The distribution of dorsal root axons to laminae IV,V, and VI of the macaque spinal cord: A quantitative electron microscopic study

The projections of dorsal root axons to the deeper laminae (IV, V, and VI) of the Macaque spinal cord were examined by the use of experimentally induced degeneration following dorsal rhizotomy or by injection of dorsal root ganglia with tritiated amino acids followed by light and electron mi-croscopic autoradiography. Following dorsal rhizotomy, neurofilamentous degeneration of synaptic profiles occurs in each of the three deep laminae, more commonly in laminae IV and V than in lamina VI. The neurofilamentous degeneration is seen both in central glomerular (C) profiles and in many of the round vesicle (R) profiles. Neurofilamentous degeneration occurs as early as 18 hours following rhizotomy and the degenerating terminals are most numerous at 3–4 days postrhizotomy. None are seen after 7 days survival. The neurofilamentous profiles form axodendritic and, occasionally, axosomatic synapses with neurons of the dorsal horn. They are also seen to be postsynaptic to flat vesicle (F) profiles in axoaxonal synapses. A second type of degeneration, electron-lucent degeneration, is seen in laminae V and VI, and only occasionally in lamina IV. The lucent degeneration occurs somewhat later after rhizotomy than does the neurofilamentous degeneration and reaches its peak at 5 days postrhizotomy. No lucent terminals are seen after 7 days survival. Electron-dense degeneration, so common in lamina II, is not seen in the deeper dorsal horn. Autoradiographic techniques show that both C and R terminals are labelled in the deeper dorsal horn. Both of these terminals form axodendritic synapses and a significant number are found to be postsynaptic in axoaxonal synapses. Most of the C terminals degenerate following rhizotomy or are labelled following injection of the parent dorsal root ganglia with tritiated amino acids. Approximately one-fifth of the R profiles are derived from dorsal roots. F profiles do not appear to be of dorsal root origin in any case. It is concluded that neurofilamentous alterations represent the degeneration of larger-diameter (Aβ) axons which distribute to the deeper dorsal horn and that electron-lucent degeneration represents the termination of Aδ fibers. Electron-dense degeneration thought to represent the termination of nonmyelinated axons (C fibers) in the superficial dorsal horn is not seen in the deeper dorsal horn and it is concluded that C fibers do not project to the deeper laminae.     

Substance P- and GABA-like immunoreactivities are co-localized in axonal varicosities in the superficial laminae of cat but not rat spinal cord

In the present study, we applied a combination of pre-embedding peroxidase-based immunocytochemistry and post-embedding immunogold staining to examine the synaptic interactions of substance P (SP) and γ-aminobutyric acid (GABA) in the superficial laminae of the dorsal horn of cat and rat spinal cord. We demonstrate for the first time the co-existence of SP and GABA immunoreactivities in axonal boutons in laminae I–III of cat spinal dorsal horn. In cat, most SP + GABA immunoreactive (IR) axonal boutons stablished synapses with SP-IR or non-IR dendrites. These synapses were exclusively symmetric. Quantitative analysis showed that the percentage of SP/GABA double labelled bouton profiles was higher (7%) in lamina I but was considerably lower in laminae IIo, IIi and III. Similarly, the density (number of bouton profiles per 100 μm²) of SP + GABA-IR bouton profiles was highest in lamina I. However, in agreement with previous studies, the co-localization of SP and GABA immunoreactivities was never detected in the rat dorsal horn. In both species, SP + GABA-IR or GABA-IR axonal bouton profiles were never seen presynaptic to SP-IR boutons. These findings provide a morphological basis for the interaction of excitatory and inhibitory agents in the nociceptive circuits in the dorsal horn of the cat and rat spinal cord.     

The fine structure of laminae I, II and III of the macaque spinal cord.

