Differential termination of large-diameter and small-diameter primary afferent fibers in the spinal dorsal gray matter as indicated by labeling with horseradish peroxidase

Primary afferent fibers, collaterals, and terminations in the spinal dorsal horn were labeled by the orthograde transport of horseradish peroxidase. Lesions of fine afferent fibers comprising the lateral division of dorsal rootlets resulted in the staining of almost no boutons in the marginal zone and substantia gelatinosa but of many boutons in the nucleus proprius. Lesions of large-diameter fibers comprising the medial division restricted staining to a very few boutons in the nucleus proprius while many were stained in the marginal zone and the substantia gelatinosa.     

The action of capsaicin on primary afferent central terminals in the superficial dorsal horn of newborn mice

Capsaicin was injected subcutaneously (50 mg/kg) into 10 mice on days 2 or 3 after birth, and 12 h, 3 and 5 days later the distribution and structure of degenerated primary afferent central axons or terminals (C-terminals) in the lumbar spinal dorsal horn were examined by electron microscopy. Degenerated terminal axons with dense or lamellar bodies or higher electron density were conspicuous 12 h after treatment with capsaicin. Severely degenerated unmyelinated axons, including dense or lamellar bodies engulfed by microglial cells, were numerous in the most superficial (marginal) layer, but rarely seen in the substantia gelatinosa. Two types of primary afferent central terminals in the substantia gelatinosa showed various extents of degeneration: small dark C-terminals (CI-terminals) with densely packed agranular synaptic vesicles, and large light ones (CII-terminals) with less dense agranular synaptic vesicles and a few granular synaptic vesicles. Thus, many central axon terminals of dorsal root ganglion (DRG) neurons that are sensitive to capsaicin enter the marginal layer and substantia gelatinosa. Degenerated primary afferent central axons or terminals markedly decreased in the superficial dorsal horn 3 and 5 days after capsaicin treatment, still, there were many degenerating DRG neurons at this time as shown by our previous study. Previously we also reported that fewer slightly degenerating unmyelinated dorsal root axons and small DRG neurons appear at 12 h and larger DRG neurons degenerate later than smaller ones after treatment with capsaicin. As a result, the discovery of many severely degenerated terminal axons in the superficial dorsal horn soon after treatment supports the idea that capsaicin first acts on the central terminals and that this is followed by damage to larger DRG neurons.     

Substance P-immunoreactive elements in laminae I and II of the chicken spinal cord: a light- and electron-microscopic study

Substance P (SP)-immunoreactive (IR) elements were studied in laminae I and II of the chicken spinal cord in conjunction with an anti-SP monoclonal antibody at light- and electron-microscopic levels by means of the indirect antibody peroxidase-antiperoxidase technique. At the light-microscopic level, SP-IR elements were most intensely observed in the dorsolateral portion of the dorsal horn, laminae I and II. Electron-microscopically, SP-IR boutons contained large spherical dense-cored vesicles (diameter range: 60-125 nm) and spherical clear vesicles. They were subdivided into two groups: large SP-IR boutons, which were the central terminals in synaptic glomeruli, and small SP-IR boutons. In the synaptic glomerulus, two kinds of non-IR presynaptic profiles made axo-axonic synapses with the SP-IR central terminal: one was the presynaptic profile containing pleomorphic clear vesicles and the other was the presynaptic profile containing large dense-cored vesicles. A 'septate junction'-like structure was observed between large SP-IR boutons in synaptic glomeruli. The present results suggest that SP-containing primary afferents are modulated presynaptically by two different neurotransmitter or modulator systems.     

Early morphological changes of primary afferent neurons and their processes in newborn mice after treatment with capsaicin

