Phagocytosis of myelin sheath fragments by dendrites

Summary In serial ultrathin sections of the frog spinal cord, profiles of dendritic appearance were identified that contained myelin fragment inclusions and received synaptic contacts. In a number of cases it could be established that the inclusions were derived from adjacent myelin sheaths. It is suggested that the phenomenon may refer to a turnover of the myelin sheath in which the detached myelin fragments are eliminated by dendrites.     

Morphohistochemical changes of the blood cells in the hibernating frog (Rana esculenta L.)

Some morphological features (degree of nuclear segmentation in neutrophils and eosinophils), histochemical patterns (DHFR, LDH, G6PDH) and, in addition, the nucleic acid distribution (DNA, RNA with acridine orange) in the peripheral blood cells of Rana esculenta, during the hibernation phase were investigated. All the observed parameters varied significantly in the frog during hibernation in comparison with the active period. The most evident changes were an increase in the nuclear segmentation of the neutrophils and a lower activity of the histochemically demonstrable DHFR and LDH, probably due to cell ageing. These findings suggest that, in hibernating frogs, there is an increase in the life span of the peripheral blood cells as a consequence of a reduced metabolic activity and a slowing of haematopoiesis.     

The fine structure of “non-specific” grey matter (Laminae V and VII) in the cat spinal cord

Summary Quantitative electron microscopical observations have been made on the afferent synaptology of neurones in laminae V and VII of the cervical spinal cord of the cat. Both laminae contain large bouton-covered polydendritic neurones of the type seen in the brainstem reticular formation. Each lamina contains two other categories of neurone. One of these, in lamina V, is peculiar in having a very high proportion of afferent boutons filled with dense-core vesicles; similar neurones exist in lamina VII, but their presynaptic afferents do not contain unusual quantities of dense-core vesicles. The third type of neurone in laminae V and VII seems to be identical with an oligodendritic neurone type found in the brainstem reticular formation and parafascicular-centromedian complex.Work performed during tenure of a Royal Society European Programme Fellowship.     

Electron microscopic identification of postsynaptic dorsal root terminals: a possible substrate of dorsal root potentials in the frog spinal cord

Summary Dorsal root fibers were labeled with cobaltous chloride iontophoresis for electron microscopic investigations. In the base of the dorsal horn, where most of the coarser collaterals of dorsal root fibers terminate, many dorsal root terminals were found in postsynaptic relation to synapsing profiles. According to their morphological characteristics, three kinds of presynaptic terminals could be discerned in these complex synapses: axon terminals with spheric vesicles, axon terminals with flattered vesicles and presynaptic dendrites. These latter terminals contained relatively few flattened vesicles accumulated adjacent to a short synaptic articulation surface, and they were rich in cytoplasmic organelles. The functional significance of these structural specializations in the mediation of dorsal root potentials and recurrent inhibition is discussed.     

Large granular vesicles in the frog spinal cord

Summary Two types of large granular vesicles have been demonstrated in the spinal cord of the frog Rana esculenta. Each type is contained in different fibres located in the neuropil of the ventral horn.The two types of vesicles differ in their diameter and quantity, in the smoothness of their limiting membrane and the electron density of their core.The possibility that they contain peptides is discussed.     

Astroglial pattern in the spinal cord of the adult barbel (Barbus comiza)

