Neurons in laminae III and IV of the rat spinal cord with the neurokinin-1 receptor receive few contacts from unmyelinated primary afferents which do not contain substance P

We have previously demonstrated that neurons in laminae III and IV of the spinal dorsal horn which possess the neurokinin-1 receptor and have long dorsal dendrites receive a major synaptic input from substance P-containing primary afferents and a more limited input from myelinated afferents. In the present study we have carried out a quantitative analysis of the contacts which cells of this type receive from two other classes of unmyelinated primary afferent: those which contain somatostatin and those without neuropeptides. We found that although boutons belonging to both of these types of afferent do form contacts with neurons of this type, the contacts are far less numerous than those formed by substance P-containing afferents. In laminae I and II, the density of contacts which dendrites of these cells received from somatostatin-containing afferents was 1.2/100 microm and that from non-peptidergic C afferents was 2.0/100 microm, which is far lower than our previous estimate of 18.8/100 microm from substance P-containing fibres in these laminae. These results indicate that although the dendrites of large neurons in laminae III and IV which possess the neurokinin-1 receptor pass through regions of the dorsal horn in which many types of primary afferent terminate, their synaptic input from primary afferents is organized in a highly selective manner.     

The morphology of hair follicle afferent fibre collaterals in the spinal cord of the cat

1. The enzyme horseradish peroxidase (HRP) was injected into single axons that innervated hair follicle receptors to study the morphology of their collaterals in the dorsal horn of the cord. The axons were impaled near the dorsal root entrance zone in the lumbosacral spinal cord of anaesthetized cats and HRP injected by passing current through the intra-axonal micro-electrode. The morphology was revealed by subsequent histochemistry.2. Thirteen hair-follicle afferent fibres were stained including six that innervated tylotrichs (type T hair follicle afferent units) and one that innervated guard hairs (type G unit). The remaining six axons were not classified according to hair type, but, on the basis of their axonal conduction velocities, would have been either type G or T.3. Eleven axons could be traced back into the dorsal roots. Eight of these, upon entering the cord, turned and ran towards the brain. They did not divide into rostral and caudal branches. Three of the eleven did divide and gave rise to both rostral and caudal branches.4. Sixty-three collaterals were given off the thirteen stained axons. All well-filled collaterals had a strikingly similar morphology. They descended through laminae I-III of the dorsal horn into the deeper parts of lamina IV or into lamina V, before turning and ascending back into superficial lamina IV and lamina III where they branched profusely to give rise to their terminal arborizations. Terminal boutons, most commonly of the ;en passant' type, were numerous in lamina III, but were also seen in the dorsal part of lamina IV and in ventral lamina II. None were observed in dorsal lamina II or near the junction of the grey and white matter (lamina I) or in lamina V.5. The terminal arborizations of collaterals from a single hair follicle afferent fibre were in line with one another in the longitudinal axis of the cord. In the better-stained preparations the terminal arborizations of adjacent collaterals from a single axon formed a continuous longitudinal column through the dorsal horn. There was a gradual shift of the column of arborizations from lateral to medial as the more rostral collaterals were given off.6. The hair-follicle afferent fibre collaterals are now identified as the ;collaterales grosses et profondes de la substance de Rolando' of Ramón y Cajal (1909) which give rise to the ;flame-shaped arbors' of Scheibel & Scheibel (1968).7. The importance of the longitudinal organization of the terminal arborizations for an understanding of the topographical properties of dorsal horn neurones is discussed.     