Degenerating figures of dorsal root ganglion (DRG) neurons and their central and peripheral processes (dorsal root and saphenous nerve) and terminals (central terminals in the superficial dorsal horn and cutaneous nerve of the hind paw dorsal skin) of neonatal mice were examined 30 min, 1, 2 and 5 h, and 2, 3, 5, and 10 days after subcutaneous injection of capsaicin on post-natal day 2. Many small DRG neurons showed degeneration 1 h after treatment. Scarcely any features of degeneration were seen in the DRG and dorsal root 10 days after treatment. The degenerating aspects of terminal axons in the marginal layer of the superficial dorsal horn were characterized by enlarged round axons with closely packed osmiophilic materials, lamellar bodies, and loss of axoplasmic organelles. Two types of central terminals (C-terminals) showed degeneration in the substantia gelatinosa from 30 min after treatment onward. One type consisted of small, round, sinuous or slender dark terminals (CI-terminals), and the other of large, pale, round or angular terminals (CII-terminals). Those that degenerated markedly had homogeneously electron-dense axoplasm with dilated synaptic vesicles and inclusion bodies. Extensive degeneration of terminal axons in the marginal layer occurred 5 h after treatment, whereas conspicuous degeneration of C-terminals occurred from 30 min to 10 days after treatment in the substantia gelatinosa. CI-terminals showed marked degeneration during the first 3 days, whereas marked degeneration of CII-terminals occurred between 5 and 10 days after treatment. This time difference between the peaks of degeneration of CI- and CII-terminals indicates an important difference in the origins of these two types of capsaicin-sensitive, nociceptive fibers in the superficial dorsal horn; CI-terminals are derived from small DRG cells, whereas CII-terminals are derived from larger DRG cells. Unmyelinated axons in the dorsal root, saphenous nerve, and dorsal skin of the hind paw showed similar degeneration patterns 2 h after treatment to those of terminal axons in the marginal layer. Thus, the degenerating profiles in the marginal layer suggest that these axons arose from collaterals of unmyelinated primary axons descending or ascending within the marginal layer. Numerous enlarged degenerating axons showing vacuolation were conspicuous in the dorsal skin 3 days after treatment. The synchronous degeneration of the smaller DRG neurons, their central and peripheral processes, and their CI-terminals in the substantia gelatinosa supports the idea that the smaller DRG neurons are directly influenced by capsaicin, and that their degeneration is followed by centrifugal degeneration.     

Glycine-containing terminals in the rat dorsal vagal complex

The present study examined the distribution of glycine and glycine-receptors in the dorsal vagal complex using pre-embedding immunocytochemistry. Glycine-immunoreactive terminals were present in moderate densities in the medial, intermediate, interstitial, commissural and ventrolateral subnuclei of the nucleus tractus solitarii. The dorsolateral nucleus tractus solitarii and the dorsal vagal motor nucleus contained only very few, scattered glycine-containing terminals. Glycine terminals appeared to be concentrated in regions of the dorsal vagal complex receiving primary vagal afferents, though previous studies have suggested that glycine is not present in these afferents. A conspicuously high concentration of glycine terminals was observed in the medial nucleus tractus solitarii where a population of cholinergic neurons has been identified previously. Ultrastructurally glycine immunoreactivity was principally associated with terminals containing flattened, pleomorphic vesicles and forming symmetrical synaptic contacts, mostly with dendrites. Glycine receptor immunoreactivity was present throughout the dorsal vagal complex with little evidence of subnuclear localization. With electron-microscopic examination, glycine receptor immunoreactivity was associated with dendritic membranes and was associated presynaptically with terminals containing flattened pleomorphic vesicles.

Overall, the present data provide evidence consistent with a neurotransmitter role for glycine in the dorsal vagal complex. The presence of glycine in regions of the dorsal vagal complex receiving vagal afferents suggests a prominent role for this neurotransmitter in autonomic regulation.     

The median and lateral substantia gelatinosa in the cervical cord of the musk shrew (Suncus murinus) and its synaptic composition

Summary The extent and laminar arrangement of the substantia gelatinosa (SG) were examined in the cervical spinal cord of the shrew. Between C1 and C6 or C7 the three dorsal layers on either side of the gray matter were confluent at the commissura posterior grisea in shrews of both sexes. Lamina I was thin with no large marginal cells. Lamina II comprised the major part of the SG, consisting of outer cellular and inner neuropil layers. Lamina III was composed of a meshwork of axon terminals, dendritic profiles and myelinated fibers. Unlike the situation in other mammals, in shrew the incidence of axon terminals with round vesicles was similar in the three uppermost layers, but the occurrence of terminal profiles with flat vesicles was significantly greater in deeper laminae. Lamina IV was restricted to the dorsal horn and did not extend through the midline.     

