Search

The ventral or inner region of spinal substantia gelatinosa (SG; lamina II(i)) is a heterogeneous sublamina important for the generation and maintenance of hyperalgesia and neuropathic pain. To test whether II(i) neurons can be hyperpolarized by the mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of mu-opioid-evoked inhibition of II(i) neurons, we combined in vitro whole-cell, tight-seal recording methods with fluorescent labeling of the intracellular tracer biocytin and confocal microscopy. Twenty-one of 23 neurons studied had identifiable axons. Nine possessed axons that projected ventrally into laminae III-V; six of these were hyperpolarized by DAMGO. Three of four neurons with identifiable axons that projected to lamina I were hyperpolarized by DAMGO. Most neurons could be classified as either islet cells or stalked cells. Five of nine labeled islet cells and only two of seven stalked cells were hyperpolarized by DAMGO. Three were stellate cells: one resembled a spiny cell and three could not be classified. DAMGO hyperpolarized each of the stellate cells, the spiny cell, and 1 of the unclassified cells. Our data support the hypothesis that part of the action of mu-opioid agonists involves the inhibition of interneurons that are part of a polysynaptic excitatory pathway from primary afferents to neurons in the deep and/or superficial dorsal horn.

Electrical and chemical stimulation given in the ventral medullary raphe nuclei inhibits spinal nociceptive reflexes and spinal nociceptive transmission; serotoninergic receptors have been demonstrated to partially mediate that inhibition. In the present study, the termination patterns of raphespinal fibers in the rat lumbar spinal cord demonstrating serotonin-like immunoreactivity were examined by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with immunohistochemistry. Fibers and terminations from the ventral medullary raphe nuclei (raphe magnus and raphe pallidus) demonstrating both PHA-L- and serotonin-like immunoreactivity were identified in all laminae of the dorsal horn and the ventral horn. Networks of large fibers, characterized by large boutons, and which did not demonstrate serotonin-like immunoreactivity, were identified in deeper laminae of the dorsal horn. The heterogeneous morphology of raphespinal fibers identified in the dorsal horn suggests that these fibers also may be heterogeneous in neurochemistry and function. Medial medullary sites outside the raphe nuclei were found to innervate the ventral horn and all laminae of the dorsal horn, with the exception of lamina I. Descending fibers and terminations also demonstrating serotonin-like immunoreactivity were identified in deep laminae (III, IV, V, VI) of the dorsal horn and in the ventral horn. Similarly, large fiber networks were identified which did not demonstrate serotonin-like immunoreactivity.

The receptive fields (RFs) of polymodal nociceptor units of canine testis consist of small fascicles of branching axons ending as clusters within the thin vascular layer overlying the seminiferous tubules. This propitious arrangement enabled serial reconstruction of electron micrographs of a flat, punctate zone, identified during recording of impulse discharge of a single physiologically characterized nociceptor "unit." The RFs showed multiple axons, with some branching and sequential vesicle-containing swellings, similar to what have been called terminals and assumed to constitute impulse generating sites in other tissues. Consistent with this interpretation is the local presence of: (1) clusters of small mitochondria, (2) glycogen granules, and (3) sectors of axolemma denuded of Schwann cell processes and thus "free" endings directly contacting the epithelial basal lamina. The most distal sectors showed additional morphologic properties: (1) zones of vesicles embedded in an extensive granular matrix, (2) preterminal arrays of "axonal reticulum" derived from the smooth endoplasmic reticulum displacing axonal cytoskeletal elements, (3) a variety of sizes and electron lucency in clear, spherical vesicles and a few granular or dense-core vesicles, and (4) specialization in the last Schwann cell of the fascicle. These latter features may reflect differences in the specialized receptor mechanisms of nociceptors that are difficult to detect without extensive serial electron microscopic analysis. Alternatively, these features may constitute a regional specialization of the testis. The term free nerve ending is perhaps an insufficient and inaccurate descriptor of the morphology of nociceptors. These findings are considered in the context of their possible relation to the sensitized vanilloid receptor mechanism unique to nociceptors.