Altered distribution of dorsal root fibers in the rat following neonatal capsaicin treatment

Distribution of primary afferent fibers was studied in intact and neonatally capsaicin treated rats by the application of horseradish peroxidase to the central branch of the transected lumbar dorsal roots. Coarse primary afferent fibers entered the spinal cord through the larger medial portion of the rootlet and arborized in the deeper part of the dorsal horn (laminae III and IV). Fine fibers reached the spinal cord through the smaller lateral portion of the rootlet and arborized in the superficial portion of the dorsal horn (lamina I and outer portion of lamina II). The technique used was inadequate to stain fine, unmyelinated primary afferent fibers terminating in the larger inner portion of lamina II. After neonatal capsaicin treatment (50 mg/kg) the flame-shaped arborizations of thick primary afferent fibers terminating in intact rat in laminae III and IV spread dorsally and occupied the inner portion of lamina II in the larger lateral sector of the dorsal horn. Medially the dense arborization of a different type of thick primary afferent fibers sprouted up to the white-gray border. The border between the lateral and medial sector was sharp and only slightly varied in localization from experiment to experiment. The sprouting fibers established complicated synaptic contacts with dendrites and axon terminals. The rearrangement of primary afferent fibers after neonatal capsaicin treatment confirmed earlier results and revealed a mediolateral difference in the fiber organization of the dorsal horn indicating differences in the projection from hairy vs non-hairy skin areas.     

Morphology and synaptic relationships of physiologically identified low-threshold dorsal root axons stained with intra-axonal horseradish peroxidase in the cat and monkey

The arborizations and synaptic relationships of intra-axonally stained horseradish peroxidase- (HRP) labeled primary afferent fibers to the dorsal horn of the cat and monkey spinal cord have been studied by light and electron microscopic methods. The light microscopic arborizations of the afferent fiber types (hair follicle afferents, pacinian corpuscle afferents, type I and type II slowly adapting afferents) are similar to those described by Brown and his colleagues (1) in the cat. The synaptic profiles formed by labeled afferents contain rounded synaptic vesicles. In serial thin sections, it was found that single dorsal root axons may make hundreds or thousands of synapses with neuronal structures of the dorsal horn. The vast majority of synaptic contacts are on the dendritic trees of dorsal horn neurons. The synapses made by these low-threshold afferent axons are almost all in the deeper laminae (III-VI) of the dorsal horn. The hair follicle afferent axons and the pacinian corpuscle afferents have numerous vesicle-containing structures that synapse on them to form either axoaxonal synapses or dendroaxonal synapses. The slowly adapting afferent axons are less often found to be postsynaptic to axons or dendrites. It is concluded that different physiological classes of primary afferent axons have different morphological characteristics, both at the light and electron microscopic level.     

An electron microscope study of glycine-like immunoreactivity in laminae I-III of the spinal dorsal horn of the rat

The ultrastructural distribution of glycine-like immunoreactivity in laminae I-III of rat spinal dorsal horn was examined by using pre-embedding immunocytochemistry. Immunoreactive axons, dendrites and cell bodies were observed in all three laminae, but were most common in lamina III. The axons were presynaptic at axodendritic and axosomatic synapses, but also at axo-axonic synapses in laminae II and III, where the postsynaptic boutons frequently resembled the terminals of myelinated primary afferents. Some vesicle-containing dendrites in lamina II also showed glycine-like immunoreactivity. Immunoreactive dendrites in laminae II and III were postsynaptic to the central axons of type II, but not type I glomeruli, which suggests that glycinergic neurons receive a major monosynaptic input from myelinated primary afferents. These results support the suggestion that GABA and glycine co-exist in some neurons in laminae I-III of rat dorsal horn, and confirm that glycine is involved in somatosensory processing involving low threshold myelinated cutaneous primary afferents.     

The central projections of primary afferent neurons of greater splanchnic and intercostal nerves in the rat