The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat

Summary The terminal degeneration in the substantia gelatinosa of the rat was studied with the Fink-Heimer silver technique following dorsal root section. Providing the survival time of the animal was in the range of 1–4 days, a massive degeneration was seen in lamina I, II and III of Rexed. The light microscope findings were corroborated by electronmicroscopic observations of degenerating boutons. Spinal cord material examined with silver methods one week after dorsal root section showed few signs of degeneration in the substantia gelatinosa. Although a significant dorsal root distribution to the substantia gelatinosa was found also in the cat, the terminal degeneration in lamina II showed considerable regional variations in this species.     

FRAP-positive and capsaicin-sensitive terminals in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis.

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.     

Endomorphin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons in vitro

Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the mu-opiate receptor. Opioid compounds expressing mu-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunuoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective mu-opiate receptor antagonist beta-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.     

Synaptic organization of the nucleus raphe dorsalis of the cat

Summary This electron microscopic study describes the different types of synaptic terminals found in the nucleus raphe dorsalis of the adult cat. Serial section analysis was used extensively to confirm the nature of the synaptic contact established by the various classes of terminals.Five different classes of terminals are identified according to the shape and packing density of the synaptic vesicles and type of contact they establish. The most common class (RDI-type) contains densely packed, round, agranular synaptic vesicles and establishes asymmetrical synaptic contacts. A second class (RDII-type) also contains spherical synaptic vesicles, but establishes symmetrical synaptic contacts with dendrites of all sizes. Most of the terminals in these two classes contain a few dense-cored synaptic vesicles, but a small sub-group contains many dense-cored vesicles. A third, less frequent, class (RSI-type) contains sparsely packed spherical synaptic vesicles and the majority of these terminals have asymmetrical contacts. A fourth terminal class contains pleomorphic synaptic vesicles (P-type), contacts dendrites of all sizes, and usually establishes symmetrical synaptic contacts. Finally, boutons thought to be the vesicle-filled excrescences of dendrites (postsynaptic dendrites) are found and in some cases the dendritic origin of these profiles was confirmed by serial sectioning. Such boutons containing pleomorphic vesicles are presynaptic to other such dendrites as well as conventional dendrites, and are postsynapticto the other terminal types described.Somata within the nucleus exhibit somatic spines but receive few synaptic contacts. Most axo-somatic terminals contain either round or pleomorphic vesicles and have postsynaptic thickenings intermediate to the symmetric and asymmetric types.     

The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala

Vasoactive intestinal polypeptide (VIP) neurons within the central nucleus of the rat amygdala were examined using light and electron microscopic immunocytochemical techniques. Vasoactive intestinal polypeptide-immunoreactive neurons were located in the ventral part and less frequently in the central part of the central nucleus. Vasoactive intestinal polypeptide positive terminals were distributed throughout the medial part of a cytoarchitectonically distinct central zone of the central nucleus. Three types of terminals formed synaptic contacts on VIP-immunoreactive neurons: type A containing round vesicles, type B containing many pleomorphic vesicles and type C containing fewer pleomorphic vesicles. All VIP-immunoreactive boutons observed were of type A variety, and made asymmetrical and symmetrical synaptic contacts on both VIP-immunoreactive and nonreactive neurons within the central nucleus.     

GABAergic neurons in the mouse superficial dorsal horn with special emphasis on their relation to primary afferent central terminals

Immunocytochemical studies were carried out on the morphological relation between primary afferent central terminals (C-terminals) and GABAergic neurons in the mouse superficial dorsal horn. The superficial dorsal horn is composed of many synaptic glomeruli comprising two types: Type I with centrally located CI-terminals surrounded by several dendrites and few axonal endings, and Type II with centrally located CII-terminals surrounded by several dendrites and a few axonal endings. The CI-terminals are sinuous or scalloped with densely packed agranular synaptic vesicles, a few granular synaptic vesicles and mitochondria, and show an electron dense axoplasm, whereas the CII-terminals are large and round or rectangular with evenly distributed agranular synaptic vesicles, a number of granular synaptic vesicles and mitochondria, and show an electron opaque axoplasm. The immunoreaction of GABA was remarkable in the superficial laminae of the dorsal horn. Many interneuronal somata in the substantia gelatinosa showed GABAergic immunoreactivity. The immunoreaction was seen in the entire GABAergic neuroplasm, but not in the nucleus and its envelope. Most GABAergic features appeared as dendrites making postsynaptic contact with CI- or CII-terminals; i.e., numerous C-terminals made presynaptic contact with GABAergic dendrites. GABA immunoreactivity was seen over round synaptic vesicles and mitochondrial membranes. A few CII-terminals made presynaptic contact with GABAergic interneuronal somata. Previous physiological and anatomical studies have suggested that not only the cutaneous nociceptive primary afferent C-terminals but also mechanoreceptive primary afferent C-terminals make presynaptic contact with the GABAergic dendrites, boutons and soma. The presynaptic relation of these primary afferents with GABAergic neurons seems to provide morphological support for the essential feature of the gate control theory: primary afferent fibers may play a part in the modulation of nociceptive information via GABAergic neurons in the superficial dorsal horn. Small GABAergic terminals were found to make contact with blood capillaries suggesting the release of GABA into circulation.     