In order to determine the regions within the parabrachial nucleus that receive synaptic input from nociceptive regions of the spinal cord and medulla in the rat, we analyzed the "Golgi-like" labeling produced by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) from discrete iontophoretic injections confined to either the superficial dorsal horn of the lumbar spinal cord or to the superficial dorsal horn of the trigeminal nucleus at the level of the obex. Labeled fibers from both the spinal cord and the medulla ascended through the ventral lateral pons and coursed with the ventral spinocerebellar tract toward the parabrachial nuclei. Spinal cord injections led to labeling of fine caliber fibers and en passant and terminal enlargements in the rostral part of the contralateral lateral parabrachial nucleus (PBL), mostly in the central lateral and dorsal lateral subnuclei. Medullary injections revealed fiber and enlargement labeling primarily in the ipsilateral caudal PBL, mostly in the central lateral, external lateral, and medial subnuclei. Injections in both regions resulted in labeled terminations in the Kolliker-Fuse nucleus. These results indicate that the nociceptive regions of the spinal cord and medulla terminate in regions of the parabrachial nucleus that have been associated with autonomic functions because of their interconnections with the hypothalamus, brainstem cardiovascular and respiratory control centers, and the amygdala.

The spinal cord terminations of two electrophysiologically identified single C-fibers (one identified as a C-nociceptor) were intraaxonally labeled with horseradish peroxidase and analyzed with both light and electron microscopy. Serial section ultrastructural analysis and postembedding immunocytochemical techniques for calcitonin gene-related peptide (CGRP), substance P (SP), and GABA were used to study the synaptology, and neuropeptide content. All C-terminal synapses were in laminae I and II. The terminals sampled (n = 73) from these two C-fibers rarely established glomerular synaptic complexes, but rather, simple terminals, usually measuring 1–4 μm in length and 1–3 μm in diameter. They most often established 1 or 2 (range 1 to 5) quite large asymmetric axodendritic synaptic contacts. Postsynaptic structures included dendritic spines and shafts with and without vesicles. C-terminals were filled with small round synaptic vesicles (45–60 nm) and also contained variable numbers of large dense-core vesicles (LDCVs, 80–110 nm). LDCVs inside identified C-terminals frequently displayed CGRP immunoreactivity. We were unable to detect SP immunoreactivity inside our sample of C-fiber LDCVs. C-terminals were never found postsynaptic to other profiles. Thus, the C-fiber terminals sampled in this study have simple synaptology, do not receive presynaptic control and contain CGRP immunoreactivity. They differ greatly from the terminals of Aδ nociceptors studied previously by our group that had glomerular endings, often received presynaptic input and did not contain CGRP immunoreactivity. This suggests the existence of different processing mechanisms, at the level of the first synapse, for nociceptive inputs arriving to lamina I and II through different types of primary afferents. © 1993 Wiley-Liss, Inc.

Classically the raphespinal system has been regarded as a serotoninergic system; inhibition of spinal nociceptive transmission produced by stimulation of the medullary raphe nuclei is mediated partially by spinal serotoninergic receptors. However, recent evidence suggests that the raphe nuclei are not homogeneous populations of serotoninergic cells. The objective of the present study was to re-examine, in the rat, the serotoninergic raphespinal projection to the lumbar spinal cord, and to determine the relative contribution of serotoninergic raphespinal neurons to the total population of raphespinal neurons. Microinjections of wheat-germ agglutinin horseradish peroxidase conjugate coupled to colloidal gold into the lumbar spinal cord resulted in the retrograde labeling of 53% and 59% of the serotoninergic neurons in the raphe nuclei and in the para-raphe zone, respectively. Conversely, 47% and 28% of the retrogradely labeled neurons in the raphe and para-raphe zone, respectively, demonstrated serotonin-like immunoreactivity. Thus, contrary to previous reports, the present results suggest 1) that only about half of the serotoninergic neurons in the raphe nuclei and in the surrounding para-raphe zone project to the lumbar spinal cord, and 2) that a large proportion of the neurons in the raphe nuclei (53%) and in the surrounding para-raphe zone (72%) that project to the lumbar spinal cord are not serotoninergic.