Summary The central projections of primary afferent fibers of the greater splanchnic nerve of the rat were investigated using the transganglionic horseradish peroxidase transport technique. In addition, the corresponding spinal ganglion cells and the preganglionic sympathetic neurons were demonstrated. For comparing visceral and somatic afferents, intercostal nerve afferents were labelled by the same technique.Splanchnic afferent dorsal root ganglion cells were found at segments T3 to T13 ipsilaterally, with the greatest density at T8 to T12. Labelled cells represented about 10%–15% of all neurons in the ganglia at maximal projection levels. They were randomly distributed within individual ganglia. The great majority were medium to small sized and round to slightly oval in shape.In the spinal cord, labelled visceral afferent axons were found maximally at T8 to T11, but could be detected in decreasing density up to T1 and down to L1. They were distributed over Lissauer's tract and the dorsal funiculus to a medial and lateral collateral pathway (MCP and LCP, respectively). The MCP, somewhat more prominent than the LCP, was destined primarily to clustered presumptive terminal fields in medial lamina I and outermost lamina IIa. Only a few axons continued further to laminae V and X. Splanchnic afferent axons, most likely derived from the MCP, formed a longitudinal bundle ventral to the central canal. The LCP consisted of more or less well-defined axon bundles emanating from the lateral Lissauer's tract and curving round the lateral edge of the dorsal horn and through the dorsolateral funiculus. Presumptive terminal sites of LCP axons are the lateral laminae I and IIa, the nucleus of the dorsolateral funiculus and the dorsal part of lamina V. A few LCP axons were seen in the vicinity of lateral dendrites of preganglionic sympathetic axons. Visceroafferent terminals were absent from laminae IIb–IV and VII. The possible consequences of the MCP/LCP duality for the central connections of splanchnic afferents are discussed. Some splanchnic afferents ascended to the gracile and cuneate nuclei, and rarely to the spinal trigeminal nucleus.These results fit into the general concept of visceroafferent terminal organization that has emerged during the last few years. Differences to other reports in the detailed arrangement of fibers and terminals are discussed.Somatoafferent cell bodies represented the vast majority of neurons in the respective spinal ganglia. Cell sizes encompassed the whole range from very small to very large without a clear predominance of one particular size class. Cell shapes of somatic neurons were more variable than those of visceral afferent neurons. Somatic afferent fibers and presumptive terminals in the spinal cord are distributed ipsilaterally to dorsal horn laminae I–V, most heavily II–IV, to the nucleus dorsalis Clarke, to the ventral horn, and also sparsely to the dorsal horn contralaterally.Labelled preganglionic sympathetic neurons were found in segments T3–T13. The vast majority was located in the intermediolateral nucleus. Fewer neurons occurred in the intercalated nucleus, and occasionally a neuron was labelled in the dorsal grey commissure.Parts of this study have been presented in abstract form at the 8th ENA meeting in Den Haag, September 1984Dedicated to Prof. Dr. W. Zenker on occasion of his 60th birthday     

The large synaptic complexes of the substantia gelatinosa

Summary A Golgi and electron microscope study, using also secondary degeneration after dorsal root transection and chronically isolated dorsal horn preparations, were undertaken with the objective to clarify the large (glomerulus-like) synaptic complexes in lamina II of the dorsal horn. The large sinusoid axon terminals forming the centers of these synaptic complexes are of intraspinal origin and are thought to arise from the hitherto unknown type of pyramid-shape nerve cells, situated at the border between laminae III and IV. The sinusoid axon terminals establish axo-dendritic synapses with substantia gelatinosa neurons and abundant axo-axonic synapses with smaller terminals that could be identified (at least partly) as endings of primary sensory afferents. The central sinuous axon terminals of the synaptic complexes are always presynaptic to the smaller axons and thus might be considered as a device for 1. presynaptic inhibition of impulse transmission from primary afferents to substantia gelatinosa neurons, and 2. as the anatomical basis for primary afferent depolarization.This study has been carried out and the paper has been prepared largely before the recent publications of the paper by Scheibel and Scheibel (1968), which accounts for some overlap between the Golgi informations presented.     

