Neuropeptide Y-immunoreactive synapses in the intermediolateral cell column of rat and rabbit thoracic spinal cord

Neuropeptide Y (NPY)-immunoreactive nerve fibers in the intermediolateral cell column of rat spinal cord segments T2-T3 and T8-T10 and rabbit segments T3-T6 were studied with light and electron microscopic immunocytochemistry. Plexuses of NPY-immunoreactive nerve fibers were found by light microscopy. NPY-positive synapses were present electron microscopically but non-immunoreactive synapses greatly outnumbered NPY-immunoreactive ones. In the lateral horn of rat T9, 30% of the vesicle-containing NPY-positive axon profiles formed synapses, 95% of which were axodendritic. These synaptic connections may mediate the effects of brainstem NPY neurons on the activity of sympathetic preganglionic neurons.     

Periterminal synaptic organization of primary afferents in laminae I and IIo of the rat spinal cord, as shown after anterograde HRP labelling

Summary The fine structure and periterminal synaptology of the primary afferent terminations in laminae I and IIo are examined in the rat, following anterograde labelling with horseradish peroxidase applied to the right C₅-dorsal root. Labelled varicosities observed along the terminal arbors in parasagittal thick sections were relocated in ultrathin sections by electron microscopy. The labelled terminal profiles generated by the three primary afferent plexuses which can be identified by light microscopy in laminae I-IIo had similar fine structural features, except that axo-axonal contacts, although rare, were more frequent in the medial network plexus. Primary boutons were packed with agranular spherical vesicles and some large granular vesicles, and were mostly presynaptic to profiles of dendritic trunks of marginal cells. Unlabelled axonal profiles, either light with some flattened vesicles, or dense with round vesicles, were also presynaptic at symmetrical or asymmetrical contacts, respectively, to those dendritic profiles. It is suggested that such knobs of intrinsic origin are responsible for postsynaptic modulation of the primary noxious input. Although the 20 m wide lamina IIo belongs cytoarchitectonically to lamina II and can be distinguished from lamina I by a decreased amount of myelinated fibres and large dendritic profiles, the periterminal synaptology was here found to be the same as in lamina I.     

Somatostatin immunoreactive cell bodies in the dorsal horn and the parasympathetic intermediolateral nucleus of the rat spinal cord

Relative changes in the amount of galactocerebroside (GC) during development were measured in mouse brain cell cultures at different stages of development. For this purpose we used an ¹²⁵I-labelled protein A indirect assay modified in the respect that the total amount of cellular proteins was evaluated before counting the radioactivity. The amount of GC greatly increased between the 10th and the 14th day of culture, then a steady state was reached between the 14th and the 20th day of culture. This change correlates well with the dynamics of the number of oligodendrocytes we observed earlier. These data suggests that the increase of the GC amount in culture during development corresponds to the increase in the number of GC-positive oligodendrocytes rather than to the increase in the number of GC molecules per cell.     

Ultrastructure of monoaminergic terminals in the intermediolateral nucleus of the cat spinal cord

Monoaminergic innervation of the intermediolateral nucleus of the cat spinal cord was investigated by fluorescence histochemistry and electron microscopy. Large numbers of monoaminergic terminals were labeled by prior administration of the false neurotransmitter 5-hydroxydopamine (5-OHDA). Ultrastructurally, 5-OHDA-labeled terminals fell into three types. Type I, which made up 55% of the labeled terminals, contained abundant, large and densely labeled vesicles and only a few small and unlabeled vesicles. This type was "bouton de passage". Type II, which made up 40% of the terminals, made asymmetrical synaptic contacts with typical postsynaptic structures. This type contained many small vesicles, some of which were labeled, and a few large dense-core vesicles. Type III, which made up 5% of the terminals, made close contact with presynaptic nerve endings containing abundant small unlabeled clear vesicles. The type III terminals contained many large and densely labeled vesicles and a few small flattened vesicles, most of which were unlabeled.     