The anterograde transport of both wheat germ agglutinin conjugated to horseradish peroxidase and the kidney bean lectin Phaseolus vulgaris leucoagglutinin was utilized to investigate the projection of primary sensorimotor corticospinal tract axons to the superficial laminae of the spinal dorsal horn in the rat. Both methods yielded qualitatively similar patterns of connectivity. Corticospinal tract axons were found to terminate within all laminae on the side contralateral to the injection site. Labeling was most dense within laminae I11 and IV and medial portions of laminae I, 11, and V in the cervical and lumbar enlargements. Labeling in the ventral horn, though present, was relatively less dense. I? vulgaris leucoagglutinin-labeled axons within laminae I and I1 exhibited boutons en passant and terminaux; many of these axons also terminated or were collaterals of axons that terminated in deeper dorsal horn laminae. Results are discussed with reference to the somatotopic organization of the spinal cord and to a possible role for the cortex in the modulation of nociception within the spinal cord. For a number of years it has been apparent that the corticospinal tract (CST) arising from the somatosensory cortex in the cat and monkey, and from the sensorimotor cortex in the rat and opossum, projects primarily to laminae 111-VI in the spinal dorsal horn (Chambers and Liu, '57; Kuypers, '60; Nyberg-Hansen and Brodal, '63; Liu and Chambers, '64; Goodman et al., '66; Martin and Fisher, '68; Petras, '68; Harting and Noback, '70; Brown, '71; Berrevoets and Kuypers, '75; Coulter and Jones, '77; Donatelle, '77; Flindt-Egebak, '77). Recent studies with more sensitive histological techniques that demonstrate the presence of anterogradely transported horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) show that the corticospinal projections to the dorsal horn of the spinal cord not only reach laminae 111-VI but also

Single primary afferent myelinated fibers from cutaneous receptors of cat and monkey were functionally identified by recording from the spinal cord with micropipettes filled with horseradish peroxidase (HRP). Relatively slowly conducting fibers (less than 40 m/sec) from high threshold mechanoreceptors (mechanical nociceptors) and two types of low threshold mechanoreceptor (D-hair and field) were selected for staining. Iontophoresis of the HRP and subsequent histochemical reaction stained the axons recorded from and their collaterals, including terminations, for several millimeters. The termination patterns in the two species proved essentially identical. Ipsilaterally, the mechanical nociceptor fibers terminated principally in the dorsal horn's marginal zone and in the ventral parts of the nucleus proprius (lamina V in the cat). Some of these nociceptors also had terminals in the midline just dorsal to the central canal, contralaterally in the marginal zone, and at the base of the opposite nucleus proprius. In contrast, the D-hair primary afferent axons terminated in the dorsal part of the nucleus proprius overlapping into the innermost portion of the substantia gelatinosa. The field receptor fibers terminated predominantly in the middle part of the nucleus proprius. These results suggest that there is a highly specialized central projection of primary afferent endings which is related to sensory function and not to fiber diameter. The marginal zone and most dorsal parts of the substantia gelatinosa receive direct projections from cutaneous nociceptors but do not have direct input from cutaneous receptors transmitting activity initiated by innocuous stimulation.