Localization of serotonin- and substance P-like immunofluorescence in the caudal spinal trigeminal nucleus of the rat

A double immunofluorescence labeling method was used to study the localization of serotonin (5-HT)- and substance P (SP)-like immunoreactivities within neuronal fibers in the caudal spinal trigeminal nucleus of the rat. The 5-HT- and SP-immunoreactive fibers share extensive topographical overlap within the superficial laminae of the caudal trigeminal nucleus; however, the majority of stained fibers are immunoreactive for either 5-HT or SP, but not both simultaneously. A small population of fibers in which 5-HT and SP are co-localized is present and restricted to the marginal zone as well as the inner and outer layers of the substantia gelatinosa. The results of the present study suggest that fibers containing coexistent 5-HT and SP may be involved in the processing of nociceptive somatic sensation in the medullary dorsal horn of rat.     

An ultrastructural morphometric study of developing rat substantia gelatinosa

A morphometric analysis has been done on developing rat substantia gelatinosa of the lower cervical and upper thoracic levels of the spinal cord starting on the 15th day of gestation. The following parameters were measured: cell body diameter, cytoplasmic/nuclear areas, synaptic density, synaptic type and vesicle morphology of the presynaptic terminal in axodendritic synapses. Cell body size and cytoplasmic/nuclear areas of gelatinosal cells increase until the 15th day postnatally and then decrease somewhat to the adult values. The first synapses are seen on gestation day 17. Synaptic density increases linearly until the third day postnatally. Axodendritic synapses are most common throughout development and in the adult, while the proportion of axoaxonic synapses increases and axosomatic synapses decreases during development. Most of the terminals in axodendritic synapses contain clear-spherical vesicles but the occurrence of clear-flat vesicles and dense-cored vesicles in the terminals increases during development. It appears that these morphological parameters provide a stable index of development in the substantia gelatinosa which can be correlated with functional development of the area. Hopefully, they will provide a means to assess subtle anomalies induced by nonteratogenic drugs or other environmental changes.     

Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord

Light- and electron-microscopic localization of substance P in the monkey spinal cord was studied by the peroxidase anti-peroxidase technique with the particular aim of examining types of interactions made by substance P-positive boutons with other neuronal elements in the dorsal horn. By light-microscopy dense labeling for immunoreactive substance P was found in laminae I, II (outer zone) and V (lateral region), consistent with findings in other mammalian species. By electron-microscopy, substance P-positive staining was mostly in unmyelinated and in some thinly myelinated small diameter fibers. Substance P-positive terminals contained both large granular vesicles (80-120 nm diameter), which were filled with reaction product, and clear round vesicles (40-60 nm). Substance P-positive large granular vesicles were sometimes observed near presynaptic sites and in contact with dense projection there. Immunoreactive substance P boutons were small to large in size (1-4 micron), formed synapses with somata and large dendrites and were the central axons of synaptic glomeruli where they were in synaptic contact with numerous small dendrites and spines. Substance P-labeled axons frequently formed synapses with dorsal horn neurons which were also postsynaptic to other types of axons. Substance P-positive profiles participated in numerous puncta adhaerentia with unlabeled cell bodies, dendrites and axons. Only rarely, some suggestive evidence was obtained indicating that axons might synapse onto substance P-containing boutons. Biochemical analysis of monkey spinal cord tissue extracts, undertaken to characterize more precisely the immunoreactive substances, indicated that only substance P and its oxide derivative were detected with the antiserum used in the immunocytochemistry. These morphological findings show that substance P is contained within a class of axon terminals, many of which have been shown previously in the monkey to originate from the dorsal root. The results suggest that modulation of substance P primary afferents terminating in the outer dorsal laminae of the monkey spinal cord occurs in part via axonal inputs onto dorsal horn neurons postsynaptic to the primary afferent.     