Structure-function relationships in rat medullary and cervical dorsal horns. I. Trigeminal primary afferents

Intracellular recording and horseradish peroxidase (HRP) labeling were used to examine structure-function relationships in the medullary dorsal horn (MDH) and rostral cervical dorsal horn. In Nembutal-anesthetized rats, 78 trigeminal (V) primary afferent fibers were physiologically characterized and injected with HRP. Axons were sufficiently well stained to reconstruct all of their collaterals in the MDH. Many also extended into the cervical dorsal horn. Except for four axons, which responded best to noxious stimuli, all responded at short (mean = 0.50 ms) latencies to V ganglion shocks and to innocuous stimulation. Forty-five of our recovered fibers were associated with facial vibrissae and responded in either a rapidly adapting, slowly adapting type I, slowly adapting type IIa, or slowly adapting type IIb fashion. The adequate stimuli consisted of either slow deflection, high-velocity deflection, or a noxious pinch of the vibrissa follicle. The collaterals of all of the above-described mystacial vibrissa primary afferents proceeded directly to their region of arborization in a plane perpendicular to the lateral border of the medulla to collectively form a largely continuous, circumscribed terminal column. This longitudinally oriented column of terminal and en passant boutons angled from lamina V rostrally to lamina III caudally. In the magnocellular laminae of the MDH, all mystacial vibrissa primary afferents gave rise to similarly shaped arbors, regardless of their functional classification. While morphological variability was observed both within and between individual axons, variance between functional classes was no greater than that within a class. Moreover, number of collaterals, number of boutons, or bouton size did not distinguish functional classes. Nonmystacial vibrissa afferent arbors, with more caudal peripheral fields, had their primary arbor focus in C1 and C2 dorsal horn. These arbors had relatively little rostrocaudal overlap with mystacial vibrissa afferents, though they exhibited the same lamina V-to-III shift as they descended through the cervical cord. Unlike mystacial vibrissa afferents in the MDH, their collaterals followed a tortuous course and often occupied laminae II-V in one transverse section. The relative location of each vibrissa afferent's terminal field could be predicted by the particular vibrissa innervated. Dorsal vibrissae afferents had ventrolateral terminations and ventral vibrissae afferents terminated dorsomedially. Rostral vibrissae were represented in the rostral MDH, whereas caudal vibrissae were represented in the caudal MDH and rostral cervical dorsal horn.(ABSTRACT TRUNCATED AT 400 WORDS)     

The neuropeptide tyrosine Y1R is expressed in interneurons and projection neurons in the dorsal horn and area X of the rat spinal cord

The localization of the neuropeptide tyrosine Y1 receptor was studied with immunohistochemistry in parasagittal and transverse, free-floating sections of the rat lumbar spinal cord. At least seven distinct Y1 receptor-positive populations could tentatively be recognized: Type 1) abundant small, fusiform Y1 receptor-positive neurons in laminae I-II, producing a profuse neuropil; Type 2) Y1 receptor-positive projection neurons in lamina I; Type 3) small Y1 receptor-positive neurons in lamina III, similar to Type 1 neurons, but less densely packed; Type 4) a number of large, multipolar Y1 receptor-positive neurons in the border area between laminae III-IV, with dendrites projecting toward laminae I-II; Type 5) a considerable number of large, multipolar Y1 receptor-positive neurons in laminae V-VI; Type 6) many large Y1 receptor-positive neurons around the central canal (area X); and Type 7) a small number of large Y1 receptor-positive neurons in the medial aspect of the ventral horns (lamina VIII). Many of the neurons present in laminae V-VI and area X produce craniocaudal processes extending for several hundred micrometers. Retrograde tracing using cholera toxin B subunit injected at the 9th thoracic spinal cord level shows that several Type 5 neurons in laminae V-VI, and at least a few Type 2 in lamina I and Type 6 in area X have projections extending to the lower segments of the thoracic spinal cord (and perhaps to supraspinal levels). The present results define distinct subpopulations of neuropeptide tyrosine-sensitive neurons, localized in superficial and deep layers of the dorsal, in the ventral horns and in area X. The lamina II neurons express somatostatin [The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons. Eur J Neurosci 11:2211-2225] and are presumably glutamatergic [Todd AJ, Hughes DI, Polgar E, Nagy GG, Mackie M, Ottersen OP, Maxwell DJ (2003) The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn. Eur J Neurosci 17:13-27], that is they are excitatory interneurons under a Y1 receptor-mediated inhibitory influence. The remaining Y1 receptor-positive spinal neurons need to be phenotyped, for example if the large Y1 receptor-positive laminae III-IV neurons (Type 5) are identical to the neurokinin (NK)1R-positive neurons previously shown to receive neuropeptide tyrosine positive dendritic contacts [Polgár E, Shehab SA, Watt C, Todd AJ (1999) GABAergic neurons that contain neuropeptide Y selectively target cells with the NK1 receptor in laminae III and IV of the rat spinal cord. J Neurosci 19:2637-2646]. If so, neuropeptide tyrosine could have an antinociceptive action not only via Y1 receptor-positive interneurons (Type 1) but also projection neurons. The present results show neuropeptide tyrosine-sensitive neuron populations virtually in all parts of the lumbar spinal cord, suggesting a role for neuropeptide tyrosine signaling in many spinal functions, including pain.     

