Enkephalin-containing neurons in substantia gelatinosa of spinal trigeminal complex: Ultrastructure and synaptic interaction with primary sensory afferents

The ultrastructural morphology and synaptic associations between enkephalin-containing neurons and sensory afferents was examined in the caudalis portion of rat trigeminal complex, the region corresponding to Rexed's layer II of the dorsal horn^12,23. Specific antiserum to Met⁵-enkephalin was localized by the peroxidase-antiperoxidase technique in normal adult rats and in rats having electrolytic lesions of the trigeminal ganglia at 2, 4 and 6 days prior to sacrifice. In both normal and lesioned animals, the enkephalin-like immunoreactivity (ELI) was diffusely distributed throughout the cytoplasm of neuronal perikarya, dendrites, myelinated and unmyelinated axons, and axon terminals. The axons terminals were characterized by the presence of both small (40–60 nm) clear, and large (60–100 nm), dense-core vesicles and by the formation of predominantly axodendritic synapses.Optimal visualization of degenerating terminals occured at 4 days after lesions of the trigeminal ganglia. Approximately one-third of the degenerating afferents were in either direct contact or were separated from an enkephalin-containing process by an intervening unlabeled dendrite. The occurrence of a well defined membrane density between a degenerating and enkephalin-labeled bouton was rare. Most commonly, the processes showing ELI were either unassociated or formed a triad arrangement with the degenerating afferents. Within the triad, the degenerating bouton formed a junction with an unlabeled dendrite which in turn was synaptically connected at the same level to an enkephalin-labeled process, usually a dendrite. We conclude that an intermediary neuron may be involved in interaction between primary sensory afferents and enkephalin-containing neurons in spinal trigeminal complex.     

Altered responses of nociceptive cat lamina I spinal dorsal horn neurons after chronic sciatic neuroma formation

The activity of lumbar spinal dorsal horn lamina I neurons with afferent drive from the sciatic nerve was studied in intact cats and in cats with acute sciactic nerve transection or chronic sciatic nerve transection with neuroma formation. The majority (51 of 75) of neurons recorded in lamina I ipsilateral to a neuroma had no receptive field and could only be identified by their responses to electrical stimulation of the sciatic nerve. The remainder could be activated by the sciatic nerve, but their responses to mechanical stimulation were irregular in comparison to the stable responses of cells recorded in control animals and to the responses of cells contralateral to chronic nerve lesions. Animals with acute nerve transections demonstrated as loss sciatic nerve-innvervated cells with receptive fields except for those cells located on the lateral edge of the dorsal horn, which had normal, proximal receptive fields and response characteristics. In addition, the characteristic somatotopy of lamina I cells was not observed in some cats with chronic neuromata. The mediolateral distribution of cell types indicated that some cells had altered receptive fields following chronic nerve transection. The data presented for lamina I neurons agrees with the observation of spinal cord plasticity first presented for cat dorsal horn cells. Since there is no evidence for a redistribution of intact afferent fibers following chronic nerve transection in adult mammals, the mechanism of altered somatotopy may involved alterations in synaptic efficacy at existing synapses.     

Golgi studies in the substantia gelatinosa neurons in the spinal trigeminal nucleus.

This Golgi study identifies three neuronal cell types in the substantia gelatinosa (SG) layer of the spinal trigeminal nucleus. The SG neurons are distinguished from each other based on: (1) dendritic branching pattern, (2) denritic spine distribution, (3) geometric shape of the denritic tree, (4) laminar distribution of the dendrites, (5) axonal branching pattern and (6) laminar distribution of the axonal arbor. The islet cell is found in small clusters and its dendrites and axonal arbor are confined within the SG layer. Its dendrites span the full width of the SG layer and extend up to 500 mum in the long axis of the layer. Dendritic spines are generally sparse with small clusters of spines found on the higher order dendritic branches. The islet cell axon extends for at least 1 mm in the long axis of the layer. Each of its collaterals divide every 50-100 mum with one branch doubling back in the direction of the cell body and the other branch continuing on in the direction of its parent. In this manner each islet cell generates a profuse axonal plexus in the SG layer. The stalked cell is found individually within the SG layer. Its cell body is usually found in the inner half of the SG layer and its sinuous dendrites cross the SG layer and enter the marginal layer. The stalked cell dendrites emit numerous fine stalk-like branches and dentritic spines. Its axon emits branches in the SG and marginal layers. The spiny cell is found singly between groups of islet cells. Its extensive dendritic tree spans up to 500 mum rostrocaudally and mediolaterally crossing into both the marginal and magnocellular layers. Spiny cells have evenly distributed dendritic spines along their dendrites in the SG layer. The spiny cell axon sends branches into all three layers of nucleus caudalis. Numerous branches enter the outer 300 mum of the magnocellular layer where they undergo further branching with some branches returning in recurrent fashion toward the SG layer. The three neuronal cell types of the SG layer satisfy all of the morphological criteria for Golgi type II interneurons. Their highly branched axons generate many collaterals within the confines of their dendritic trees and do not project out of nucleus caudalis. The SG neurons are considered to be inhibitory interneurons interposed between V nerve primary afferent axons which arborize in the SG layer and second order neurons of nucleus caudalis.     