The neuronal and synaptic organization of the upper three laminae of the macaque monkey spinal cord have been examined by light and electron microscopy. Using a variety of light microscopic techniques it is possible to demonstrate a laminar pattern in the upper spinal cord which is comparable to that originally defined by Rexed ('52) in the cat. Lamina I contains an array of neurons and neuronal processes oriented chiefly in a horizontal pattern, parallel to the dorsal surface of the spinal cord. Most of the neurons of the lamina are small, 10-15 μ in diameter, but an occasional cell exceeds 20 μ in diameter. The dorso-ventral width of this lamina varies from 40-150 μ, medially to laterally. Lamina II corresponds to the substantia gelatinosa and is readily recognized owing to its paucity of myelinated axons and the abundance of small neurons measuring 8-10 μ. This layer varies in dorso-ventral width from 150-250 μ, medially to laterally, and may be divided cytoarchitecturally into an outer zone in which the cells are densely packed and an inner zone containing somewhat more dispersed neurons. The immediately subjacent lamina III contains a meshwork of small and medium sized axons which clearly separate it from the adjacent substantia gelatinosa. The neurons are about the same size as those of lamina II. The ventral border between lamina III and lamina IV is poorly defined as the differentiation between the two laminae is rimarily determined by the appearance of larger cells scattered in lamina IV. The dorso-ventral width of lamina III is approximately 250 μ. The synaptic populations, analyzed by counts of more than 10,000 synapses, were quantitatively determined in the upper three laminae of the seventh lumbar segment of three normal monkeys. Synaptic profiles with round synaptic vesicles were found to be the dominant synaptic population in lamina I and outer lamina II and gradually decrease in number in inner lamina II and throughout lamina III. Conversely, flat vesicle profiles are not numerous in lamina I but increase in numbers throughout lamina II until they become the dominant synaptic population in lamina III. Synaptic profiles with an admixture of large granular vesicles and small agranular vesicles are common in lamina I and outer lamina II, then decrease markedly in number in inner II and throughout lamina III. Synaptic profiles forming the central axon in synaptic glomeruli are present in all three laminae. The vast majority of synapses are axodendritic; axosomatic synapses are generally found only on larger neurons. Axoaxonal synapses are not common in lamina I, but are frequently seen in laminae II and III. Dendritic processes containing synaptic vesicles are present, but not in large numbers. It is concluded that there are significant morphological differences in the neuronal and synaptic populations in various subdivisions of the three laminae of the upper dorsal horn. The differences in synaptic populations reinforce the subdivision of the upper dorsal horn into horizontal laminae, and the separation of the substantia gelatinosa (lamina II) from the subjacent lamina III. It is likely that these morphological variations underly differences in neuronal functions of the upper three laminae.     

Distribution of the tract of Lissauer and the dorsal root fibers in the primate spinal cord.

The tract of Lissauer receives small caliber dorsal root fibers in addition to axons arising from dorsal horn neurons. The termination of Lissauer's tract and dorsal root fibers was examined in the C7 segment of the rhesus monkey spinal cord. The distribution of normal dorsal root afferents was mapped by labelling the C7 dorsal root ganglion with tritiated amino acids, and then compared with the degeneration of C7 dorsal root fibers following an intradural dorsal rhizotomy. To focus on the distribution of the small afferents, the degeneration following a Lissauer tractotomy was compared with the degeneration following dorsal rhizotomy and following selected lesions involving the large afferents. The survival times following the lesions and rhizotomies were varied to facilitate identification of groups of fibers and terminals which might degenerate at different rates. Both large and small diameter dorsal root afferents were found to exhibit the same rostro-caudal topography within the dorsal horn. The C7 root axons and terminals distribute throughout the mid-C7 dorsal horn grey. Proceeding rostrally through C6, the majority of the C7 root fibers ending in laminae I-IV shift to a lateral position. Proceeding caudally through C8, the C7 root fibers shift medially. Few of the small diameter C7 afferents entering via Lissauer's tract extend above C6 or below C8. Large diameter C7 afferents, arising as dorsal column collaterals, can extend several segments above and below C7. Autoradiography revealed label in all dorsal horn laminae, the heaviest always occurring in the substantia gelatinosa. After one day, label was absent over dorsal column and Lissauer's tract axons, suggesting that the label was mainly associated with fine axonal branches or possibly terminals. After six to ten days many axons were labelled and could be traced into the dorsal and ventral horn. Degeneration from the rhizotomies and lesions, as demonstrated with Fink-Heimer and Nauta methods, depended on the survival time. No degeneration products were present before three days. The large afferents begin to degenerate within the dorsal horn after three to four days and mainly terminate in laminae IV-VI; by 12 days they can also be traced into the intermediate and ventral grey. The small afferents, which include those serving pain and temperature sensibility, arise from the tract of Lissauer and distribute to laminae I, II and III. The tract of Lissauer consists of two populations, each containing small afferents. One population degenerates at three to five days and distributes mainly to laminae II and III (substantia gelatinosa); the other degenerates around 12 days and distributes mainly to lamina I and the outer zone of II. It is suggested that the exclusive termination of the small afferents to laminae I, II and III may be correlated with certain unique histochemical properties (e.g., high substance P and high opiate receptor binding levels) of these same dorsal horn areas...     