Calcitonin gene-related peptide containing primary afferent fibers synapse on primate spinothalamic tract cells

Spinothalamic tract (STT) cells were identified by intracellular injection or retrograde labeling with horseradish peroxidase (HRP) in the primate spinal cord. Using immunohistochemical techniques, a population of primary afferents containing calcitonin gene-related peptide (CGRP) is demonstrated in synaptic contact with these neurons. Large glomerular type CGRP terminals with morphology considered typical of primary afferent fibers are occasionally observed in contact with STT profiles in laminae I and IIo; however, this morphological type targets STT profiles chiefly in the deeper dorsal horn (laminae III, IV and V). In contrast, the majority of CGRP terminals contacting STT profiles in the superficial dorsal horn (laminae I and IIo), are small, round or oblong shaped terminals. Thus, evidenced by these data, the absence of large, glomerular type terminals does not rule out primary afferent input.     

Structure-function relationships in identified afferent neurones

Summary The review deals with structure-function relationships in primary afferent and spinal cord neurones that were intracellularly injected with a marker substance (mostly HRP) after physiological identification. At the level of dorsal root ganglion (DRG) cells, there is a significant correlation between soma size and conduction velocity (or diameter) of the afferent fibre for most subpopulations of DRG cells, but the scatter of data is considerable, so that the size of a DRG cell soma cannot be predicted from the diameter of its axon or vice versa. The spinal terminations of primary afferent fibres are the best example of a relationship between structure and function, since most of the afferent units possess characteristic patterns of spinal arborization, e.g. the flame-shaped arbors of hair follicle afferents in lamina III of the dorsal horn, or the projection of nociceptive afferents onto lamina I. The morphological features of spinal cord neurones can be used only to a limited extent for functional identification. Thus, many SCT neurones can be recognized by their triangular dendritic tree and STT cells in lamina VII/VIII by their dendritic projection into the white matter. It is still not possible, however, to distinguish a nociceptive STT cell from a low-threshold mechanoreceptive one on the basis of morphological criteria.     

大鼠延髓背角Ⅱ层神经元向臂旁区、延髓尾侧腹外侧区和脊髓的投射

将逆行追踪剂荧光金分别注入大鼠一侧臂旁区、延髓尾侧腹外侧区和颈髓第四节段 ,观察到延髓背角浅层 ( 、 层 )向上述部位均有投射 , 层外侧部的投射神经元多于 层内侧部。臂旁区接受双侧延髓背角浅层的投射 ,但以同侧为主 ;延髓尾侧腹外侧区和第 4颈髓接受同侧延髓背角浅层神经元的投射但延髓背角浅层向第 4颈髓投射的神经元数量较少。结合免疫荧光组织化学研究表明 , 层向臂旁区和延髓尾侧腹外侧区投射的神经元部分呈 calbindin-D2 8k样阳性 ,而未见呈 parvalbumin样阳性者。向第 4颈髓投射的 层神经元数量很少 ,未见有呈 calbindin-D2 8K和 parvalbumin样阳性者。本研究结果进一步支持 层有传出投射的观点 ,也提示 层的 calbindin-D2 8K样和 parvalbumin样阳性神经元可能分别属于不同的细胞亚群。     