The distribution and the structural, ultrastructural and immunohistochemical characteristics of the astroglial cells in the spinal cord of the adult barbel (Barbus comiza) have been studied by means of metallic impregnations (Golgi and gold-sublimate), immunohistochemical (GFAP and vimentin) and electron microscopic techniques. GFAP-positive cells were mainly distributed in the ependyma and in the periependymal region, but they have also been observed at subpial level in the anterior column. The ependymocytes were heterogeneous cells because they showed different immunohistochemical characteristics: GFAP-positive, vimentin-positive or non-immunoreactive cells. The radial astrocytes showed only GFAP immunoreactivity, and their processes ended at the subpial zone forming a continuous subpial glia limitans. Desmosomes and gap junctions between soniata and processes of radial astrocytes were numerous, and a relationship between radial astroglial processes and the nodes of Ranvier was also described. The perivascular glia limitans was poorly developed and it was not complete in the blood vessels of the periependymal zone; in this case, the basal lamina was highly developed. An important characteristic in the barbel spinal cord was the existence of a zone with an abundant extracellular space near the ependyma. The presence of radial astroglial somata at subpial level, the existence of vimentin-positive ependymocytes and the abundant extracellular space in the periependymal zone is discussed in relation to the regeneration capacity and the continuous growth showed by fish. Moreover, the abundance of gliofilaments and desmosomes leads us to suggest that mechanical support might be an important function for the astroglial cells in the barbel spinal cord.     

Ultrastructure and cytochemistry of circulating erythrocytes during the annual cycle of Rana esculenta L.

Morphometrical (image analysis of cell and nuclear shape factor), morphological (electron microscopy) and cytochemical (acridine orange fluoro-chromasia and propidium iodide-DNA flow cytometry) features of circulating red blood cells were investigated during two periods of the annual cycle of Rana esculenta: the active phase (July) and the underground hibernating phase (January).The results showed that the hibernating phase is marked by more homogeneity of the red cells, both at nuclear (strongly condensed chromatin) and cytoplasmic level (loss of intact organelles and acridine orange fluorochromasia). The almost complete disappearance of the immature erythrocytes from the circulation, during the hibernating phase, should be related to a decrease of haemopoietic activity and to an increase of life span accompanied by uncommon storage in different organs both at vascular and intracellular level.     

Continous electrophysiological measurements of changes in cell volume of motoneurons in the isolated frog spinal cord

One type of ion-sensitive micro-electrode (K⁺ ligand Corning 477317) is sensitive to large quaternary ammonium ions such as choline or tetramethylammonium (TMA⁺). We have now used such electrodes for continuous electrophysiological measurements of changes in cell volume of motoneurons in the isolated frog spinal cord. The electrodes were double-barrelled with tip diameters of 1 m. The reference barrel was filled with 100 mM choline or 100 mM TMA⁺ in 1 M Mg²⁺-acetate, the sensitive barrel contained the Corning K⁺ ligand. After the impalement of a motoneuron, choline or TMA⁺ diffused into the cell and about 1 h later, a steady-state concentration of these ions in the range of 10–20 mM was reached. Following this period, the motoneurons were activated by repetitive electrical stimulation or by application of amino acids via the bathing solution. All these stimuli led to a transient rise of the intracellular concentrations of choline or TMA⁺ (indicating a cell shrinkage of 3–10% difference to control volume).     

Circumferential cells of the developing Rana catesbeiana lumbar spinal cord

Summary Neural elements in the lumbar enlargement of the developing Rana catesbeiana spinal cord were labelled by placing chips of dessicated horseradish peroxidase (HRP) into various lesions of the spinal cord. Of the elements labelled in the lumbar enlargement, a population of cells circumjacent to the gray matter was seen to be distinct from all others on the basis of their morphology, position and their putative embryonic origin. One cell type not previously described was a large circumferential cell (LCC) with primary processes completely circumscribing the gray matter. The ventral process crosses the midline and ascends or descends in the ventral funiculus. The dorsal primary process was observed to extend to the midline and turn ipsilaterally in a rostro-caudal direction in the dorsal funiculus. LCC's were present at early stages of larval development (stage III, Taylor and Kollros 1946) but could not be labelled in juvenile frogs. LCC's were only observed in the lumbar enlargement and could only be labelled through HRP applications at that level. They receive abundant synaptic input from the ipsilateral lateral funiculus. Possible roles for the LCC in the early function and development of the ranid lumbar spinal cord are discussed.     

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 m transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of -aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing -motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei cocontained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of -motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26–42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5–9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface. A colocalization of GABA and glycine immunoreactivities in putative nerve terminals could be shown both in the neuropil and in close relation to cell bodies of motoneurons. These results suggest that among the studied amino acids probably only three, namely GABA, glycine, and glutamate, can be considered to be neurotransmitter candidates in the ventral horn of the cat spinal cord.     

