Substance P receptor-like immunoreactive neurons in the caudal spinal trigeminal nucleus send axons to the gelatinosus thalamic nucleus in the rat.

By means of substance P receptor (SPR) immunofluorescence histochemistry combined with Fluoro-Gold (FG) fluorescent retrograde labeling, SPR-like immunoreactive neurons in the caudal spinal trigeminal nucleus of the rat were observed to send their axons to the gelatinosus thalamic nucleus with a clear ipsilateral dominance. FG/SPR double-labeled neurons were distributed mainly in the ventral part of lamina I at the rostral level of the caudal spinal trigeminal nucleus. The percentages of FG/SPR-LI neurons in the total number of SPR-LI neurons and FG-labeled neurons are 10.5% and 31.1%, respectively. The present results suggest that trigemino-gelatinosus thalamic projection neurons with SPR-LI in the caudal spinal trigeminal nucleus might receive SP-containing, nociceptive primary afferent fibers from the orofacial region and transmit nociception to the gelatinosus thalamic nucleus.

Evidence of synaptic contacts of nociceptive primary afferent central terminals on GABAergic interneurons in the substantia gelatinosa.

Recently, we showed that capsaicin induced the degeneration of not only glomerular CI terminals but also of non-glomerular CI terminals making presynaptic contact with interneuronal soma. Studies of the nature of interneurons making contact by nonglomerular CI terminals should provide important information to facilitate our understanding of the processing of nociceptive impulses in the substantia gelatinosa. The most likely candidate molecule involved in this process in these interneurons is gamma-aminobutylic acid (GABA). Therefore, ultrastructural relationships between nonglomerular CI terminals land GABAergic interneuronal soma in the mouse substatia gelatinosa were examined by an immunocytochemical method using an antibody to GABA. Terminals with the same profiles as the CI terminals, i.e., slender, sinuous and scalloped terminals filled with clear synaptic vesicles, were found to make synaptic contacts with GABA-immunoreactive somata. Thus, nociceptive primary afferents are suggested to modulate pain transmission by themselves via GABAergic neurons in the substantia gelatinosa.

The subnuclear distribution of 5-HT3 receptors in the human nucleus of the solitary tract and other structures of the caudal medulla.

The distribution of 5-HT3 receptors was examined in the human medulla using [3H]LY278584, a highly selective 5-HT3 receptor antagonist. The highest density of 5-HT3 receptors was found in the substantia gelatinosus subnucleus of nucleus of the solitary tract (NTS) throughout its rostrocaudal extent, followed by the dorsal subnucleus, the area postrema (AP), the commissural subnucleus, the medial subnucleus, and in an arc corresponding to the pars gelatinosus of the spinal trigeminal nucleus (nSp5). The distribution of 5-HT3 receptors in the brain may help explain some of the reported CNS activities of 5-HT3-selective drugs. The anti-emetic and antinociceptive activities of 5-HT3 antagonists may be mediated by receptors in sensory areas of the brainstem.

Spinal cord transection produces a long-term increase in GABAB binding in the rat substantia gelatinosa.

Quantitative autoradiography was used to determine changes in GABAB receptor binding in the substantia gelatinosa of the lumbar spinal cord at 4 days and at 6 weeks after a midthoracic spinal transection in rats. In the 4 day lesion animals, there was no significant change in either the density or the affinity of the GABAB binding. At 6 weeks, however, there was a 35% increase in binding density, with no significant change in affinity. The results suggest that alterations in spinal synaptic mechanisms can slowly evolve following loss of descending input to the spinal cord.

Neurons in the substantia gelatinosa rolandi (lamina II) project to the caudal ventrolateral reticular formation of the medulla oblongata in the rat.

Following injections of cholera toxin subunit B in the caudal ventrolateral reticular formation, in an area between the lateral reticular nucleus and the ventrocaudal tip of the spinal trigeminal nucleus, pars caudalis, large numbers of retrogradely labelled cells occurred in lamina II, amounting to 28% and 21% of all labelled neurons in cervical and lumbar enlargements, respectively. Most lamina II cells presented dendritic arbors distributed as narrow radial sheets, resembling the central cells of Ramòn y Cajal. It is suggested that the ventrolateral bulbar reticular formation is the only significant supraspinal target of lamina II tract neurons.

Direct projection of the corticospinal tract to the superficial laminae of the spinal cord in the rat.

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 III and IV and medial portions of laminae I, II, and V in the cervical and lumbar enlargements. Labeling in the ventral horn, though present, was relatively less dense. P. vulgaris leucoagglutinin-labeled axons within laminae I and II 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.

Localization of glutamate in trigeminothalamic projection neurons: a combined retrograde transport-immunohistochemical study.