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord.

Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms.     

The origin, distribution and synaptic relationships of substance P axons in rat spinal cord.

Substance P positive (SP⁺) immunoperoxidase reaction product has been localized in light and electron microscopic preparations of rat lumbar spinal cord using an immunocytochemical method. SP⁺ reaction product was found to be highly concentrated in dorsal horn laminae I, II, portions of III, Lissauer's tract, s small nucleus in the dorsal part of the lateral funiculus, and in a longitudinal bundle of fibers just ventral to the central canal. Moderate accumulations of SP⁺ reaction product were observed in portions of laminae III-V, lamina X and in a narrow zone bordering fasciculus gracilis which expanded in its ventral aspect to include nucleus cornucommissuralis dorsalis and nucleus dorsalis. Remaining portions of the spinal gray matter exhibited extremely sparse staining. Light microscopic observations indicated that SP⁺ product was concentrated in fine axons and collaterals, which displayed densely-stained varicosities and terminal puncta. These varicosities and puncta were closely associated with neurons and blood vessels. Dorsal rhizotomy produced a marked reduction in the number of SP⁺ fibers in Lissauer's tract, dorsal horn laminae I-III and the nucleus of the dorsolateral funiculus. However, dorsal root fibers were not the only source of SP⁺ structures in these regions since some SP⁺ fibers remained following dorsal root resections. In addition, this residual population of SP⁺ fibers did not appear to be altered by a combination of dorsal rhizotomy and ipsilateral transverse hemisection of the spinal cord. In conjunction with the results of other investigators, this finding suggests that the SP⁺ fibers which remain after dorsal rhizotomy are derived from local circuit interneurons of the spinal cord. Electron microscopic observations revealed that certain axons and axon terminals contained concentrations of SP⁺ product which were associated with large, granular vesicles, while lesser amounts were in the terminal cytoplasm and associated with the exterior surfaces of small, agranular synaptic vesicles. SP⁺ terminals formed axodendritic, axosomatic and axoaxonal synapses, but the deposition of SP⁺ product often was not concentrated precisely at the “active sites” of these synaptic junctions. Some SP⁺ axons also made rudimentary contacts with astrocytic processes including those surrounding blood vessels. Since many of the SP⁺ terminals are similar in several respects to neuroendocrine terminals, it is possible that substance P may be released at several different types of non-synaptic sites as well as at conventional synaptic junctions. Thus our findings suggest that substance P could participate in a variety of neural functions ranging from those which are limited to synaptic junctions to those which are more generally distributed via an involvement of axonal plexuses as well as the glial, vascular and ventricular systems.     

Dorsal root projections to dorsal horn neurons in the cat spinal cord

The projection of dorsal root fibers to the dorsal horn of the spinal cord of the cat has been studied by electron microscopy. Two distinct types of synaptic degeneration are seen with the electron microscope: small knobs which exhibit a marked increase in electron density, and large knobs which fill with neurofilaments. Variations in the distribution of degenerating knobs to the six laminae of the dorsal horn are observed: dense degenerating knobs are found throughout the horn, but are quite rare in laminae I and II. Lamina III exhibits the most dense knobs, and a great many are also present in laminae IV and VI with a somewhat lesser number seen in lamina V. Knobs undergoing neurofilamentous degeneration are found only in laminae V and VI, and are much Jess frequently seen than are dense knobs. The types of synaptic contacts made by degenerating dorsal root fibers also vary from one region of the dorsal horn to another. Dense degenerating knobs synapse primarily with small dendrites in laminae I, II and III, but not at all with large dendrites or nerve cell bodies. In lamina III, dense knobs are seen in axoaxonal synapses, and are always the pre-synaptic component of the synapse. In laminae IV, V and VI dark knobs are commonly seen to synapse upon cell bodies of large and medium sized neurons and their proximal dendrites, but not upon small cell bodies Dense knobs upon small dendrites are common. In degenerating axoaxonal synapses, the dense knob is always the post-synaptic component. Degenerating neurofibrillar knobs are only seen to synapse with cell bodies or larger dendrites in V and VI and not at all other synaptic knobs. The results are correlated with findings by light microscopy. Comparisons are made with some of the known physiological properties of dorsal horn neurons.     