A-fibres sprouting from lamina I into lamina II of spinal dorsal horn after peripheral nerve injury in rats

We have examined the labeling pattern in the spinal dorsal horn by an intra-sciatic nerve injection of cholera toxin B subunit conjugated horseradish peroxidase (HRP) after transection of the posterior cutaneous nerve and inferior gluteal nerve, and found that the cholera toxin B subunit conjugated HRP labeling in lamina I was expanding into lamina II and there was a shrinking gap between lamina I and lamina III. This result suggests that A-fibre sprouting arise after peripheral nerve injury, but mainly from small calibre Adelta-fibres which terminate in lamina I.     

Functional reorganization in adult monkey thalamus after peripheral nerve injury.

Large changes in somatotopic organization can be induced in adult primate somatosensory cortex by cutting peripheral afferents. The role, if any, of the thalamus in these changes has not been investigated previously. In the present experiments, electrophysiological recording in the ventroposterior lateral nucleus (VPL) has revealed that not only can reorganization occur in the thalamus, but it may be as extensive as that revealed in the cortex of the same monkeys. Thus, for at least some types of deafferentation, the reorganization revealed in the cortex may depend largely on subcortical changes.     

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.     

Synaptic action of vestibular nerve afferents on reticular neurons of the isolated medulla oblongata of the goldfish

Excitatory post-synaptic potentials (EPSPs) were evoked in reticular neurons of isolated goldfish brian stem by stimulation of the ipsilateral vestibular nerve or by direct intra-axonal activation of single vestibular fibres. Composite EPSPs displayed usually two components. Ca2+-deficient, Mn2+-containing solution abolished the delayed chemical components of EPSPs, but did not affect the short latency responses suggesting dual (electrical-chemical) mode of transmission between some vestibular afferents and reticular neurons.     

Synaptic detachment from hypoglossal neurons after different types of nerve injury in the cat.

Quantitative electron microscopy was used to examine synaptic numbers and total relative synaptic coverage on hypoglossal neuronal perikarya following transection or crush of the hypoglossal nerve in the cat. In cats subjected to nerve transection there was a statistically significant decrease in total synaptic coverage as well as in number of synaptic contacts/soma profile. After nerve crush there was a statistically significant decrease in total synaptic coverage but not in the number of synaptic contacts. These results indicate that the degree of synaptic detachment on motor neurons in the cat is influenced by the type of nerve injury.     

Synaptic innervation of midbrain dopaminergic neurons by glutamate-enriched terminals in the squirrel monkey

The excitatory amino acid, glutamate, has long been thought to be a transmitter that plays a major role in the control of the firing pattern of midbrain dopaminergic neurons. The present study was aimed at elucidating the anatomical substrate that underlies the functional interaction between glutamatergic afferents and midbrain dopaminergic neurons in the squirrel monkey. To do this, we combined preembedding immunocytochemistry for tyrosine hydroxylase and calbindin D-28k with postembedding immunostaining for glutamate. On the basis of their ultrastructural features, three types (so-called types I, II, and III) of glutamate-enriched terminals were found to form asymmetric synapses with dendrites and perikarya of midbrain dopaminergic neurons. The type I terminals accounted for more than 70% of the total population of glutamate-enriched boutons in contact with dopaminergic cells in the dorsal and ventral tiers of the substantia nigra pars compacta as well as in the ventral tegmental area, whereas 5–20% of the glutamatergic synapses with dopaminergic neurons involved the two other types of terminals. The major finding of our study is that the glutamate-enriched boutons were involved in 70% of the axodendritic synapses in the ventral tegmental area. In contrast, less than 40% of the boutons in contact with dopaminergic dendrites were immunoreactive for glutamate in the dorsal and ventral tiers of the substantia nigra pars compacta. Approximately 50% of the terminals in contact with the perikarya of the different populations of midbrain dopaminergic neurons displayed glutamate immunoreactivity. In conclusion, our findings provide the first evidence that glutamate-enriched terminals form synapses with midbrain dopaminergic neurons in primates. The fact that the proportion of glutamatergic boutons in contact with dopaminergic cells is higher in the ventral tegmental area than in the substantia nigra pars compacta suggests that the different groups of midbrain dopaminergic neurons are modulated differently by extrinsic glutamatergic afferents in primates. © 1996 Wiley-Liss, Inc.     

