Ultrastructural analysis of the synaptic connectivity of TRPV1-expressing primary afferent terminals in the rat trigeminal caudal nucleus

Trigeminal primary afferents that express the transient receptor potential vanilloid 1 (TRPV1) are important for the transmission of orofacial nociception. However, little is known about how the TRPV1-mediated nociceptive information is processed at the first relay nucleus in the central nervous system (CNS). To address this issue, we studied the synaptic connectivity of TRPV1-positive (+) terminals in the rat trigeminal caudal nucleus (Vc) by using electron microscopic immunohistochemistry and analysis of serial thin sections. Whereas the large majority of TRPV1+ terminals made synaptic contacts of an asymmetric type with one or two postsynaptic dendrites, a considerable fraction also participated in complex glomerular synaptic arrangements. A few TRPV1+ terminals received axoaxonic contacts from synaptic endings that contained pleomorphic synaptic vesicles and were immunolabeled for glutamic acid decarboxylase, the synthesizing enzyme for the inhibitory neurotransmitter γ-aminobutyric acid (GABA). We classified the TRPV1+ terminals into an S-type, containing less than five dense-core vesicles (DCVs), and a DCV-type, containing five or more DCVs. The number of postsynaptic dendrites was similar between the two types of terminals; however, whereas axoaxonic contacts were frequent on the S-type, the DCV-type did not receive axoaxonic contacts. In the sensory root of the trigeminal ganglion, TRPV1+ axons were mostly unmyelinated, and a small fraction was small myelinated. These results suggest that the TRPV1-mediated nociceptive information from the orofacial region is processed in a specific manner by two distinct types of synaptic arrangements in the Vc, and that the central input of a few TRPV1+ afferents is presynaptically modulated via a GABA-mediated mechanism.     

Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei

Little is known about the central projection patterns of trigeminal afferent neurons expressing the vanilloid receptor TRPV1 and their coexpression of neuromodulatory peptides. To address these issues, we examined the distribution of TRPV1-positive neurons in the trigeminal ganglion (TG) and trigeminal sensory nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc) in the rat via light and electron microscopy. In addition, we studied the colocalization of TRPV1-positive neurons with substance P (SP) and calcitonin gene-related peptide (CGRP) via confocal microscopy. In TG, only small and medium-sized neurons were immunopositive for TRPV1. The staining for TRPV1 was found in axon collaterals in the dorsal parts of Vp, Vo, and Vi and in terminals and fibers throughout lamina I and the outer zone of lamina II (IIo) of Vc. With electron microscopy, TRPV1-positive fibers in the ascending and descending trigeminal tracts were found to be unmyelinated. Almost all TRPV1-positive terminals in Vc contained numerous large dense-core vesicles and formed synaptic contacts with single small dendrites. Multiple immunofluorescence revealed a high degree of colocalization of TRPV1 with SP and CGRP in TG neurons as well as in fibers and terminals confined to laminae I and IIo of Vc. These results suggest that the central projections of unmyelinated (C) afferents sensitive to noxious heat and capsaicin are organized differently between Vc and the rostral trigeminal nuclei and that Vc may play a role in the development of hyperalgesia.     

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.     

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat

Trigeminal primary afferents expressing P2X(3) receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X(3)-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X(3) (+) terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN.     

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

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

ATP potentiates neurotransmission in the rat trigeminal subnucleus caudalis

Ionotropic purine receptors (P2X) have been implicated in nociceptive neurotransmission. In this study, we examine the actions of the P2X receptor agonist alpha,beta methylene adenosine 5'-triphosphate on excitatory neurotransmission in neurons in the deep and superficial laminae of the trigeminal spinal subnucleus caudalis (Vc), which receives nociceptive inputs from the craniofacial region. Alpha, beta methylene adenosine 5'-triphosphate caused an increase in spontaneous excitatory neurotransmission (miniature excitatory postsynaptic currents) in neurons in deep but not superficial laminae of Vc; this effect could be inhibited by the P2X receptor antagonist 2,3-O-2,4,6-trinitrophenyl-ATP. Conversely, the TRPV1 agonist capsaicin caused an increase in miniature excitatory postsynaptic currents in neurons in the superficial but not deep laminae. These data suggest that alpha,beta methylene adenosine 5'-triphosphate acts on presynaptic terminals to increase glutamatergic neurotransmission in deep Vc neurons.     

Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat

Serotoninergic projections to the spinal dorsal horn are implicated in the modulation of nociceptive transmission. However, morphological evidence indicating that serotoninergic projection fibers make synapses on nociceptive neurons in the medullary dorsal horn is still meager. Thus, we examined whether axonal varicosities with serotonin (5-HT)-like immunoreactivity (5-HT-LI) might make synapses on nociceptive projection neurons in the caudal spinal trigeminal nucleus (Vc) of the rat. Projection neurons were retrogradely labeled with tetramethylrhodamine-dextran amine (TMR-DA) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) that was injected into the parabrachial or thalamic region. Vc neurons in which c-fos protein-like immunoreactivity (Fos-LI) was induced by subcutaneous injection of formalin into the lip were considered nociceptive. Vc neurons in direct contact with axonal varicosities that bind isolectin I-B4 were also considered nociceptive. Triple labeling for 5-HT, TMR-DA, and Fos as well as that for 5-HT, TMR-DA, and I-B4 were done by using the immunofluorescence and fluorescence histochemical techniques. Confocal laser-scanning microscopy revealed that axonal varicosities with 5-HT-LI were in close apposition to TMR-DA-labeled neurons showing Fos-LI in lamina I and the outer part of lamina II (lamina IIo), and that both axonal varicosities with 5-HT-LI and those binding I-B4 were in close apposition to single neuronal profiles labeled with TMR-DA. The presumed nociceptive neuronal profiles in close apposition to axon terminals with 5-HT-LI were mainly those of laminae I and II neurons as well as dendrites of lamina III neurons. Electron microscopy confirmed that axon terminals with 5-HT-LI and those with I-B4 binding activity in laminae I and II made synapses on somatic and dendritic profiles that were labeled with WGA-HRP. The results indicate that serotoninergic neurons project directly on nociceptive projection neurons in the Vc. J. Comp. Neurol. 384:127-141, 1997. © 1997 Wiley-Liss, Inc.     

Electron microscopy of immunoreactivity patterns for glutamate and γ-aminobutyric acid in synaptic glomeruli of the feline spinal trigeminal nucleus (subnucleus caudalis)

We studied the ultrastructure of the synaptic organization in the feline spinal trigeminal nucleus, emphasizing specific neurotransmitter patterns within lamina II of the pars caudalis/medullary dorsal horn. Normal adults were perfused, and Vibratome sections from pars caudalis were processed for electron microscopy. Ultrathin sections were reacted with antibodies for the excitatory neurotransmitter glutamate (Glu) and for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) by using postembedding immunogold techniques. Both single- and double-labeled preparations were examined. Results with single labeling show that Glu-immunoreactive terminals have round synaptic vesicles and form asymmetric synaptic contacts onto dendrites. GABA-immunoreactive axon terminals and vesicle-containing dendrites have pleomorphic vesicles, and the axon terminals form symmetric contacts onto dendrites and other axons. Double labeling on a single section shows glomeruli with central Glu-immunoreactive terminals that are presynaptic to dendrites, including GABA⁺ vesicle-containing dendrites. These Glu⁺ terminals are also postsynaptic to GABA⁺ axon terminals, and these GABA-immunoreactive terminals may also be presynaptic to the GABA⁺ vesicle-containing dendrites. Quantitative analyses confirm the specificity of the Glu and GABA immunoreactivities seen in the various glomerular profiles. The results suggest that a subpopulation of Glu-immunoreactive primary afferents (excitatory) may be under the direct synaptic influence of a GABA-immunoreactive intrinsic pathway (inhibitory) by both presynaptic and postsynaptic mechanisms. © 1996 Wiley-Liss, Inc.     