Trigeminothalamic projection neurons are important components of the pathways for conscious perception of pain, temperature, and tactile sensation from the orofacial region. The neurotransmitters utilized by trigeminal neurons projecting to the thalamus are unknown. By use of a monoclonal antibody specific for fixative-modified glutamate and a polyclonal antiserum against glutaminase, we recently identified neurons in the trigeminal sensory complex that contain glutamate-like immunoreactivity (Glu-LI) and glutaminase-like immunoreactivity. In the present study, we utilized combined retrograde transport-immunohistochemical techniques to localize putative glutamatergic trigeminothalamic neurons. Following injection of the retrograde tracer, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP), into the ventroposterior medial thalamus (VPM), the number of neuronal profiles that were double-labeled with WGA:HRP and Glu-LI was greatest in principal sensory nucleus (Pr5), followed by subnuclei interpolaris (Sp5I) and caudalis (Sp5C). The average percentages of projection neurons double-labeled with Glu-LI were approximately 60-70% in Pr5 and Sp5I and 40% in Sp5C. The majority of double-labeled profiles in Sp5C were located in the magnocellular layer, as opposed to the marginal and substantia gelatinosa layers. A large injection site that spread into the intralaminar thalamic nuclei and nucleus submedius--areas implicated in the processing of nociceptive information--resulted in an increase in the ratio of single-labeled to double-labeled projection profiles in Sp5C. These results suggest that glutamate may be the neurotransmitter for a majority of trigeminothalamic projection neurons located in Sp5I and Pr5. However, on the basis of anatomical association, glutamate does not appear to be the major transmitter for neurons in Sp5C that forward nociceptive information to the thalamus.

Origin and central projections of rat dorsal penile nerve: possible direct projection to autonomic and somatic neurons by primary afferents of nonmuscle origin.

Cell number, size, and somatotopic arrangement within the spinal ganglia of the cells of origin of the rat dorsal penile nerve (DPN), and their spinal cord projections, were studied by loading the proximal stump of the severed DPN with horseradish peroxidase (HRP). The DPN sensory cells were located entirely in the sixth lumbar (L6) dorsal root ganglia (DRG), in which a mean of 468 +/- 78 cells per side were observed, measuring 26.7 +/- 0.8 microns in their longest axis (range 10-65 microns) and distributed apparently randomly within the ganglia. Within the spinal cord, no retrograde label was found, i.e., no motoneurons were labeled, indicating that in the rat the DPN is formed exclusively of sensory nerve fibers. Although labeled fibers entered the cord only through L6, transganglionically transported HRP was evident in all spinal segments examined, i.e., T13-S2. Labeled fibers projected along the inner edge of the dorsal horn (medial pathway) throughout their extensive craniosacral distribution. However, laminar distribution varied with spinal segment. In the dorsal horn, terminals or preterminal axons were found in the dorsal horn marginal zone (lamina I), the substantia gelatinosa (lamina II), the nucleus proprius (laminae III and IV--the most consistent projection), Clarke's column (lamina VI), and the dorsal gray commissure. In the ventral horn, terminals were found in lamina VII and lamina IX. Label apposed to cell somas and dendrites in lamina VII may represent direct primary afferent projections onto sympathetic autonomic neurons. In lamina IX, labeled terminals delineated the somas and dendrites of cells that appeared to be motoneurons. This is the first description of an apparently monosynaptic contact onto motoneurons by a primary afferent of nonmuscle origin.

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord.

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.

Superficial dorsal horn of the adult human spinal cord.

Golgi studies in the adult human spinal cord reveal 10 cell types in the first three laminae. Five are Golgi Type II or ipsilateral proprioneurons of short or long range--the latter including Waldeyer cells. Several of the cells in this group have dendrites that help to form interlaminar boundaries on the gray-white boundary. Two of the four cell types in Lamina II have dendritic fields that correspond exactly to the primary afferent terminal axonal fields described in the cat by Rethelyi (1977). Three cell types, one in each lamina, can be tentatively homologized with monkey spinothalamic cells described by other authors. Our previously described classification method based on dendritic patterns suggests that the Golgi Type II interneurons and ipsilateral proprioneurons belong to two different cell families (and Waldeyer cells to a third), whereas the putative spinothalamic neurons are all different cell types.

Neuronal connections of the area postrema.

The literature dealing with the neuronal connections of the area postrema (AP) is reviewed. Although it has been suggested that many regions of the central nervous system are directly connected with the AP, convincing evidence exists for only a few such regions. Afferent projections have been described from specific components of the vagus and glossopharyngeal nerves, and efferent connections have been reported to the nucleus tractus solitarii and to the parabrachial nuclei. In addition, an indirect projection from the AP to the dorsal motor nucleus of the vagus nerve exists. Further study is necessary to establish whether other important connections occur, and to determine if the connectivity of the AP is consistent in various species.