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.     

TRPA1‐expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole‐cell patch‐clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C‐ (but not Aδ‐) fiber‐evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.     

Peripheral innervation patterns of vestibular nerve afferents in the bullfrog utriculus

Vestibular nerve afferents innervating the bullfrog utriculus differ in their response dynamics and sensitivity to natural stimulation. They also supply hair cells that differ markedly in hair bundle morphology. To examine the peripheral innervation patterns of individual utricular afferents more closely, afferent fibers were labeled by the extracellular injection of horseradish peroxidase (HRP) into the vestibular nerve after sectioning the vestibular nerve medial to Scarpa's ganglion to allow the degeneration of sympathetic and efferent fibers. The peripheral arborizations of individual afferents were then correlated with the diameters of their parent axons, the regions of the macula they innervate, and the number and type of hair cells they supply. The utriculus is divided by the striola, a narrow zone of distinctive morphology, into medial and lateral parts. Utricular afferents were classified as striolar or extrastriolar according to the epithelial entrance of their parent axons and the location of their terminal fields. In general, striolar afferents had thicker parent axons, fewer subepithelial bifurcations, larger terminal fields, and more synaptic endings than afferents in extrastriolar regions. Afferents in a juxtastriolar zone, immediately adjacent to the medial striola, had innervation patterns transitional between those in the striola and more peripheral parts of the medial extrastriola. Most afferents innervated only a single macular zone. The terminal fields of striolar afferents, with the notable exception of a few afferents with thin parent axons, were generally confined to one side of the striola. Hair cells in the bullfrog utriculus have previously been classified into four types based on hair bundle morphology (Lewis and Li: Brain Res. 83:35–50, 1975). Afferents in the extrastriolar and juxtastriolar zones largely or exclusively innervated Type B hair cells, the predominant hair cell type in the utricular macula. Striolar afferents supplied a mixture of four hair cell types, but largely contacted Type B and Type C hair cells, particularly on the outer rows of the medial striola. Afferents supplying more central striolar regions innervated fewer Type B and larger numbers of Type E and Type F hair cells. Striolar afferents with thin parent axons largely supplied Type E hair cells with bulbed kinocilia in the innermost striolar rows. © 1994 Wiley-Liss, Inc.     

Synaptic inputs to medullary respiratory neurons from superior laryngeal afferents in the cat.

Synaptic inputs from afferents in the superior laryngeal nerve (SLN) to medullary respiratory neurons (n = 154) in the dorsal respiratory group (DRG), ventral respiratory group (VRG) and the region of the B?tzinger complex (BOT) were studied in anesthetized cats. Single pulse stimulation of the SLN-evoked monosynaptic EPSPs in most inspiratory bulbospinal (I-BS) neurons in the DRG, and disynaptic or oligosynaptic chloride-dependent IPSPs in other I-BS neurons in the DRG and VRG. Stimulation of laryngeal afferents also inhibited oligosynaptically expiratory bulbospinal neurons in the VRG, and all types of respiratory neurons recorded in the BOT region. Oligosynaptic potentials (usually EPSPs) were recorded in inspiratory and expiratory laryngeal motoneurons. These results provide evidence of a processing of SLN-evoked synaptic responses by all tested groups of medullary respiratory neurons. The pathways mediating these synaptic responses are discussed.     

Neonatal capsaicin treatment induces invasion of the substantia gelatinosa by the terminal arborizations of hair follicle afferents in the rat dorsal horn

Capsaicin, administered on the day of birth, was found to alter laminar distribution, but not the receptive field properties or the morphology of the collateral arborizations of hair follicle afferents (HFAs) intra-axonally injected with horseradish peroxidase (HRP). Of the 65 HFA terminal arbors in capsaicin treated rats, 46 (71%) were found to enter the substantia gelatinosa (in control rats, 44/165, 27%). All of the collaterals projected to somatotopically normal areas of cord. Dorsal horn shrinkage (21%), as estimated by planimetric measurements of Nissl and acetylcholinesterase-stained material, was only a partial explanation of this result. This idea was supported by the statistically significant increase (27%, P less than 0.05) in the absolute dorsoventral length of collaterals. The results show that the destruction of unmyelinated fibres during the early postnatal period by capsaicin induces HFA invasion into the area that C fibres normally occupy. This invasion suggests that the laminar termination sites for different primary afferent fibres are not altogether specified and that intact neonatal primary afferents have the capacity to sprout into denervated regions of spinal cord.     

