracic, L1 L2 and L3, the pattern was constant throughout the segment. Eleven motor columns were traced in the human spinal cord. Column 1 belonged to the medial division and columns 2–11 to the lateral division of the ventral grey horn. Columns 1 and 2 were the most extensive as they were traceable from the lower medulla to S3 segment. Columns 3–8 were confined to cervical segments (including T1), while columns 9–11 were traced in lumbosacral segments. In general, motor columns followed a definite mode for their appearance and disappearance. Many of them showed rotation from a dorsal to a ventromedial direction.     

Differential sensitivity of spinal neurones to amino acids: an intracellular study on the frog spinal cord

Intracellular recordings from in vitro neurones of the frog spinal cord slice preparation were performed in order to examine the mechanism of action of gamma-aminobutyrate and glutamate on two distinct neuronal populations in the same region of the central nervous system. Amino acids were superfused at fast rate and low temperature (7 degrees C) to reduce their uptake process. On interneurones, the inhibitory action of gamma-aminobutyrate was characterized by a large input conductance increase while on motoneurones the conductance change was much smaller. Glutamate excited interneurones which greatly increased their input conductance and showed burst firing; motoneurones were also excited by glutamate but usually did not fire repeatedly nor showed large conductance changes. In spite of these differences the amplitude of depolarization in the presence of the same concentration of glutamate was similar for motoneurones and interneurones. It is suggested that amino acids (particularly glutamate) may act through different membrane mechanisms on two neuronal populations in the same region of the spinal cord.     

Organophosphate pesticide DDVP-induced alterations in the rat cerebellum and spinal cord -- an electron microscopic study.

DDVP is an organophosphate pesticide whose neurotoxicity, in the form of cholinesterase inhbition, is well-known. Rats, weighing 200-225 g were given intraperitoneal injections of 3 mg/kg. DDVP daily for 10 days. Following perfusion-fixation, ultrathin sections of the cerebellum and spinal cord were examined with an electron microscope. A large number of electron-dense bodies exhibiting electron-lucid vacuoles were discernible in the perikarya of cerebellar neurons. Aggregations of mitochondria were also seen. Myelin-figures in dendrites and axons of the spinal cord were detected. Evidence of oedema was evident in the spinal cord.     

Scanning electron microscopic study of spinal arachnoid plaques

Calcified or ossified plaques within the spinal arachnoid membranes are often reported at autopsy and are occasionally incriminated as the cause of neurological symptoms in patients undergoing surgery. Histological studies of plaques have been done, but to our knowledge no studies using the scanning electron microscope (SEM) have been reported. In this study, portions of the spinal cord with plaques were removed from adult human cadavers in the dissecting laboratory and processed for SEM and energy-dispersive x-ray analysis (EDAX). The surfaces of the plaques appeared to be fibrous in some areas, studded with irregularly shaped projections or nodules in others, and smooth in yet other areas. Cells were seen in lacunae-like depressions on the surfaces of some plaques and EDAX revealed the presence of calcium on and near the cells. In some areas calcium and phosphorus were both detected, but not in the ratios seen in bone. No calcium was seen on smooth or fibrous areas. It is suggested that the plaques sampled in this study were composed largely of fibrous tissue encrusted in some areas with calcium salts. There was no evidence that any of the plaques contained bone.     

A study of the interaction between motoneurones in the frog spinal cord

1. A short-latency interaction between motoneurones has been studied with intracellular and root potential recordings from the isolated spinal cord of the frog. Antidromic stimulation of one ventral root causes brief depolarization (VR-EPSP) of the motoneurones of adjacent, non-excited motoneurones. The summed activity of many such VR-EPSPs can be seen as a brief depolarization (VR-VRP) passing out an adjacent ventral root.

2. Both intracellular and root-recorded signs of this interaction are graded in amplitude.

3. It was found that this interaction decreased with increasing temperature. This is in contrast to the behaviour of the ventral root potential resulting from dorsal root stimulation (DR-VRP) or the dorsal root potentials resulting from either dorsal root (DR-DRP) or ventral root (VR-DRP) stimulation, all of which increased in amplitude from below 10 to about 17° C.