Miniature excitatory postsynaptic potentials in embryonic motoneurons grown in slice cultures of spinal cord,dorsal root ganglia and skeletal muscle

Summary Miniature excitatory postsynaptic potentials (mEPSPs) were recorded in motoneurons grown in organotypic cocultures of embryonic rat spinal cord, dorsal root ganglia and muscle in the presence of TTX. The motoneurons were electrically compact with a mean electrotonic length of 0.6. Spontaneous EPSPs were found in most of these motoneurons. With TTX the large EPSPs disappeared, whereas in more than half of the experiments mEPSPs persisted with a range in size of 1 to 4 mV (mean: 2.1 mV), probabely originating from the spontaneous release of single vesicles. The net inward charge transfer at the soma ranged from 0.12 to 0.34 pC. The mEPSPs were heterogeneous in size even within pools of potentials that were homogeneous in shape. They had similar shapes and amplitudes as the smallest spontaneous unitary EPSPs mediated by presynaptic impulses, suggesting that for the smallest afferents not more than one vesicle was released per afferent impulse. Both the miniature and the TTX-sensitive EPSPs were readily blocked by the glutamate antagonist DNQX.     

Extra-lysosomal localization of acid phosphatase in the spinal cord of the rat

Summary The Rolando substance of the rat spinal cord was investigated by means of standard electron microscopy and by light-and electron microscopic histochemical demonstration of acid phosphatase. In accordance with previous studies, electron dense, large sinusoid terminals in the Rolando substance, constituting the central element of Rolando glomeruli, are shown to establish numerous axo-axonal and axo-dendritic synapses. Acid phosphatase reaction of the Rolando substance, resistant to 0.2 mM sodium fluoride inhibition, is located in and around the large sinusoid terminals. By using various incubation parameters and cryo-protection, the most plausible physiological localization of this enzyme, probably related to transmission processes appears to be in the greater membrane (glycocalyx) of large sinusoid terminals.     

Lithium distribution across the membrane of motoneurons in the isolated frog spinal cord

Lithium sensitive microelectrodes were used to investigate the transmembrane distribution of lithium ions (Li⁺) in motoneurons of the isolated frog spinal cord. After addition of 5 mmol·l^–1 LiCl to the bathing solution the extracellular diffusion of Li⁺ was measured. At a depth of 500 m, about 60 min elapsed before the extracellular Li⁺ concentration approached that of the bathing solution. Intracellular measurements revealed that Li⁺ started to enter the cells soon after reaching the motoneuron pool and after up to 120 min superfusion, an intra — to extracellular concentration ratio of about 0.7 was obtained. The resting membrane potential and height of antidromically evoked action potentials were not altered by 5 mmol·l^–1 Li⁺.     

The effects of gallamine on field and dorsal root potentials produced by antidromic stimulation of motor fibres in the frog spinal cord

Summary The effects of gallamine on the intraspinal field potentials and the dorsal root potentials produced by antidromic stimulation of motor fibres were studied in the isolated frog spinal cord preparation. After gallamine (10^-3 M), the duration of the negative field potential produced by antidromic activation of motoneurons (N₁ response) was increased often without changing its amplitude. This resulted in an increased passive spread of the antidromic action potential towards the dorsal dendritic regions, where afferent fibres terminate.In the untreated spinal cord, stimulation of motor axons produced a late negative dorsal root potential (VR-DRP) which was depressed after gallamine administration. Abolition of the VR-DRP was frequently associated with the appearance of a short latency, conducted response, in the dorsal roots (EVR-DRP). The earliest component of the EVR-DRP had a latency ranging between 0.5 and 2.5 ms measured after the peak of the N₁ response recorded at the motor nucleus. Such a brief latency of the EVR-DRP suggests that this response results from electrical interaction between motoneurons and afferent fibres. After gallamine, the primary afferent depolarization produced by orthodromic stimulation of sensory nerves facilitates the EVR-DRP without necessarily increasing the amplitude or duration of the N₁ response. Also, gallamine appears to increase directly the excitability of the afferent fibre terminal arborizations.The nature of the electrical interaction between motoneuron dendrites and afferent fibre terminal arborizations is discussed in terms of two hypotheses: interaction by current flows and by electrical coupling.     