大鼠脊髓神经肽Y、神经降压肽免疫反应神经元向结合臂旁核的投射

用HRP逆行束路追踪法结合免疫细胞化学双标记技术,研究了大鼠颈、胸、腰、骶各段脊髓神经肽Y(NPY)、神经降压肽(NT)免疫反应神经元向结合臂旁核的投射。HRP逆行标记细胞分布于两侧脊髓灰质第Ⅰ、Ⅱ、Ⅳ、Ⅴ、Ⅶ层、中央管背侧灰质连合及外侧颈核、外侧脊髓核中,以对侧为主。NPY免疫反应阳性神经元分布于两侧后角第Ⅰ层、第Ⅱ层浅部、外侧颈核、外侧脊髓核及骶髓后连合核中。NT免疫阳性神经元分布于两侧后角第Ⅰ层及第Ⅱ层中。在后角第Ⅰ层、外侧颈核和外侧脊髓核中,可观察到部分NPY-HRP双标记神经元;在后角第Ⅰ层可观察到部分NT-HRP双标记神经元。本研究结果提示,NPY和NT神经元可能参与脊髓-结合臂旁核的痛觉传递。     

Inflammation reduces the contribution of N-type calcium channels to primary afferent synaptic transmission onto NK1 receptor-positive lamina I neurons in the rat dorsal horn

N-type calcium channels contribute to the release of glutamate from primary afferent terminals synapsing onto nocisponsive neurons in the dorsal horn of the spinal cord, but little is known of functional adaptations to these channels in persistent pain states. Subtype-selective conotoxins and other drugs were used to determine the role of different calcium channel types in a rat model of inflammatory pain. Electrically evoked primary afferent synapses onto lumber dorsal horn neurons were examined three days after induction of inflammation with intraplantar complete Freund's adjuvant. The maximal inhibitory effect of the N-type calcium channel blockers, ω-conotoxins CVID and MVIIA, were attenuated in NK1 receptor-positive lamina I neurons after inflammation, but the potency of CVID was unchanged. This was associated with reduced inhibition of the frequency of asynchronous-evoked synaptic events by CVID studied in the presence of extracellular strontium, suggesting reduced N-type channel contribution to primary afferent synapses after inflammation. After application of CVID, the relative contributions of P/Q and L channels to primary afferent transmission and the residual current were unchanged by inflammation, suggesting the adaptation was specific to N-type channels. Blocking T-type channels did not affect synaptic amplitude under control or inflamed conditions. Reduction of N-type channel contribution to primary afferent transmission was selective for NK1 receptor-positive neurons identified by post hoc immunohistochemistry and did not occur at synapses in laminae II_o or II_i, or inhibitory synapses. These results suggest that inflammation selectively downregulates N-type channels in the terminals of primary afferents synapsing onto (presumed) nociceptive lamina I NK1 receptor-positive neurons.     

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.     

Distribution of and organisation of dorsal horn neuronal cell bodies that possess the muscarinic m2 acetylcholine receptor