4. Pharmacological evidence suggests that the interaction between motoneurones is not chemically mediated. The VR-VRP was not affected by a large variety of transmitter blocking agents, including curare, dihydro-β-erythroidine, atropine, succinylcholine, hexamethonium and DOPA, while the VR-DRP, which probably originates with the release of ACh from an axon collateral, was consistently blocked.

5. Mg²⁺ suppressed the VR-VRP more slowly than the other potentials, and this suppression was increased by adding Ca²⁺, rather than reversed, as in the case of the other root potentials, which are presumably mediated by chemical transmission.

6. The interaction between motoneurones is strongly facilitated by orthodromic depolarization of the motoneurones being antidromically stimulated. Extracellular recordings within the cord support the conclusion that this facilitation is a result of the enhancement of antidromic invasion, perhaps especially of the dendrites, by slight depolarization.

7. One VR-VRP (or VR-EPSP) first suppresses response to another (for about 10 msec), then facilitates response to the second, with maximum effect around 20-40 msec. This is the case whether both stimuli go to the same or to different ventral roots, although occlusion is less and facilitation greater in the latter case. Occlusion of the VR-EPSP also results from full excitation of the cell in which recording is being done.

8. The mechanism of this interaction remains uncertain, but it would seem likely that overlapping dendrites of adjacent motoneurones interact with each other electrically through close apposition or specialized contacts. Occlusion would result from the refractoriness of strongly depolarized dendrites, facilitation from the enhancement of invasion of antidromically stimulated motoneurones by the weaker (or residual) depolarization occurring after earlier activity of motoneurones or their dendrites.

     

Distribution of GABA immunoreactivity in the optic tectum of the frog: a light and electron microscopic study

GABA immunoreactivity was studied in the optic tectum of the frog, Rana esculenta, by postembedding immunohistochemical methods at the light and electron microscopic levels. Nearly one-third of the total population of tectal cells appeared to be GABA-immunoreactive. The proportion of stained neurons was highest in layer 9 (61%), and they occurred less frequently in layers 7 (21%) and 6 (27%). Stained perikarya represented a population of small neurons with a diameter of 8-10 microns. Large cell bodies in layer 7 or at the top of layer 6, and cells of origin of the mesencephalic trigeminal tract in layer 2, were devoid of labelling. Axon terminals and dendrites displaying immunoreactivity for GABA were observed in all of the plexiform layers. On the basis of ultrastructural characteristics two types of GABA-positive axon terminals and two variations of GABA-immunoreactive dendrites were distinguished. Synaptic relations of GABA-immunoreactive and GABA-negative axons as well as dendrites were also studied. Besides a wide variety of axodendritic synapses, dendrodendritic synaptic appositions were also revealed. The results suggest that various inhibitory mechanisms are involved in tectal circuits, which have to be incorporated into future neuronal models concerning visual information processing in the optic tectum of the frog.     

Proximodistal gradients of the postjunctional folds at the frog neuromuscular junction: a scanning electron microscopic study

Frog endplates were studied with the scanning electron microscope following the removal of the presynaptic terminal by collagenase and acid treatments. Endplates had 2-14 branches of primary cleft. The longest branches were parallel to the muscle fiber. Short branches oblique or perpendicular to the muscle fiber were also present near the central region of the endplates. The openings of postjunctional folds in the primary cleft were clearly visible at the bottom of the primary cleft and could be counted and measured. The longest primary cleft branches of each endplate were divided into segments of 20 microns (length corrected for shrinkage). The number of postjunctional folds per micrometer of primary cleft, the average postjunctional fold length (i.e. across the primary cleft) and the total postjunctional fold's length per micrometer of primary cleft were evaluated for each 20-microns segment of primary cleft. Negative proximodistal gradients were observed for these three parameters for the long branches of primary cleft, i.e. values were higher in the proximal region (near the motor axon) than in the distal region. These postsynaptic gradients probably reflect similar or smaller proximodistal presynaptic gradients for the active zones along the nerve.