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

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

The termination pattern of sciatic nerve afferents in the substantia gelatinosa of neonatal rats.

A study was carried out to determine at what stage of development the adult pattern of primary afferent projections to the substantia gelatinosa (SG) is first established. Horseradish peroxidase conjugated wheat-germ agglutinin (WGA-HRP) was applied to cut ends of sciatic nerves at mid-thigh level in adult and neonatal rats aged 1-16 days. The distribution of transganglionically transported label in primary afferent terminals in SG was reconstructed from transverse sections of spinal cord segments L1 to S1. The distinctive U-shaped projection field of the sciatic nerve in SG of the adult was found to exist in miniature form already in the second postnatal day 24 h after WGA-HRP had been applied. The highly specific somatotopic projections of primary afferents to SG are probably established before birth. Any delay in the maturation of functional specificity in the dorsal horn of the neonate cannot be attributed to the absence or disorganization of anatomical projections to SG of primary afferent inputs.

[Horseradish peroxidase labeled sources of descending propriospinal tracts in the cat]. / Mechennye peroksidazoi khrena istochniki niskhodiashchikh propriospinal'nykh putei u koshki.

Propriospinal interneurons with descending axons were labelled after unilateral horseradish peroxidase microinjections to the cat spinal cord at different segmental levels. Main part of long propriospinal units were bilaterally localized in the ventral quadrants (laminae VII, VIII) of cervical segments; some neurons with long ipsilateral projections were also found in the marginal layer (lamina I) and lateral regions of the dorsal horn basis (laminae IV-VI). Propriospinal neurons with short axons were distributed in wider regions of the spinal gray matter. In the lumbar segments a certain trend for concentration of such neurons was found: ipsilaterally--in the lateral part of the dorsal horn basis, intermediate gray and ventral horn and contralaterally--in the ventromedial parts of gray matter. In the cervical segments short neurons with both ipsilateral and contralateral projections are concentrated in lateral parts of gray matter. Some interneurons in the marginal layer, gelatinous substance and intermediolateral sympathetic nuclei were also identified as sources of short descending propriospinal pathways. A certain part of propriospinal neurons has axons of intermediate length; location of such units was to a certain extent similar to the location of long neurons. Correlation of location of propriospinal neuron populations and patterns of synaptic inputs to them as well as correlation of structural and functional characteristics of the neurons are discussed.

Light and electron microscopic autoradiographic study of the dorsal root projections to the cat dorsal horn.

The distribution of terminals arising from dorsal root primary afferents was examined in the lumbar spinal cord of cats using light- and electron-microscopic autoradiography. Tritiated proline or leucine was injected into either the L6 or L7 dorsal root ganglion. The light-microscopic spinal cord distribution of radioactivity in the ganglia was independent of the type of amino acid used. Likewise, the length of the survival time after injection had no effect. The projections to the substantia gelatinosa and the marginal zone were consistently densest. However, the topography of the dorsal horn distribution, relative to the segment of entry, varied significantly especially in the gelatinosa, depending upon the ganglia injected. Those to the substantia gelatinosa were largely limited to the segment of entry; those to the marginal zone and nucleus proprius extended many segments beyond the level of entry. At all levels the projection was exclusively ipsilateral to the side of injection. At the electron-microscopic level the distribution of radioactivity was determined in each of the three easily recognizable areas of the dorsal horn: the marginal zone, the substantia gelatinosa and the nucleus proprius. In each dorsal horn area the total terminal population was divided into four basic categories. Each of these areas was found to contain a characteristic distribution of these four terminal categories. The difference between areas arose, primarily, as a consequence of the dorsal to ventral decreasing frequency gradient of two types of terminal: those containing large, dense-cored vesicles and the increasing gradient of those containing flattened vesicles. The terminals with small pleomorphic vesicles and those with large round vesicles were frequently encountered in all three areas without a detectable frequency gradient. Similarly the primary afferent terminal population that is the subset of the total terminal population labelled after dorsal root ganglion injection, was also characteristic of the area, and each area was dominated by a different terminal-type. In the marginal zone the terminals containing large dense-cored vesicles dominated. In the substantia gelatinosa the terminals with pleomorphic vesicles (which included the so-called 'C' type terminals) dominated. And the terminals containing large round vesicles dominated the primary afferent population in the nucleus proprius. The terminals containing flattened vesicles were never found to be specifically labeled in any of the areas examined.

Neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat: I. Light microscopic studies of cell bodies and proximal dendrites.