Cholinergic systems in the dorsal horn are involved in antinociception but little is known about the organisation of receptors that mediate this process. In this study we examined immunocytochemical properties of dorsal horn neuronal cell bodies that express the m2 muscarinic acetylcholine receptor. Tissue was examined with confocal laser scanning microscopy and quantitative analysis performed. Immunoreactive cells were found throughout the dorsal horn and in lamina X. Quantitative analysis revealed that 22% of neuronal somata in the dorsal horn possess the receptor. The greatest concentration of cells was found in deeper laminae (IV-VI) and around lamina X. A proportion of cholinergic cells (labelled with an antibody against choline acetyltransferase) were immunoreactive for the receptor (approximately, 40% of dorsal horn cells and 44% of lamina X cells). Populations of presumed inhibitory interneurons also displayed immunoreactivity for the receptor. Between 27-34% of cells immunoreactive for GABA, nitric oxide synthase and the somatostatin receptor(2A) expressed the receptor but only 8% of parvalbumin-immunoreactive cells displayed receptor immunoreactivity. Cells labelled with neurotensin, which belong to a subgroup of excitatory neurons, displayed no receptor immunoreactivity. A small number neurokinin-1 receptor-immunoreactive cells in lamina I possessed m2 immunoreactivity but 42% of laminae III/IV neurokinin-1 cells possessed it.This study shows that a significant proportion of cell bodies in the dorsal horn express the muscarinic m2 acetylcholine receptor. The receptor is present on some cholinergic neurons and therefore may function as an autoreceptor. It is associated with inhibitory local circuit neurons and may have a role in the modulation of specific inhibitory systems. It is also found on a proportion of projection cells that possess the neurokinin-1 receptor. This could be the basis of some of the antinociceptive actions of acetylcholine.     

Relationship between capsaicin-evoked substance P release and neurokinin 1 receptor internalization in the rat spinal cord

The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3-7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 microM producing significantly less release. The effective concentration for 50% of response (EC(50)) for capsaicin, calculated from its stimulatory phase, was 2.3 microM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC(50)=0.37 microM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC(50)=1.9 microM) and lamina IV (EC(50)=14.5 microM), suggesting that neurokinins released in laminae I-II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I (R(2)=0.82) and III (R(2)=0.78), but it was poor (R(2)=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.     

Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn

To clarify the circuitry through which opioid compounds modulate spinal and trigeminal nociceptive transmission, we have examined the synaptic associations formed by leucine-enkephalin-containing (enkephalin) neurons in the superficial dorsal horn of the cat. As described previously, punctate enkephalin immunoreactivity is concentrated in the marginal layer (lamina I) and in both the outer and inner layers of the substantia gelatinosa (lamina IIo and IIi). In colchicine treated cats, enkephalin perikarya are most numerous in lamina I and at the border between laminae I and II. Ultrastructural analysis reveals that enkephalin cells receive a diverse afferent input. The majority of afferent inputs are presynaptic to the enkephalin dendrites; few axosomatic synapses are seen. Among these presynaptic axonal profiles are unlabeled axons which resemble primary afferent terminals, including the characteristic central axonal varicosity. Enkephalin dendrites are also postsynaptic to enkephalin immunoreactive axons. Two types of enkephalin axonal profiles appear in the superficial dorsal horn. Class I profiles are only found in lamina I. These are large profiles which form few synapses; those synapses made are axodendritic. Class II enkephalin axons are smaller and are distributed in both layers I and II. While Class II axons most commonly form axo-dendritic synapses, they also form axo-axonic synapses with flat vesicle-containing profiles; the latter are generally presynaptic to the enkephalin terminals. Serial analysis further revealed that both the enkephalin and the flat vesicle-containing profile synapse onto a common dendrite. Although enkephalin axons frequently lie adjacent to round vesicle-containing profiles, anatomical evidence that opioid axons form synapses with this type of ending was not found. An additional type of enkephalin vesicle containing-profile is found in layer IIi; its morphological features do not clearly distinguish its axonal or dendritic origin. These endings are typically postsynaptic to unlabelled central endings, and provide minimal presynaptic input to other elements in the neuropil. Like some class II axons, these labelled profiles contain vesicles which cluster at the membrane immediately adjacent to unlabelled central axons. These results indicate that spinal enkephalin neurons receive a variety of synaptic inputs. These include inputs which may derive from primary afferent axons. Enkephalin neurons, in turn, influence nociceptive transmission predominantly through postsynaptic mechanisms. Finally, while we did not observe enkephalin terminals presynaptic in an axoaxonic relationship, the possibility that enkephalin neurons modulate the excitability of fine fiber nociceptive and nonnociceptive afferents via "nonsynaptic interactions" is discussed.     