Single myelinated axons were recorded in the dorsolateral funiculus of the cat and physiologically characterized as descending from the midline medulla or midline pons. Following further physiological characterization (e.g., conduction velocity, adequate stimulus, receptive field, activation by stimulation of periaqueductal gray), the axons were labeled with horseradish peroxidase that was iontophoretically ejected from the recording micropipette. Histochemical reaction allowed visualization of the stained axons and their arborizations in the spinal gray matter. The conduction velocities of the sampled axons ranged from 7.3 to 117.2 m/second with a mean of 35.5 m/second. However, unmyelinated axons could not be sampled with the technique employed here. Descending axons could be divided into two groups: (1) those which terminated in laminae I, II, V, and X, and (2) those which terminated in laminae V, VII, and X. Axons from both groups had myelinated parent axons, were activated by periaqueductal gray stimulation, and responded to noxious pinch of their receptive field. Terminal collaterals from both groups of axons were generally transversely oriented. These results suggest heterogeneous functions for these descending axons which may include modulation of nociceptive input to higher centers.

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.

Primary afferent fibers in the lumbar, sacral, and caudal spinal segments of several mammals (rat, cat, monkey) were stained by applying horseradish peroxidase to the proximal part of cut dorsal rootlets and reacting the tissue histochemically after several hours of survival. The stained fibers' pattern of termination in the dorsal horn was similar in all three species, with many bouton-like enlargements in the ipsilateral marginal zone, substantia gelatinosa, and nucleus proprius, as well as a few projections at each level to the dorsal commissure and contralaterally to the ventral border of the nucleus proprius. Partial lesions of dorsal rootlets in monkey revealed that the thin fibers comprising the lateral division end principally in the marginal zone and substantial gelatinosa, while the thick fibers of the medial division terminate in the nucleus proprius and deeper regions, contributing little to the substantia gelatinosa and marginal zone. On the basis of the termination patterns observed for whole and partly sectioned rootlets, the superficial dorsal horn can be divided into at least four regions. (1) The marginal zone (lamina I of cat) appears to receive terminations from intermediate (smaller myelinated) fibers; (2) the outer substantia gelatinosa (outer lamina II) receives many terminations from the very finest afferent fibers; (3) the inner substantia gelatinosa (inner lamina II) receives endings from some of the finest fibers and also from intermediate (smaller myelinated) fibers; and (4) the superficial part of the nucleus proprius (lamina III) receives endings from intermediate and large diameter dorsal root fibers.

Functional characteristics of spinal neurons located in the marginal zone (lamina I) and substantia gelatinosa (lamina II) were compared to their structural features by intracellularly staining the source of unitary potentials with horseradish peroxidase (HRP) in unanesthetized, spinal cats. The responses of postsynaptic units to graded electrical volleys in intact dorsal roots and to physiological stimulation revealed that the peripheral excitatory input to neurons of the region is dominated by slowly conducting afferent fibers; often, the input to a given element is largely from a particular class of receptors. One type commonly seen received its principal peripheral excitation from low threshold mechanoreceptors with Aδ or C afferent fibers. Mechanoreceptive elements often exhibited a marked, prolonged habituation and many were not excited by afferent volleys. Other units were predominantly excited by nociceptors with myelinated or unmyelinated fibers, or by thermoreceptors with unmyelinated fibers. A few units (principally the thermoreceptive) showed substantial ongoing activity which was modulated by sensory stimulation, but most had little or none. The HRP staining revealed neuronal morphology in fine detail. No relationship between neuronal configuration and physiological response was discerned. Soma location was not always linked to afferent input, although the cell bodies of nonciceptive and thermoreceptive neurons tended to be in lamina I or outer lamina II (SGo) while those of the inocuous mechanoreceptive neurons tended to be in inner lamina II (SGi). The locus of a neuron's major dendritic arborization was more closely related to the source(s) of peripheral excitation. Cells excited by nociceptors with myelinated fibers had major dendritic projections in the marginal zone. Cells excited by nociceptors or thermoreceptors with unmyelinated fibers had important dendritic branching in the SGo. Innocuous mechanoreceptive neurons had primary dendritic aranching in the SGo. Innocuous mechanoreceptive neurons had primary dendritic arborization in the SGi when the input derived from unmyelinated fibers, or in the SGi and extending into the outer nucleus proprius (lamina III) when the afferent drive came from Aδ fibers. These findings support the concept that laminae I and II constitute a major termination region for thin primary afferent fibers, myelinated fibers from nociceptors ending principally in lamina I and unmyelinated fibers from nociceptors, thermoreceptors, and mechanoreceptors terminating predominantly in lamina II. Substantial integrative and distributive functions can be expected of such an afferent termination zone.