Ultrastructural analysis of dynorphin B-immunoreactive cells and terminals in the superficial dorsal horn of the deafferented spinal cord of the rat

Light microscopic studies have demonstrated important differences in the distribution of enkephalin and dynorphin cells and terminals in the dorsal horn. Most importantly, dynorphin neurons are located in regions almost exclusively associated with the transmission and/or control of nociceptive messages (laminae I, IIo, and V); enkephalin neurons, although located in the same regions, are also found in areas involved in the transmission of nonnociceptive messages, e.g., laminae IIi and III. To determine whether there are also differences in the synaptic organization of the two opioid peptides, we have examined the distribution of dynorphin B immunoreactivity at the ultrastructural level. The studies were performed in colchicine-treated rats that underwent dorsal rhizotomy so that the relationship of dynorphin terminals and cells to primary afferent terminals could be established. Dynorphin B-immunoreactive cell bodies and dendrites in laminae I and IIo receive convergent primary and nonprimary afferent input, which suggests that dynorphin neurons receive a small-diameter, nociceptive input. Dynorphin terminals predominantly contain round, agranular vesicles; some terminals also contain a few dense core vesicles. Most dynorphin terminals are presynaptic to unlabelled dendrites; both asymmetric and symmetrical axonal contacts were noted. Dynorphin-immunoreactive boutons are also presynaptic to unlabelled cell bodies and spines. Twenty-nine percent of dynorphin terminals were associated with axonal profiles, including degenerating primary afferent terminals; only rarely could a synaptic density be detected. Although some degenerating primary afferent terminals were clearly presynaptic to dynorphin-immunoreactive terminals, in most cases, the polarity of the relationship between primary afferents and dynorphin terminals could not be established. These data indicate that synaptic interactions made by and with dynorphin-immunoreactive cells and terminals in the superficial dorsal horn are not very different from those that were previously reported for enkephalin cells and terminals. Thus, it is unlikely that dynorphin terminals provide a significant presynaptic input to primary afferent fibers. On the other hand, the presence of a primary afferent input to dynorphin cell bodies and dendrites in the superficial dorsal horn suggests that dynorphin cells receive a direct input from small-diameter, nociceptive primary afferents. That connection might contribute to the increased levels of dynorphin message and peptide that have been reported in rats experiencing a chronic inflammatory condition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic ultrastructure of functionally and morphologically characterized neurons of the superficial spinal dorsal horn of cat

Recordings of neuronal unitary discharges evoked by primary afferent input were made in the superficial part of the spinal cord's dorsal horn, the marginal zone and substantia gelatinosa (also known as laminae I and II), using fine micropipette electrodes filled with HRP. After physiological characterization with respect to primary afferent input, HRP was injected intracellularly iontophoretically into the recorded neuron. Following histochemical processing, the neurons so delineated were studied at the light and electron microscopic levels. No clear relationship between function and either general cellular configuration or synaptic ultrastructure appeared in these analyses, although the concentration of dendritic distribution could be related to the nature of primary afferent excitation. Nocireceptive cells had dendrites mostly branching and ending in lamina I and IIo, while the dendrites of innocuous mechanoreceptive cells arborized principally in lamina II and III. Glomerular synaptic complexes (large, complex arrays of axonic and dendritic profiles with synaptic interconnections) were found to contact a few neurons of both the nocireceptive and mechanoreceptive classes. All neurons received large numbers of simple axonic contacts (small axonic boutons with only 1 or 2 synaptic contacts with a single postsynaptic profile). A degree of specificity in the presynaptic articulations appeared to be reflected by the observations that (1) nocireceptive neurons were never found to receive synaptic contacts from boutons which resembled the known ultrastructure of peripheral innocuous mechanoreceptors, and (2) mechanoreceptive neurons were never seen to receive synaptic contacts from boutons which resembled the known ultrastructure of primary afferent nocireceptors. The axons of the labeled neurons of both nocireceptive and mechanoreceptive classes terminated in simple axonic synapses. All classes of neurons participated in dendrodendritic contacts; however, only some mechanoreceptive neurons had dendrites containing vesicles that were presynaptic to other profiles. No nocireceptive neurons, regardless of gross configuration, were found to have vesicles in their dendrites, but 3 nocireceptive neurons received synapses from presynaptic dendritic profiles.     