5-Hydroxytryptamine, substance P and thyrotropin-releasing hormone synapses in the intermediolateral cell column of the rat thoracic spinal cord.

Serotonin-, substance P-, and thyrotropin-releasing hormone-immunoreactive profiles were studied in the intermediolateral cell column at the thoracic level of the rat spinal cord with light- and electron-microscopic immunocytochemistry. For each transmitter, a dense immunoreactive deposit was observed with the light microscope. At ultrastructural level, morphologically identified synapses amounted to 47% of all serotonergic varicosities, to 49% for substance P and 50% for thyrotropin-releasing hormone. Synapses appeared both symmetrical and asymmetrical. In each case, these synapses were mainly axodendritic (98%). These synaptic connections could mediate the physiological influence of these 3 substances in the spinal cord on the cardiovascular system.     

Enkephalin fibers synapse on cholinergic neurons in the rat sacral intermediolateral nucleus: a double-immunostaining at the light and electron microscopic levels

Relationships between leucine-enkephalin fibers and cholinergic neurons in the rat sacral intermediolateral nucleus were examined by light and electron microscopy using double-immunostaining method. Cholinergic neurons in the sacral intermediolateral nucleus were labeled by a rat-mouse monoclonal antibody to choline acetyltransferase and stained bluish green with 5-bromo-4-chloro-3-indolyl-beta-D- galactoside reaction products using beta-galactosidase as a marker. On the same sections, leucine-enkephalin fibers were labeled by a rabbit polyclonal antiserum to leucine-enkephalin and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the sacral intermediolateral nucleus. In the same region, leucine-enkephalin-like immunoreactive cells. In the electron microscope, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products were in the form of coarse electron dense deposits in the choline acetyltransferase-like immunoreactive structures and could be distinguished from the much finer grained diaminobenzidine reaction products. Choline acetyltransferase-like immunoreactive neurons received synaptic inputs from leucine-enkephalin fibers-like immunoreactive terminals. These findings suggest that leucine-enkephalin fibers may affect the activity of cholinergic parasympathetic preganglionic neurons.     

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.     

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.     

Characterization of trkB immunoreactive cells in the intermediolateral cell column of the rat spinal cord

The objective of the present study was to characterize the trkB receptor immunoreactive (-ir) cells in the intermediolateral cell column (IML) of the upper thoracic spinal cord. Small trkB-ir cells (area = 56.1 ± 4.4 μm²) observed in the IML showed characteristics of oligodendrocytes and were frequently observed in close apposition to choline acetyltransferase (ChAT)-ir cell bodies. Large trkB-ir cells (area = 209.3 ± 25.2 μm²) showed immunoreactivity for the neuronal marker NeuN, indicating their neuronal phenotype, as well as for ChAT, a marker for preganglionic neurons. TrkB and ChAT were co-localized in IML neurons primarily in cases that had received in vivo administration of nerve growth factor (NGF). These findings reveal two different cell types, oligodendrocytes and neurons, in the IML of the spinal cord that show trkB immunoreactivity, suggesting their regulation by brain derived neurotrophic factor (BDNF) and/or neurotrophin-4 (NT-4). In addition, there is evidence that NGF may play a role in the regulation of trkB-ir preganglionic neurons in the IML.     

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)     

Preganglionic neurons in the sacral spinal cord of the rat: an HRP study.

The pelvic nerve in the rat was exposed to HRP in order to localize the cells of origin of preganglionic fibers coursing in this nerve. Labeled cells were located in spinal segments L₆-S₁, primarily in the lateral intermediate gray. Occassionally, an HRP-filled cell was observed in the ventral horn in association with somatic motoneurons.     