Projection field of primary afferent fibers innervating the ventral portion of the lumbar intervertebral disc in the spinal cord dorsal horn

In the present study, we investigated the central projection of afferent fibers innervating the lumbar intervertebral disc using the fluorescent neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). The tracer Dil was applied to the ventrolateral portion of the L5-L6 intervertebral disc in 11 adult rats. Fluorescent sites were observed microscopically on spinal cord transverse sections. Fluorescent spots in laminae I-III were plotted on the central projection map of cutaneous afferents. In six of 11 rats, Dil was restricted to the application site. Of these six rats, three showed no evident fluorescent sites. In the remaining three rats, small fluorescent spots were scattered in the dorsal horn. Fluorescent spots in dorsal horn lamina I were located in the central projection fields of the low back and groin skin. Fluorescent spots were observed, also sporadically, in Clarke's column in T12-L1 segments. The central projection of afferent fibers innervating the rat lumbar intervertebral disc was indistinct with Dil labeling. We presumed this was due to the scarcity of central terminal arbors of disc afferent fibers. Spotty projections in laminae I-IllIIere present near the central projection fields of the loin and groin, indicating that pain would be perceived in the groin.     

Physiological studies of cutaneous inputs to dorsal horn laminae I-IV of adult chickens.

1. The dorsal horn (DH) of chickens exhibits a novel pattern of cytoarchitectonic lamination among vertebrates, whereby lamina III lies medial, rather than ventral, to lamina II. Indeed, cutaneous nerves labeled with horseradish peroxidase (HRP) form two separate projections across the mediolateral axis of the superficial DH; each projection is somatotopically organized, such that two non-overlapping somatotopic maps are formed: the medial map within lamina III and the lateral map within lamina II. Interestingly, these two projections of cutaneous nerves are differentially labeled by HRP ligands. The present experiments were designed to address whether the separate subpopulations of cutaneous afferents, as demonstrated neuroanatomically, also differ physiologically on the basis of myelination, fiber diameter, and/or sensory modality. 2. Extracellular multi- and single-unit recordings were obtained in the DH at the ninth synsacral level of spinal adult chickens anesthetized with alpha-chloralose. Activity in lateral (laminae I/II) and medial DH (laminae III/IV) was studied after both electrical stimulation of the caudal femoral cutaneous nerve (CFC) and natural stimulation of the skin. Single units were characterized in terms of the conduction velocity (CV) and sensory modality of their cutaneous afferent inputs. 3. In multiunit recordings, electrical stimulation of the CFC above C-fiber intensities elicited a robust, long-latency response in lateral (laminae I/II) but not medial DH (laminae III/V). The afferents responsible for this late lateral response were C-fibers, as evidenced by a CV of approximately 1 m/s; birds were spinalized to rule out long spinal loops. In contrast, only a single, short-latency (and low-threshold) multiunit response was seen in medial DH, even after activation of C-fibers. 4. Natural stimulation of skin revealed a clear segregation of sensory modalities between medial (laminae III/IV) and lateral DH (laminae I/II). Innocuous mechanical stimuli were extremely effective at eliciting multiunit activity in medial DH, but ineffective in lateral DH. In contrast, noxious mechanical and thermal stimuli were extremely effective at eliciting multiunit activity in lateral DH, but were ineffective in medial DH. 5. In single-unit studies, primary afferent inputs to units in medial DH (laminae III/IV) had an average CV close to 43 m/s; no medial units received exclusive inputs from afferents with CVs less than 5.5 m/s. In contrast, primary afferent inputs to units in lateral DH (laminae I/II) had an average CV close to 10 m/s; 20% of the lateral units received exclusive inputs from C-fibers (CVs less than 1.7 m/s; N = 1.2 m/s).(ABSTRACT TRUNCATED AT 250 WORDS)