Cells in laminae III and IV of the rat spinal cord which possess the neurokinin‐1 receptor receive monosynaptic input from myelinated primary afferents

We have previously demonstrated that neurons which have cell bodies in laminae III or IV of the rat spinal cord, dendrites that enter the superficial laminae and which possess the neurokinin-1 receptor receive a major synaptic input from substance P-containing primary afferent axons. In this study we set out to determine whether these cells also receive monosynaptic input from myelinated primary afferents by using transganglionic transport of the B subunit of cholera toxin to identify the central terminals of myelinated afferents from the sciatic nerve. Dual-immunofluorescence and confocal microscopy revealed apparent contacts between labelled primary afferent terminals and all of the neurokinin-1 receptor-immunoreactive cells examined, although these contacts were much less numerous than those which the cells receive from substance P-containing primary afferents. By using a combined confocal and electron microscopic technique we were able to confirm that synapses were present at some of the contacts between primary afferents and neurokinin-1 receptor-immunoreactive neurons. These results suggest that cells of this type will have wide-dynamic range receptive fields, but with a relatively strong input from nociceptors.     

Fine structural survey of the rat's brainstem sensory trigeminal complex

The fine structural organization of the principal sensory trigeminal nucleus was compared with that of the spinal trigeminal nucleus (subnuclei oralis, interpolaris, and the deep layers of caudalis) in adult albino rats. Direct comparisons indicate similarities between all of the subdivisions of the brainstem trigeminal complex both in the major morphological classes of neurons present and in basic patterns of synaptic connections. Major differences between the several subdivisions occur in the relative numbers and distribution of the different cell types. The spinal trigeminal nucleus is distinguished by more numerous large (22-40 micron) polygonal neurons which give rise to long straight primary dendrites. Both the perikaryal surface and the thick primary dendrites of many of these cells are densely innervated by synaptic terminals. Especially large cells of this type are a prominent feature of subnucleus oralis. By contrast, the principal sensory nucleus is distinguished by its high density of small to medium-sized (8-20 micron) round or ovoid neurons. These smaller neurons tend to receive a sparse axosomatic innervation. In addition to these differences the spinal trigeminal neuropil is distinguished by the striking manner in which it is broken up by large rostrocaudally oriented bundles of myelinated axons. Proximal dendrites of polygonal and fusiform neurons often wrap around these large axon bundles. Morphologically heterogeneous populations of synaptic terminals with round vesicles (R terminals) and terminals with predominantly flattened vesicles (F terminals) occur in all of the subdivisions of the trigeminal complex. Both types of terminal make primarily axodendritic synapses, but both also make axosomatic synapses, and axospinous synapses with somatic as well as dendritic spines. In addition, axoaxonic synaptic contacts from F terminals onto large R terminals are seen in all subdivisions. Convincing examples of presynaptic dendrites were not observed in any of the brainstem subdivisions. Synaptic glomeruli, characteristic groupings of dendrites and synaptic terminals, are found throughout the brainstem trigeminal complex. The dendritic elements in these glomeruli tend to be small-diameter dendrites, spines, and large, spinelike appendages. Within the glomerulus these elements are postsynaptic to a single large R terminal and may also be postsynaptic to smaller F terminals. In addition, axoaxonic synaptic contacts from the F terminals onto the R terminal are a consistent feature of trigeminal synaptic glomeruli.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of gastrin‐releasing peptide in the rat trigeminal and spinal somatosensory systems

Gastrin‐releasing peptide (GRP) has recently been identified as an itch‐specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP‐immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small‐ and medium‐sized type cells. In both the TG and DRG, many GRP‐ir neurons also expressed substance P and calcitonin gene‐related peptide, but not isolectin B₄. The different proportions of GRP and transient receptor potential vanilloid 1 double‐positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP‐ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP‐ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP‐ir terminals contained clear microvesicles and large dense‐cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP‐dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn. J. Comp. Neurol. 522:1858–1873, 2014. © 2013 Wiley Periodicals, Inc.     