Ultrastructure of normal and degenerating glomerular terminals of dorsal root axons in the substantia gelatinosa of the rhesus monkey

The fine structure of primary sensory terminals within glomerular complexes of lamina II of Rexed (substantia gelatinosa Rolandi) in the spinal cord was investigated in normal adult rhesus monkeys and in monkeys subjected to thoracic or lumbosacral dorsal root transection. Three types “scalloped” primary sensory terminals were distinguished on the basis of their ultrastructural characteristics, size, and distribution of synaptic vesicle population: (1) dense sinusoid axon (DSA) terminals contain mediumsized (42–46 nm and 58–62 nm) and large (80 nm) clear synaptic vesicles; (2) large dense-core vesicles (LDCV) terminals are equipped with empty synaptic vesicles ranging from 30 to 106 nm, large, (80 nm) and very large, (100 nm) dense-core vesicles; and (3) regular synaptic vesicles (RSV) terminals contain a homogeneous population of 45–50 nm clear synaptic vesicles. Follwing transection of the dorsal roots, all three types of primary afferents degenerate and become engulfed and phagocytosed by glial cells. However, each type of terminal displays a different mode and tempo of degeneration as seen in monkeys sacrificed 36, 48, and 72 hours following rhizotomy. DSAs follow the osmiophilic degeneration pattern; LDCVs are characterized by a gradual increase in the number of “electron-dense bodies” and, less frequently, by a progressive osmiophilic process; RSVs exhibit signs of a filamentous degeneration, accompanied by clusters of synaptic vesicles. The three types of terminals are distributed in an overlapping but distinct pattern within posterior horn. Thus DSAs are present in highest numbers in lamina II where they constitute the most frequent terminal type. LDCVs also occur in lamina II in its outer half but are most concentrated in lamina I. RSVs predominate in the deepeer layers of the dorsal horn (lamina III) but are also found in the internal half of lamina II. On the basis of these ultrastructural data and a comparison with afferent profiles impregnated according to the Golgi method, it appears that DSAs and LDCVs correspond respectively to superficial and marginal collaterals of small, thin dorsal root fibers whereas RSVs represnt terminals of deep collaterals from large, thick dorsal root axons.     

The Relationship Between Glycine and Gephyrin in Synapses of the Rat Spinal Cord

In order to examine the relationship between gephyrin (the peripheral membrane protein associated with glycine receptors) and glycinergic boutons, we have carried out a post-embedding immunogold study of glycine-like immunoreactivity on sections of rat lumbar spinal cord which had previously been reacted with monoclonal antibody to gephyrin. In all three areas examined (laminae I and II, lamina III and lamina IX) the majority of profiles which were presynaptic at gephyrin-immunoreactive synapses were enriched with glycine-like immunoreactivity. It was estimated that at least 83% of profiles presynaptic to gephyrin-immunoreactive synapses in the superficial dorsal horn (laminae I and II) were glycine-immunoreactive, while for lamina III and the ventral horn (lamina IX) the proportions were at least 91% and 98% respectively. This provides strong evidence that glycine is a transmitter at those synapses where gephyrin- and glycine-like immunoreactivities are both present, but suggests that gephyrin may sometimes be expressed at non-glycinergic synapses and indicates the need for caution in using gephyrin-immunoreactivity as a marker for glycinergic synapses within the spinal cord. By reacting serial sections of dorsal horn with antisera to glycine and GABA, we have shown that many boutons in laminae I-III of the dorsal horn show both types of immunoreactivity and are therefore likely to use both amino acids as inhibitory transmitters. Many of the boutons which were presynaptic at axoaxonic synapses in the ventral part of lamina II and in lamina III were glycine- and GABA-immunoreactive and in many cases the postsynaptic element was the central axon of a type II synaptic glomerulus. Taken together with pharmacological evidence, this suggests that inhibitory intemeurons in the dorsal horn which use both GABA and glycine may be important in controlling the flow of information from hair follicle afferents to other spinal neurons.     