Single axons descending in the spinal, dorsolateral funiculus which were directly activated by stimulating in or near nucleus raphe magnus (nRM) in the rostral medulla were iontophoretically injected with horseradish peroxidase (HRP). Labeled axons and terminal arborizations in the spinal cord were demonstrated by diaminobenzidine histochemistry following fixation. Sections were processed for electron microscopy and embedded between coverslips. Following examination with the light microscope, selected boutons were sectioned in ultrathin series and examined with the electron microscope. Two arborization patterns were observed with the light microscope. One terminated dorsally in laminae I, II, V, and X, while the other terminated ventrally in laminae V, VII, and X. At the ultrastructural level, boutons predominantly contacted dendrites and occasionally contacted cell somas. Ventrally projecting axons had boutons characterizable as Gray's type I. Some dorsally projecting axons also had Gray's type I boutons, while other axons had Gray's type II boutons. Thus, these descending axons may be heterogeneous in function. No differences in physiological characteristics were found between axons with Gray's type I boutons versus axons with Gray's type II boutons.

Although numerous studies have discussed serotoninergic pathways projecting from the brainstem to the spinal cord, the intraspinal course of these descending axons remains a matter of debate. One of the difficulties with traditional immunohistochemical methods is that the interpretation of results has been subjective. We have therefore employed a computer-assisted imageprocessing system to provide objective measurements of serotonin immunoreactivity within the rodent dorsal horn and lamina X caudal to unilateral thoracic cordotomy in order to establish the intraspinal course of descending serotoninergic fibers. Lesion analysis combined with computer-determined optical density measurements, high-power microscopic examination, and camera lucida drawings demonstrated that the predominant serotoninergic pathway terminating within the dorsal horn courses unilaterally within the dorsal portion of the dorsolateral funiculus. The thoracic dorsal horn may receive a slightly greater contribution from the contralateral side than the lumbar dorsal horn. Lamina X receives serotoninergic innervation both from fibers descending within the ipsilateral dorsolateral funiculus and from more ventrally located ipsilateral pathways, and it also contains serotonin derived from other sources.

Horseradish peroxidase was applied to proximal ventral roots of the coccygeal and sacral spinal cord of cats. Subsequent histochemical reaction resulted in extensive staining of spinal cord neurons that had processes in the ventral roots. This procedure was used to study four issues concerning ventral root neurons. (1) Extensive transverse dendritic arborizations were revealed for large and small neurons presumed to be alpha and gamma motoneurons respectively. Dendrites from these neurons were found to project heavily into the ipsilateral white matter, both laterally and ventrally. Dendrites also projected extensively through the anterior commissure, attaining the contralateral grey and white matter. (2) Medially-located efferent neurons were found to contribute the contralateral dendrites as well as some dorsally-directed dendrites. Laterally-located neurons projected dendrites extensively into the lateral and ventral white matter. (3) Stained neurons were found in the intermediolateral cell column, and were presumed to be preganglionic efferent neurons. Some of these neurons projected dendrites into the marginal zone of the dorsal horn, while others sent dendrites medially toward the central canal. (4) Stained fibers, presumed to be primary afferents, were found to enter from the ventral roots and course to the dorsal horn. Most of these fibers were small in diameter and distributed boutons predominantly to the substantia gelatinosa. A few large ventral root afferent fibers were observed that distributed boutons mostly to the nucleus proprius.