Tight junctions in the ependyma of the spinal cord of the urodele Pleurodeles waltlii

Summary The ependymal junction pattern in the spinal cord of postmetamorphic ribbed newts has been studied, using transmission electron microscopy of ultrathin sections of normal animals and of animals perfused through the IVth ventricle with lanthanum. Contrary to what has been observed in mammalian CNS, the ependyma of the urodelan spinal cord is furnished with tight junctions that seal the luminal border of the terminal bars. These occludens junctions are made up of two to seven punctate fusions of the plasma membranes. Lanthanum tracer remains restricted inside the lumen of the central canal, being stopped at the first punctate fusion on its way through the intercellular clefts. Beyond this point, the extracellular space contains no tracer material. Besides tight junctions, intermediate, desmosomal and gap junctions are also present. Gap junctions and desmosomes are not present in CSF-contacting neurons. It is suggested that ependyma with occluding junctions (special ependyma) overlay the regions of the CNS where the ependymal cells significantly modify the composition of both intercellular and cerebrospinal fluids, through secretory, transporting and permeability control activities.Dedicated to Dr. Jean Emmanuel Gruner     

Distribution of calretinin-like immunoreactivity in the brain of Rana esculenta

The distribution of calretinin-like immunoreactivity has been analyzed in the brain of Rana esculenta. Several neurons of nuclei belonging to sensory pathways, subhabenular area and left habenula were immunopositive. Immunoreactivity was present in fibers of motor and sensory pathways, thalamus, tegmentum and isthmus. The immunolabeling pattern partially overlapped that previously described in the rat. However, in comparison with the rat, fewer cells and fibers were immunoreactive and there were less positive brain nuclei, especially in the pallium, septum and striatum, that were totally negative. Taking into consideration that these regions are rather simple in the frog, the presence of calretinin seems to be consistent with the degree of complexity of brain areas and segregation of different nuclei.     

腺病毒介导的神经营养素-3基因能够在大鼠脊髓前角运动神经元内过表达神经营养素-3

目的观察腺病毒介导的神经营养素-3(NT-3)基因在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元的过表达。方法在坐骨神经内直接注射含有绿色荧光蛋白(GFP)基因(报告基因)的NT-3基因重组腺病毒(Ad-NT-3-GFP),7d后应用免疫荧光组织化学染色技术,在荧光显微镜下观察脊髓前角运动神经元的NT-3过表达。结果 GFP表达组(对照组)和NT-3加GFP表达组两组动物的L4和L5脊髓段横切片上,有绿色荧光蛋白阳性标记的细胞。在NT-3加GFP表达组,还可以观察到NT-3阳性标记的细胞,这种细胞能与绿色荧光蛋白阳性标记的细胞重合,是过表达NT-3的前角运动神经元。与GFP表达组的前角运动神经元形态比较,NT-3加GFP表达组的过表达NT-3的前角运动神经元呈现更富有分支的突起。结论腺病毒介导的NT-3基因能够在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元内过表达NT-3,这为下一歩应用NT-3基因治疗策略修复实验性脊髓损伤提供初歩的实验资料。     

Coexistence of substance P and calcitonin gene-related peptide in the frog spinal cord

The localization of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the untreated spinal cord of the frog using single or double immunohistochemical stainings. SP and CGRP appear to coexist in the primary afferent fibers and in the marginal and submarginal dorsal horn zones, as well as in the dorsolateral zone. In other parts of the spinal cord CGRP immunoreactivity was scanty while diffuse SP systems were seen, suggesting that the coexistence of the two peptides is restricted to primary afferent fibers.