Bulbospinal projections in the primate: a light and electron microscopic study of a pain modulating system

The projections of the nucleus raphe magnus (NRM) and the immediately adjacent reticular formation were studied in the macaque monkey following injections of the rostroventral medulla with 3H-leucine and examination of the resultant labeled axons and terminals by light and electron microscopic autoradiography. Five monkeys had accurately placed injections, which resulted in fiber pathway labeling that coursed caudally, laterally, and dorsally to project to laminae I, II, and V of subnucleus caudalis of the trigeminal and then traveled in the dorsolateral funiculus of the cord and terminated in similar laminae of the spinal dorsal horn at cervical levels. The pathway was only lightly labeled caudal to the cervical enlargement and could not be readily discerned above background in the thoracic or lumbar cord. Electron microscopy revealed that axons and terminals serving this system constitute a heterogeneous population. Large-diameter myelinated axons (3-6-micron diameter), small myelinated axons (0.75-3-micron diameter), and clusters of nonmyelinated axons were labeled. Terminals in laminae I, II, and V contained mixtures of clear round and granular vesicles or clear pleomorphic and granular vesicles or formed the central element in synaptic glomeruli. The labeled profiles formed asymmetrical or symmetrical synapses on medium and small dendrites; labeled axosomatic synapses were not observed. In rare instances there were contacts between labeled profiles and vesicle-containing structures, which were probably dendritic, but whether the NRM axon was pre- or postsynaptic to such structures could not be determined. It was concluded that the NRM in the monkey is organized in a manner quite similar to that previously described in the cat. The wide variety of fiber types and synaptic terminals serving this system suggests that different classes of neurons participate in it, probably using several transmitter substances that result in varying postsynaptic effects on neurons located in the trigeminal complex and dorsal horn.     

Neurons in the superficial dorsal horn of the rat spinal cord projecting to the medullary ventrolateral reticular formation express c-fos after noxious stimulation of the skin

The nociceptive nature of the neurons of the superficial dorsal horn (laminae I–III) which project to the medullary ventrolateral reticular formation is studied in the rat. Medullary injections of Fluoro-Gold showed exclusive retrograde labeling of laminae I–III cells when the tracer filled a zone intermediate between the lateral tip of the lateral reticular nucleus and the spinal trigeminal nucleus, pars caudalis. This zone is here called VLMlat. Following noxious mechanical or thermal stimulation of the skin, double-labeled neurons, which stained retrogradely and were Fos-immunoreactive, prevailed in laminae I and IIo. Double-labeled neurons were few in lamina IIi after thermal stimulation and entirely lacking in lamina III after the two kinds of stimulation. Findings in lamina I confirm previous electrophysiological data (see Menétrey et al.,J. Neurophysiol., 52 (1984) 595–611) showing that lamina I cells projecting to the ventrolateral reticular medulla convey noxious messages. The occurrence of numerous double-labeled cells in lamina IIo suggests that this lamina is also involved in nociceptive transmission to the VLMlat.     

Synaptic reorganization in the substantia gelatinosa after peripheral nerve neuroma formation: aberrant innervation of lamina II neurons by Abeta afferents.

Intracellular recording and extracellular field potential (FP) recordings were obtained from spinal cord dorsal horn neurons (laminae I-IV) in a rat transverse slice preparation with attached dorsal roots. To study changes in synaptic inputs after neuroma formation, the sciatic nerve was sectioned and ligated 3 weeks before in vitro electrophysiological analysis. Horseradish peroxidase labeling of dorsal root axons indicated that Abeta fibers sprouted into laminae I-II from deeper laminae after sciatic nerve section. FP recordings from dorsal horns of normal spinal cord slices revealed long-latency synaptic responses in lamina II and short-latency responses in lamina III. The latencies of synaptic FPs recorded in lamina II of the dorsal horn after sciatic nerve section were reduced. The majority of monosynaptic EPSPs recorded with intracellular microelectrodes from lamina II neurons in control slices were elicited by high-threshold nerve stimulation, whereas the majority of monosynaptic EPSPs recorded in lamina III were elicited by low-threshold nerve stimulation. After sciatic nerve section, 31 of 57 (54%) EPSPs recorded in lamina II were elicited by low-threshold stimulation. The majority of low-threshold EPSPs in lamina II neurons after axotomy displayed properties similar to low-threshold EPSPs in lamina III of control slices. These results indicate that reoccupation of lamina II synapses by sprouting Abeta fibers normally terminating in lamina III occurs after sciatic nerve neuroma formation. Furthermore, these observations indicate that the lamina II neurons receive inappropriate sensory information from low-threshold mechanoreceptor after sciatic nerve neuroma formation.     