A biochemically distinct sub-population of neurons in the human substantia gelatinosa. Study with G-6-PD histochemistry

A method for localization of glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate: NADP+ oxidoreductase; E.C. 1.1.1.49) activity has been applied to human nervous tissue. Intensely staining cells, not definable by conventional histologic techniques, have been identified in the human spinal cord, with highest numbers present in the substantia gelatinosa of the sacral region. The cells have a neuron-like morphology and express neuronal-specific antigen but are heterogeneous in size and shape. They are not detectable in infant spinal cord, but stain heavily in adults. We propose that these cells are homologous to the G-6-PD-active dorsal medullary cells first noted by Sakharova et al. (1979) and together with the latter group, may comprise a hitherto unrecognized system of neurons in the human central nervous system.     

Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat

In the present study, developing neurons of the substantia gelatinosa (SG) are examined at short interval sequential stages from 15 days of gestation through 20 days postpartum. Rapid Golgi preparations are utilized to examine axonal and dendritic development and toluidine blue preparations are employed to study the overall growth pattern of SG cells by measuring changes in mean cell body area. Results show that there are two maturation periods, which involve two separate groups of SG neurons. The sequence and pattern of development for each group is different. The first period occurs prenatally and involves the axonal and dendritic development of presumptive projection and propriospinal neurons. In classical terminology, these cells can be classified as limiting, large find small central, and transverse cells. These neurons have axons that enter the white matter and their dendritic arbors develop through a relatively simple process of elongation and branching. The second maturation period occurs postnatally and involves the development of presumptive nonprojection intrinsic neurons that have axons which remain within the gray matter. These neurons are identified as islet, stalk, inverted stalklike, and vertical cells. Unlike projection or propriopinal neurons, the intrinsic nonprojection neurons sprout numerous short, beaded dendrites that radiate from the cell body in a starlike fashion. Star-shaped cells undergo a metamorphosis involving a rearrangement of dendrites along adult dendritic patterns. Measurements taken from toluidine blue preparations indicate that the nonprojection intrinsic population makes up the greatest percentage of SG neurons, as evidenced by a marked increase in the size of the average SG nerve cell during the second maturation period.     

Morphological features of functionally defined neurons in the marginal zone and substantia gelatinosa of the spinal dorsal horn.

Functional characteristics of spinal neurons located in the marginal zone (lamina I) and substantia gelatinosa (lamina II) were compared to their structural features by intrcellularly 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 A delta 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 nociceptive and thermoreceptive neurons tended to be in lamina I or outer lamina II (SGo) while those of the innocuous mechanoreceptive meurons 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 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 A delta 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 mechanoreceptros terminating predominantly in lamina II. Substantial integrative and distributive functions can be expected of such an afferent termination zone.     

Cell type-specific postsynaptic effects of neuropeptide Y in substantia gelatinosa neurons of the rat spinal cord

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 μM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (～77%) but not DFNs (～8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ～5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.     

The rat's postero-orbital sinus hair: II. Normal morphology and the increase in peripheral innervation with adjacent nerve section.

The morphology and innervation of the postero-orbital (PO) sinus hair has been studied in normal rats and in adult animals in which an adjacent nerve, the infraorbital nerve, was sectioned on postnatal day 0 or day 7. The normal morphology of the follicle was similar to that of mystacial sinus hairs. However, the normal innervation differed from mystacial follicles in three respects: (1) instead of a separate innervation, the deep vibrissal nerve (DVN) and dermal plexus were supplied by a common follicle and skin nerve, named here the postero-orbital cutaneous nerve, a branch of the zygomaticofacial nerve; (2) the entry of the DVN through the capsule was highly variable; in some cases fascicles entered in close proximity, but in others they were widely distributed around the capsule; and (3) two or three small nerves, called here anastomosing nerves, were found to leave the PO follicle. These arose from the DVN after it had passed through the capsule to the cavernous sinus. The anastomosing nerves passed back through the capsule and ascended on the outer surface of the follicle to join the dermal plexus. Each nerve contained 1-4 myelinated fibres and 11-35 unmyelinated fibres. Infraorbital (IO) nerve section on day 0 caused a 19% (P less than 0.001, n = 8) increase in numbers of fibers to the DVN on the lesioned side. Most of the increase was due to unmyelinated fibres with no significant change in myelinated axons. No change in axon numbers in the DVN occurred after day 7 lesions. Labelling of the mystacial pad and the PO follicle did not result in any double labelling of cells in the trigeminal ganglion, in either normal or lesioned animals, making it improbable that the increased numbers of unmyelinated axons arose from rerouting of infraorbital fibres. It is suggested that the increased innervation of the PO follicle may arise by the rescue of ganglion cells from developmentally programmed cell death.