Immunocytochemistry of enkephalin and serotonin distribution in restricted zones of the rostral trigeminal spinal subnuclei: comparisons with subnucleus caudalis

The spinal trigeminal subnucleus caudalis processes nociceptive input from the head. However, physiological and behavioral studies in monkeys and humans indicate that painful stimuli from the central face and oral cavity also project through trigeminal nuclei rostral to the spinal subnucleus caudalis. Both enkephalin (ENK) and serotonin (5-HT) are present in rostral trigeminal nuclei and these regions receive inputs from the raphe complex. Thus, it appears that elements of pain-modulating circuitry proposed by Basbaum and Fields (Annu. Rev. Neurosci., 7:309-338, 1984) for the spinal and medullary dorsal horn may also exist in this region. In order to begin an exploration of this circuitry, the present study combines the techniques of retrograde transport of HRP from the ventral posteromedial thalamic nucleus (VPM) of the cat's thalamus to label trigeminothalamic relay cells. Secondarily, immunocytochemical techniques are employed to define the distribution patterns of ENK and 5-HT cells and terminals in relationship to both labeled and nonlabeled neurons in each of the subnuclei of the spinal trigeminal nucleus. Trigeminothalamic relay cells were observed in laminae I and II, the magnocellular region, and the interstitial nucleus (IN) of subnucleus caudalis (Vc). ENK was found in axodendritic and axosomatic terminals, together with a population of small fusiform neurons in all these same areas except the magnocellular region. ENK axosomatic contacts innervated approximately 30% of labeled relay cells, chiefly in lamina I and the IN, or small unlabeled neurons in the same area. Serotonin activity occurred principally in lamina I and the IN and was confined almost exclusively to axodendritic terminals. Examination of subnucleus interpolaris (Vi) revealed relay cells distributed throughout the length of the nucleus and increasing in numbers at rostral levels. A rostral extension of the IN was found just ventrolateral to the main body of Vi and contained numerous labeled cells. The distribution of ENK activity was restricted to the ventral part of Vi and the IN and occurred in axodendritic and axosomatic terminals. These latter elements innervated 30-40% of labeled relay cells in Vi, particularly those located in the IN. Cells containing ENK generally resembled the fusiform cells found in Vc and were distributed in ventral Vi and the IN. Some ENK cells were larger, displayed several dendrites, and occurred only in the ventral Vi. Serotonin within Vi and Vc was confined principally to axodendritic terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Craniofacial inputs to upper cervical dorsal horn: implications for somatosensory information processing

The aim of this study was to characterize the properties of somatosensory neurons in the first 2 cervical spinal dorsal horns (C1 and C2 DHs) and compare them with those previously described for the rostral subnucleus caudalis (rVc). A total of 74 nociceptive neurons classified as wide-dynamic-range (WDR) or nociceptive-specific (NS), as well as 72 low-threshold mechanoreceptive (LTM) neurons, was studied in urethane/chloralose-anesthetized rats. The majority of LTM neurons were located in laminae III/IV and had a small mechanoreceptive field (RF) that included the posterior face and cervical tissues. In contrast, the nociceptive neurons were located in laminae I/II or V/VI, and the RF of each C1 and C2 DH nociceptive neuron included a part of the face and in 47% of them the RF included a region supplied by upper cervical afferents. There was a gradual caudal shift in the neuronal RF from nasal/intraoral tissues towards the neck as recording sites progressed from rVc to C1 and C2 DHs. In contrast to LTM neurons, many C1 and C2 DH nociceptive neurons received mechanosensitive convergent afferent inputs from cervical and craniofacial deep tissues (e.g., tongue muscles or temporomandibular joint), and over 50% could be activated by hypoglossal (XII) nerve electrical stimulation. We propose that C1 and C2 DHs represent part of the caudal extension of the Vc, and that Vc and C1 and C2 DHs may act together as one functional unit to process nociceptive information from craniofacial and cervical tissues, including that from deep craniofacial tissues.     