Cells in laminae III and IV of rat spinal dorsal horn receive monosynaptic primary afferent input in lamina II

In order to determine how information conveyed by fine primary afferent fibres might reach the deeper laminae of the spinal dorsal horn, 5 Golgi-stained neurones with somata in lamina III or IV and dendrites that entered lamina II were examined by electron microscopy. Three of the cells were from animals in which 2 or 3 dorsal roots had been cut 26 or 30 hours previously. These cells received numerous synapses in lamina II, and between 13 and 16% of these (24-31% of asymmetric synapses) were from degenerating axons. Synapses with degenerating axons were found throughout the depth of lamina II, including the dorsal part, which receives primary afferent input from myelinated nociceptors and from unmyelinated axons. In addition, all 3 cells were postsynaptic to degenerating axons within lamina III. The 2 cells from unoperated animals also received many synapses within lamina II and at some of these the presynaptic axon was the central terminal of a glomerulus. Only one example of a dendrodendritic synapse involving a stained dendrite was seen. Cells of laminae III and IV may therefore not be a major target for presynaptic dendrites of cells in lamina II. It is concluded that one way in which information carried by primary afferents passes from the superficial dorsal horn to the deeper laminae is through monosynaptic contacts between these afferents and the dorsal dendrites of some cells whose somata are situated in laminae III and IV. If the axons of these cells generate local collaterals, this may account for some of the activation of cells whose dendrites do not enter lamina II.     

Morphology and ultrastructure of physiologically identified substantia gelatinosa (lamina II) neurons with axons that terminate in deeper dorsal horn laminae (III–V)

In order to determine their local circuit function, we have examined physiologically characterized, intracellularly labeled neurons in laminae I and II with light and electron microscopes. Single neurons in the spinal substantia gelatinosa (lamina II) of the cat and monkey were recorded intracellularly and characterized physiologically. Following characterization, the neurons were labeled with horseradish peroxidase that was iontophoretically ejected from the recording micropipette. After fixation and sectioning, histochemical reaction allowed visualization of the neuron soma, dendrites, and axon. The four nociceptive neurons reported here (three from cats and one from a monkey) had axons that distributed terminal collaterals to deeper laminae of the spinal cord, including laminae III, IV, and V. Electron microscopy of the axons demonstrated that the parent axons were myelinated and that the terminal collaterals established synaptic contact with neurons in the deeper laminae. These results suggest that some substantia gelatinosa neurons relay nociceptive information to neurons in deeper regions of the spinal dorsal horn via myelinated axons.     

The fine structure of neurons in the dorsal horn of the cat spinal cord

The fine structure of the dorsal horn of the cat cord has been compared with the light microscope cytoarchitectural studies. Several distinct laminae may be seen with the electron microscope. Lamina I contains marginal cells of Waldeyer and has its processes oriented in a horizontal direction. Lamina II contains small neurons and processes oriented vertically, and may be distinguished from lamina III by the rich content of myelinated axons in the latter. In laminae I–III there are numerous non-myelinated axons and many axodendritic and axoaxonal synapses but few axosomatic ones. Complex synaptic arrays made up of a central axon synapsing with other axons and dendrites are commonly seen. Laminae IV, V and VI are distinguished by their content of large neurons. They exhibit many axodendritic and axosomatic synapses but few axoaxonal ones and no complex synaptic arrays. Synaptic knobs containing rings of neurofilaments are present only in laminae IV–VI and synapse usually with somata or dendrites of neurons. There are differences in synaptic vesicle configuration and in the density of subsynaptic membranes associated with them. Differentiation of the dorsal horn into horizontal laminae is demonstrated by both light and electron microscope studies. The regional differences in neuronal organization may represent differences in functional organization as well.     

Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. III. Serotonin immunoreactivity

Enkephalinergic axons and terminals were identified by the PAP immunohistochemical method in lamina I (marginal zone) and lamina II₀ (outer substantia gelatinosa) in the dorsal horn of the monkey spinal cord. Synaptic profiles with enkephalin-like immunoreactivity (MELI) contained clear, round, vesicles, sometimes a few large granular vesicles, and usually formed asymmetrical contacts.MELI terminals forming synaptic contacts with various sizes of dendrites and with dendritic spines were the most common type of relationship found; axosomatic contacts were few. Additionally, two types of complexes were observed in which an MELI terminal formed a specialized apposition with an unlabelled terminal. The contact of often resembled a synapse and in most cases the MELI terminal was suspected to be presynaptic. One complex consisted of a MELI terminal apposing the LGV type terminal (containing large granular vesicles), which in turn was presynaptic to a dendrite. (The identify of the LGV terminal could not be determined, but it had some characteristics similar to those described for substance P terminals and for a class of primary afferents in the monkey dorsal horn). The other type of complex consisted of a MELI terminal apposing an R-type terminal (containing small, round, clear vesicles) which was in turn presynaptic to a dendrite. Often, the MELI terminal also formed a synapse onto the same dendrite.The axodendritic, axospinous and axosomatic contacts of MELI terminals in the superficial dorsal horn may produce some of the depressive postsynaptic-like effects of enkephalin iontophoresis onto dorsal horn neurons. In these cases the responses of dorsal horn neurons to both low threshold and nociceptive primary afferents is suppressed. However, the opiate receptor-dependent PAD of C-fibers observed in the dorsal horn may be mediated by the MELI complexes formed with LGV and R terminals found in lamina I.     

Organization of glutamate-like immunoreactivity in the rat superficial dorsal horn: light and electron microscopic observations

Glutamate has been shown to be a neurotransmitter in the central nervous system of vertebrates, and it has been hypothesized that glutamate is functional as a neurotransmitter in the spinal cord dorsal horn. A monoclonal antibody to fixative-modified glutamate was used in this study to examine the light microscopic and ultrastructural profiles of glutamate-like immunoreactivity in the superficial dorsal horn of the rat spinal cord. Glutamate-like immunoreactivity was observed in neurons, fibers, and terminals of both laminae I and II. Marginal zone immunoreactive neurons ranged from 10 to 30 micron in diameter and received many nonimmunoreactive somatic synapses. In substantia gelatinosa, immunoreactive neurons were observed in both inner and outer layers, ranged 5 to 10 micron in diameter, and received few nonimmunoreactive somatic synapses. Glutamate-like immunoreactive dendrites were observed in both laminae and were contacted primarily by nonimmunoreactive synaptic terminals that generally contained small clear vesicles. Both myelinated and unmyelinated immunoreactive axons were observed in Lissauer's tract. Immunoreactive terminals contained small (40 nm) clear vesicles and generally formed simple synaptic contacts with nonimmunoreactive dendrites in laminae I and II. The results of this study corroborate the importance of glutamate as a neurotransmitter in spinal sensory mechanisms.     

The location of GABAB receptor binding sites in mammalian spinal cord

GABAB binding sites in rat spinal cord have been detected by receptor autoradiography using 3H-GABA in the presence of isoguvacine. The sites could be demonstrated throughout the spinal cord grey matter. The maximum concentration of GABAB sites occurred in lamina II with substantial amounts in other laminae of the dorsal horn. Much lower levels were detected in the ventral horn. Unilateral rhizotomy reduced the number of GABAB sites in the dorsal horn without affecting levels in the ventral horn. The greatest reduction occurred in lamina II with 18% loss 2 days after surgery, 23% after 4 days, 25% after 6 days, and 48% after 15 days. The change after 15 days was comparable to that produced 4 months after neonatal capsaicin administration (50 mg/kg). The only apparent difference between rhizotomy and capsaicin treatment occurred in lamina IV, where rhizotomy produced a greater reduction than capsaicin. 3H-Neurotensin binding in sections from the same animals was unaltered after rhizotomy, indicating a lack of change in the populations of neurons containing neurotensin-binding sites. This would support the view that up to 50% of GABAB binding sites are located on nerve terminals. The greater reduction in lamina IV after rhizotomy would suggest that GABAB sites may be present on large-diameter afferent fibres that terminate in this region as well as on smaller-diameter C and A delta fibres.