Caudal end of the rat spinal dorsal horn

We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons.     

应用单宁酸增加致密核心小泡电子密度的研究

对如何应用单宁酸提高密核心小泡电子密度进行了研究，结果发现实验组动物应用单宁酸后，其延髓背角胶状质的超微结构出现了如下特征性变化；（１）膜结构清晰度增加，反差鲜明；（２）大、小致密核心小泡均被媒染，其电子密度显著提高、大致密核心小泡于突触部位胞吐入细胞间隙内的介质，被即时媒染、固定。     

Location of nerve cells producing the synaptic vesicles situated in the substantia gelatinosa of the spinal cord (1)

A search for the cells of origin of synaptic vesicles with electron-opaque cores and located in the substantia gelatinosa was attempted by subjecting cats to transection of dorsal roots and double hemisection in the upper lumbar region. The hemisections were accompanied by interruption of the dorsal rootlets to the affected segment. The cats were perfused with buffered aldehydes and processed for electron microscopy 55–71 days after the operations. Comparison of intact and operated sides of the rhizotomized cats revealed no discernible differences in numbers and location of the granulated vesicles associated with the substantia gelatinosa. In cats with double hemi-section plus rhizotomy the distribution of the granules was the same on the normal and operated sides but the numbers appeared to be reduced especially close to the surgical incisions. Dark-core vesicles were situated in association with the Golgi apparatus of substantia gelatinosa neurons, in dendrites, in small axons and in synaptic sacs in all material studied. It was concluded that most of the granulated vesicles located in the substantia gelatinosa are produced by small neurons in the immediate vicinity of their axon terminals.     

Synapses upon the axon origin of dorsal horn neurons in the rat spinal cord

Axon terminals synapsing with axon hillocks or origins of Golgi-impregnated and gold-toned neurons in the dorsal horn of the rat were shown in serial electron micrographs. Synapses occurred irrespective of the site (perikaryon or dendrite) and mode (with or without an axon hillock) of the axon origin. The synapsing axon terminals contained 3 populations of vesicles: pleomorphic and flattened synaptic vesicles and a combination of pleomorphic and dense-core vesicles. The membrane thickening in the axon-axon hillock synapses was of the symmetrical type.     

A comparison between wheat germ agglutinin-and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat

Horseradish peroxidase conjugates of either the lectin wheat germ agglutinin or choleragenoid, the binding subunit of cholera toxin, were injected into the L5 spinal ganglion of adult rats. This enabled comparison of these two conjugates as anterograde tracers in the primary sensory system. After a postoperative survival of 4 h to 30 days, the rats were perfused and frozen sections from spinal and medullary regions receiving primary afferents were processed for horseradish peroxidase histochemistry with tetramethylbenzidine as the chromogen. Additional observations were made in two adult cats. Following injection of wheat germ agglutinin-horseradish peroxidase the labelling appeared mostly as small-sized granules. The concentration of labelled primary afferents in the grey matter of the spinal cord was greatest in the marginal zone and the substantia gelatinosa and less pronounced in the deep parts of the dorsal horn. Labelling was also found in a region lateral to the central canal and in the ventral horn. Following injection of choleragenoid-horseradish peroxidase the labelling appeared mainly as larger-sized granular profiles. The concentration of labelled primary afferents was greatest in the deep part of the dorsal horn and pronounced in a region lateral to the central canal and in the ventral horn. All these regions are known to receive large calibre fibres. The marginal zone and the substantia gelatinosa, known to receive fine calibre fibres, showed almost no labelling in the rat. In the cat, however, there was somewhat more labelling in the substantia gelatinosa. Labelling of neuronal cell bodies indicating transneuronal transport was seen after injection of wheat germ agglutinin-horseradish peroxidase. Transneuronal labelling did not seem to occur after injection of choleragenoid-horseradish peroxidase. The present findings show that wheat germ agglutinin-and choleragenoid-horseradish peroxidase give rise to markedly different labelling patterns. A possible explanation for the different labelling in the marginal zone and substantia gelatinosa could be that certain primary sensory neurones lack either receptors for choleragenoid on their neuronal plasma membrane or the ability to transport the choleragenoid-horseradish peroxidase complex.