Identification of signal substances in synapses made between primary afferents and their associated axon terminals in the rat trigeminal sensory nuclei

The relationships between primary afferent terminals (PATs) and their associated presynaptic terminals in the rat trigeminal sensory nuclear complex (TSNC) were examined with special reference to amino acid transmitters glutamate (Glu) and gamma-aminobutyric acid (GABA). Primary afferent terminals anterogradely labeled from the trigeminal ganglion with the B subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) were sectioned for electron microscopy. Serial sections from the principal nucleus (Vp), dorsomedial parts of the oral and interpolar nuclei (Vdm), and lamina III/IV of caudal nucleus (Vc) were immunostained for Glu and GABA by using a postembedding immunogold technique. The tracer, CTB-HRP to the trigeminal ganglion, preferentially labeled myelinated primary afferents. Sections immunostained with Glu antiserum showed that most labeled PATs were enriched with immunoreactivity (IR) for Glu. The Glu-IR PATs contained clear, round, synaptic vesicles and formed asymmetric synaptic contacts with somata or dendrites. They were frequently postsynaptic to, unlabeled axon terminals filled with a mixture of clear, round, oval, and flattened vesicles (p-endings), with symmetric synaptic junctions. The frequency of synapses onto somata or primary dendrites per Glu-IR PAT was higher in the Vdm than in either the Vp or Vc lamina III/IV. The frequency of contacts of the p-endings per Glu-IR PAT was higher in the Vp than in the Vdm and Vc lamina III/IV. Sections immunostained with GABA antiserum showed that most axon terminals presynaptic to PATs were enriched with GABA in the three nuclei. The GABA-IR axon terminals and their postsynaptic PATs had a similar ultrastructural character to p-endings and their postsynaptic Glu-IR PATs, respectively. The present study suggests that primary afferent neurons with large-caliber fibers use glutamate as a neurotransmitter and are subject to presynaptic modulation by GABAergic fibers.     

Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS⁺) and evaluate the synaptic relationship of NOS⁺ striatal neurons with those containing parvalbumin (PV⁺). In both the single-label and double-label studies, NOS⁺ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS⁺ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS⁺ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV⁺ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV⁺ dendrites, and on NOS⁺ soma and dendrites. In contrast, NOS⁺ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV⁺ or NOS⁺ neurons. These findings suggest that NOS⁺ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV⁺ or NOS⁺ striatal interneurons. NOS⁺ neurons do, however, receive synaptic input from PV⁺ neurons.     

Immunocytochemical localization of enkephalin in the neostriatum of rat brain: A light and electron microscopic study

The peroxidase-antiperoxidase (PAP) immunocytochemical technique was used to determine the light and electron microscopic localization of antisera directed against either methionine [Met⁵]- or leucine [Leu⁵]-enkephalin in the neostriatum of brains from untreated rats. By light microscopy, neuronal perikarya and processes showing enkephalin-like immunoreactivity (ELI) were unevently distributed throughout the neostriatum. The greatest accumulation of neuronal structures showing ELI was in the ventro- and caudo- lateral portions of the nucleus. The labeled perikarya measured 10–15 μm in diameter and constituted about 15–20% of the total neurons in the enostriatum. By electron microscopy, examination of three areas from horizontal and coronal sections revealed no regional differences in types of neurons showing ELI or in their synaptic organization. All labeled neurons showed a relatively low intensity reaction product which was diffusely distributed throughout the perikarya and dendrites. The cytoplasm contained relatively few organelles, which included mitochondria, endoplasmic reticulum and numerous “alveolate” vesicles. The dendrites had many spiny processes which formed asymmetric synapses with unlabeled axon terminals containing all small clear vesicles. In contrast to the perikarya and dendrites a dense accumulation of reaction product was present in a few myelinated and numerous unmyelinated axons and axonal varicosties. Approximately 75% of the labeled varicosities did not form a specialized synaptic junction in a single plane of section. The remaining 25% of the labeled terminals formed asymmetric junctions primarily with unlabeled dendrites and rerely with unlabeled perikarya or axons. The morphology and synaptic relations of the neurons showing ELI suggest that they may belong to the general group of medium-sized spiny cells characterized in Golgi studies by Kemp and Powell ('71a). At least some of the peptide-containing neurons may also have a myelinated efferent axon.     