Direct projections from the Ediger-Westphal nucleus to the cerebellum and spinal cord in the cat: An HRP study

A vast majority of neurons in the Edinger-Westphal nucleus (EW), consisting of the anteromedian nucleus (AM) and the posterior part of the EW (EWp: so-called visceral nucleus), were labeled retrogradely with horseradish peroxidase (HRP) injected into the cerebellum in the cat. The pattern of distribution of these HRP-labeled EW neurons was similar to that of EW neurons labeled with HRP injected into the spinal cord. A tendency, however, was noted that EW neurons located in the nuclear areas close to the midline remained unlabeled in the cats injected with the enzyme into the spinal cord. It was assumed that some EW neurons might contribute fibers by way of axon collaterals both to the cerebellum and to the spinal cord. Possible function of the EW as a relay nucleus for retinal inputs to the cerebellum and the spinal cord was discussed.     

The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction

Horseradish peroxidase injections, or solid HRP placements, were made into the dorsolateral quadrant of the spinal cord in ten adult rats, processed according to the blue-reaction (benzidine dihydrochloride) protocol [6]. When the interventions involved the dorsal lateral funiculus (DLF) HRP-labelled somata were observed primarily in the ipsilateral nucleus raphe magnus (NRM), with an occasional cell in nucleus raphe obscurus (NRO) and pallidus (NRP). Other brainstem raphe nuclei consistently lacked labelled neurons. The red nucleus and paralemniscal reticular formation of the rostal pons also contained appreciable numbers of HRP back-filled perikarya. The direct NRM-spinal projection in the rat, involved in the modulation of pain transmission in the spinal cord dorsal horn, originates primarily from the expanded region of the nucleus in the rostralmost medulla.     

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.     

Ultrastructure and synaptic organization of the spinal accessory nucleus of the rat

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.     

A quantitative electron microscopic study of synaptic reorganization in the rat medial habenular nucleus after transection of the stria medullaris

Synaptic plasticity has been studied electron-microscopically in the rat medial habenular nucleus by counting the numbers of different types of synapses per unit area at various times after transection of the stria medullaris on one or both sides.Over 80% of synapses in the normal medial habenula had asymmetrical thickenings, and of these most were in contact with dendritic spines, the rest were in contact with dendritic shafts. The presynaptic terminals were complex structures frequently invaginated by slender spinules from the postsynaptic element. Each presynaptic terminal made contact with a large number of dendritic spines. The remainder of the synapses involved symmetrical contacts on dendritic shafts or neuronal somata.After transection of the ipsilateral stria medullaris the presynaptic elements of both symmetrical and asymmetrical synapses showed electron-dense degeneration. Degeneration was first seen at 24 hours after operation, reached a maximum (of 19%) at two days, and disappeared by 12 days.Non-degenerating synapses fell to around 30%, of their normal level at four days and finally recovered to around 80%, of normal by more than 100 days. Differential counts for synapses on dendritic spines, shafts and cell bodies revealed different time courses for reinnervation. Thus, synapse numbers on spines began to recover sooner than those on shafts and somata. but only the number of synapses on somata returned to almost normal (98%), whilst those on spines and shafts reached only about 70%, of normal.Membrane thickenings resembling vacant postsynaptic sites were rarely (0.3%,) found in unoperated animals. They appeared after transection of the ipsilateral stria, reached a peak of around 12%, at 4 6 days, (slightly later than the degeneration), and then fell, although not quite reaching their normal low levels even at more than 100 days.The nucleus as a whole shrank by about 17% of its normal volume. This would cause the synaptic density to increase by about 12% (calculated as³ √17²%), an effect which is not of sufficient magnitude to account for the observed recovery of synapse numbers, which involves a nearly three-fold increase —i.e. from 30% to 80% normal. The temporal coincidence between the recovery of non-degenerating synapses, and the disappearance of degenerating synapses and vacant sites favours the view that the denervated sites are reinnervated by the formation of new presynaptic terminals.Under normal circumstances the contralateral stria medullaris forms few synapses in the medial habenular nucleus (0.07%, degeneration at four days after transection). However, at long-term survivals after an ipsilateral striai lesion (when the degeneration from that lesion has been completely removed), a lesion of the contralateral stria causes appreciably more degeneration (around 2%), and a considerable fall (more than 30%,) in non-degenerating synapses. Despite the (unexplained) discrepancy between these figures, the findings still suggest that at least some of the new synapses induced after denervation by an ipsilateral strial lesion are formed by axons belonging to the contralateral stria.     