Although neurotensin perikarya have been identified in the superficial laminae of the dorsal horn, little is known regarding the cytoarchitecture of these neurotensin-containing neurons. Immunohistochemical staining of 75-microns sections in several planes through colchicine-treated rat spinal cords revealed morphologic features of neurotensinlike immunoreactive (NLI) perikarya and proximal dendrites. Populations of NLI neurons were identified in the outer one-third of lamina II, the inner one-third of lamina II, and outer lamina III. The NLI neurons in lamina II had elongated perikarya and the dendrites emerged from the rostral and caudal poles of the cell body. The proximal dendrites of NLI cells in inner laminae II were shorter and contained more spines than outer lamina II NLI cells. The NLI cells in outer lamina III did not exhibit polarity and the proximal dendrites were generally shorter than those of NLI cells in lamina II. The lamina II cells appear similar to islet cells (Gobel, '75), while the lamina III cells have many of the characteristics of the II/III border cell described by Gobel ('78). Interestingly, the inner lamina II NLI cells occurred in clusters with NLI cells in outer lamina III. The periodicity of these clusters suggests a neurochemical dimension for the organization of the superficial laminae of the dorsal horn.

Morphological identification of axo-axonic and dendro-dendritic synapses in the rat substantia gelatinosa.

Axo-axonic and dendro-dendritic synapses of the rat substantia gelatinosa Rolandi (SGR) have been studied in 14 adult rats by means of thin section and freeze-etch electron microscopy. Out of 6045 synaptic contacts we identified 54 between axon terminals, 10 between dendritic processes. Aside from vesicle shape the following parameters were used in a morphometric study; width of synaptic clefts, depth and length of postsynaptic densities. There are two main types of axo-axonic synapses, those with agranular vesicles (AA-type) and those with an increased population of large granular vesicles (AAgr-type). The former is prevailing and can be subdivided into two subgroups: AA1 and AA2. The pre- and postsynaptic terminals of AA1 contain spherical vesicles, the depth and length of postsynaptic densities as well as the width of the junctional clefts being significantly larger than that of AA2. Presynaptic terminals of AA2 synapses contain predominantly flattened vesicles, while spherical vesicles were found exclusively in the postsynaptic boutons. AAgr-type is rarely encountered; it is characterized by large granular vesicles (65-110 nm) which accumulate postsynaptically and occasionally in both pre- and postsynaptic boutons. Two types of dendro-dendritic synapses (DD1 and DD2) constitute another SGR feature. The difference between DD1 and DD2 is analogous to that between AA1 and AA2 except that pleomorphic synaptic vesicles appear in both groups. The width of subsynaptic membrane appositions turned out to be the most consistent criterion by which the two groups could be differentiated.

Immunohistochemical localization of leucine-enkephalin in the spinal cord of the cat: enkephalin-containing marginal neurons and pain modulation.

This study examined the spinal cord distribution of the endogenous opioid peptide leucine-enkephalin in the cat using immunohistochemical techniques. The distribution of nerve processes was studied in untreated cats; colchicine was administered to study the distribution and morphology of spinal enkephalin-containing perikarya. Enkephalin immunoreactive processes were greatest in laminae I and II (marginal layer and substantia gelatinosa) of the superficial dorsal horn. In many sections, the outer substantia gelatinosa (SG), lamina IIa, was discernibly less immunoreactive than I or IIb. Laminae III and IV were relatively devoid of staining. Laminae V and VII had moderate enkephalin-immunoreactivity, lamina VI somewhat less. Enkephalin immunoreactivity in lamina X, around the central canal, was very dense. Enkephalin-containing beaded varicosities coursed throughout the ventral horn. Although previous studies in the rat emphasized the enkephalin-somata of the SG, we found that in the cat the majority of superficial dorsal horn enkephalin-somata are in the marginal layer. These enkephalin-containing marginal cells were morphologically similar to a population of marginal neurons which project to the brainstem and/or the thalamus. Some light staining small SG neurons were also identified; many were located at the lamina I-II border. Considerably more cells were found ventral to the SG, in lamina III, and at the IV-V border. These latter cells had dendrites coursing dorsally, toward the SG. Numerous immunoreactive cells were found in lamina VIII, in a band across the intermediate gray. These cells fused medially with cells of lamina X. Enkephalin cells were also found in the sacral autonomic nucleus and encircling the central cervical nucleus, Clarke's column, and stilling's nucleus. Although surrounded by labeled cells, the latter regions were devoid of enkephalin-immunoreactive processes. Many of these spinal enkephalin neurons are morphologically similar to and distributed in regions known to contain projection neurons. Thus it is suggested that many spinal enkephalin neurons, generally thought to be local circuit neurons, project rostrally, to other spinal levels and perhaps to brainstem and/or thalamus.