Light and electron microscopic localization of calcitonin gene-related peptide immunoreactivity in lamina II of the feline trigeminal pars caudalis/medullary dorsal horn: A qualitative study

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is associated with a subset of primary afferent fibers and appears to have a role in nociception. The purpose of the present study was to perform a qualitative light, and especially electron microscopic (LM and EM), examination of CGRP-immunoreactivity (IR) within lamina II (substantia gelatinosa) of the feline pars caudalis/medullary dorsal horn (PC/MDH) of the spinal trigeminal nucleus. The LM investigation revealed massive CGRP-IR within lamina II outer, with fewer fibers that traversed lamina II inner. The EM preparations showed CGRP-IR in small, thinly myelinated and unmyelinated axons, preterminal axons, and in axon terminals that formed asymmetric synaptic contacts onto small dendritic profiles. The terminals with CGRP-IR were often the central element within glomeruli, where the terminal usually formed 2 or more asymmetric synaptic specializations onto 1 or more dendrites. Many of these postsynaptic dendrites contained synaptic vesicles. Other profiles were seen forming presynaptic contacts onto the terminal with CGRP-IR, and these profiles most likely represent presynaptic dendrites and/or other axon terminals of intrinsic origin. The synaptic association of terminals showing CGRP-IR with vesicle-containing dendrites, presynaptic dendrites, and/or other axon terminals suggests that terminals with CGRP-IR are especially susceptible to modulation. © 1993 Wiley-Liss, Inc.     

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.     

Noradrenaline‐mediated facilitation of inhibitory synaptic transmission in the dorsal horn of the rat spinal cord involves interlaminar communications

In the dorsal horn of the spinal cord (DH), noradrenaline (NA) is released by axons originating from the locus coeruleus and induces spinal analgesia, the mechanisms of which are poorly understood. Here, the effects of NA on synaptic transmission in the deep laminae (III–V) of the DH were characterized. It was shown that exogenously applied, as well as endogenously released, NA facilitated inhibitory [γ‐aminobutyric acid (GABA)ergic and glycinergic] synaptic transmission in laminae III–IV of the DH by activating α₁‐, α₂‐ and β‐adrenoceptors (ARs). In contrast, NA had no effect on excitatory (glutamatergic) synaptic transmission. Physical interruption of communications between deep and more superficial laminae (by a mechanical transection between laminae IV and V) totally blocked the effects of α₂‐AR agonists and strongly reduced the effects of α₁‐AR agonists on inhibitory synaptic transmission in laminae III–IV without directly impairing synaptic release of GABA or glycine from neurons. Short‐term pretreatment of intact spinal cord slices with the glial cell metabolism inhibitor fluorocitrate or pharmacological blockade of ionotropic glutamate and ATP receptors mimicked the consequences of a mechanical transection between laminae IV and V. Taken together, the current results indicate that the facilitation of inhibitory synaptic transmission in laminae III–IV of the DH by NA requires functional interlaminar connections between deep and more superficial laminae, and might strongly depend on glia to neuron interactions. These interlaminar connections and glia to neuron interactions could represent interesting targets for analgesic strategies.