Endomorphin-2 immunoreactivity in the cervical dorsal horn of the rat spinal cord at the electron microscopic level

Endomorphin-2 is a newly discovered endogenous opioid peptide with high affinity and selectivity for the micro-opioid receptor, and potent analgesic activity, particularly in the spinal cord. Using immunoelectron microscopy, we examined the ultrastructure of the endomorphin-2-like immunoreactive processes and their synaptic relationships in the spinal cord. Endomorphin-2-like immunopositive dense-cored vesicles were observed in many axon terminals, and, in a few cases, were observed together with immunonegative dense-cored vesicles. Immunopositive axons with or without myelination were also observed. The endomorphin-2-like immunoreactive axon terminals formed synapses with both immunopositive and immunonegative processes. Most synapses were asymmetrical, but symmetrical synapses were also found. Examples of axo-dendritic, axo-somatic and axo-axonic contacts were observed.This first demonstration of the ultrastructure and synaptic relationships of endomorphin-2-like immunoreactive axon terminals in the spinal cord dorsal horn provides morphological evidence that this peptide functions as a transmitter regulating pain processes.     

Regeneration of the rat spinal cord after thoracic segmentectomy: Restoration of the anatomical integrity of the spinal cord

Segmentectomy of the spinal cord (SC) was performed in 28 rats at the level of the tenth thoracic vertebra. Scar formation at the operation site was studied, along with the completeness of the anatomical integrity of the SC, in control animals (group 1) and after filling of the defect with the neural matrix Spherogel (group 2) and with Spherogel containing embryonic nerve cells (group 3). The defect in the SC in animals of the control group was filled with fibrin masses by 1–2 weeks, while connective tissues scars were already formed by this time in rats of experimental groups 2 and 3. By 10–11 weeks, these animals showed partial recovery of movement in the three lower limb joints. The scar tissue and adjacent zones of the SC showed large quantities of regenerating fine myelinated nerve fibers. These were clearly evident in the zones of the cranial and caudal margins, to the extent of the appearance of typical SC tissue. In preparations obtained from control animals, fine myelinated fibers were noted in the immediate vicinity of SC substance, while nerve fibers were few in number in scar tissue and the cellular bands of the transitional zone. __________ Translated from Morfologiya, Vol. 127, No. 2, pp. 39–43, March–April, 2005.     

胸髓半横断引起腰髓前角钙网蛋白阳性中间神经元突触联系的变化

为检测大鼠胸髓半横断后后肢运动功能状况和腰髓前角中间神经元的突触联系的变化,并分析其相关关系,本实验通过BBB评分、钙网蛋白(CR)和突触囊泡素(SYN)免疫荧光双标记及相关分析方法进行了研究。结果显示:大鼠胸髓半横断后12 h时,后肢运动功能完全丧失,BBB分值下降,从第3 d开始后肢功能逐渐恢复,BBB分值逐渐增加,至第21 d基本恢复正常;同时观察到腰髓损伤侧12 h时CR免疫反应阳性(CR-IR)神经元周围的SYN-IR降低,第3 d时恢复至正常水平,至第21 d时升高;相关分析显示后肢运动功能BBB评分与CR-IR中间神经元周围的SYN表达水平呈正相关(r=0.45,P<0.05)。上述结果提示大鼠胸髓半横断后,与腰髓CR-IR中间神经元形成的突触联系出现可塑性改变,这种可塑性变化可能是后肢运动功能自发性恢复的形态学基础。