Alz-50 recognizes epitopes in primary sensory fibres and in neurons of the substantia gelatinosa of the spinal cord. An ultrastructural study in the rat

Summary The monoclonal antibody Alz-50 has been proposed as a marker for cellular pathological changes in Alzheimer's disease. However, it has been reported that this antibody also reacts with specific epitopes in normal individuals. Furthermore, intense Alz-50 immunoreactivity has been recently described in the hypothalamus and spinal cord of rat and monkey. In the present study, we analysed the distribution pattern of Alz-50 immunostaining in the spinal cord of the adult rat. Using light microscopy, immunostained fibres and varicosities were detected mainly in laminae I-II, although some immunostaining could be detected in deeper laminae. At the ultrastructural level, immunostained axonal varicosities could be detected in lamina I and the outer two thirds of lamina II. The varicosities appeared either scalloped or dome-shaped and contained numerous agranular synaptic vesicles and a few dense-core vesicles. Most varicosities were presynaptic to dendrites. A few immunostained cell bodies and dendrites were also observed, but glial cells were never immunostained. Some ultrathin sections were processed for postembedding immunogold detection of calcitonin gene-related peptide and GABA immunoreactivities. Most of the varicosities which were immunoreactive for Alz-50 also showed calcitonin gene-related peptide immunoreactivity. In contrast, GABA immunoreactivity was never co-localized with Alz-50 immunoreactivity. These results indicate that, in the superficial dorsal horn, the epitope recognized by the Alz-50 antibody is located mainly, but not exclusively, in primary sensory fibres.     

大鼠脊髓后角内甲啡肽标记及非标记结构与C纤维间关系的免疫电镜研究

向大鼠蛛网膜下腔注射辣椒素导致一级传入纤维中的Ｃ纤维变性后，用免疫电镜技术在大鼠脊髓后角浅层内观察到：（１）大量含甲啡肽的轴突终末与未标记树突（或棘）形成对称性（少数为非对称性）突触；（２）含甲啡肽的轴突终未与变性终末共同会聚于同一甲啡肽阴性树突；（３）少量含甲啡肽轴突终末与变性终末间形成轴－轴突触或接触；（４）变性终末与含甲啡肽的树突形成非对称性轴－树突触；（５）甲啡肽阴性轴突终末与变性终末会聚于同一未标记树突并与变性终末间形成轴－轴突触或接触．以上结果表明，在脊髓后角内，脑啡肽除主要以突触后抑制方式调节Ｃ纤维传入外，也可通过轴－轴突触或接触对Ｃ纤维传入进行突触前抑制；非甲啡肽能神经元也能对Ｃ纤维传入进行突出后又突触前抑制．此外，Ｃ纤维在脊髓后角内可通过兴奋性轴－树突触直接影响甲啡肽能中间神经元的活性．     

脊髓侧角区5-HT、TH、SP和L-ENK免疫反应纤维和终末的超微结构

本文应用免疫细胞化学ABC法,在电镜下观察脊髓侧角区单胺能和某些肽能纤维及末梢的突触组合。大鼠侧角内的5-HT、TH、SP和L-ENK免疫反应纤维均为无髓纤维。在侧角细胞簇内,这些纤维穿行于胞体之间,有的与胞体相邻,但很少与胞体形成轴-体突触。这些单胺和肽类纤维也与树突伴行,在树突束内数量最多。有时一小束无髓纤维都含同一种免疫反应物质。轴-树突触是各种免疫反应纤维终末所形成突触的主要形式。各种纤维终末所含的小泡多为圆清亮小泡,或兼有少数大颗粒泡。SP和L-ENK纤维膨体内的小泡与其终末内者不同,大颗粒泡较多,有时约占半数。各种免疫反应终末所组成参与的突触,对称或非对称型均不显现优势。     

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

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

Immunoelectron microscopic analysis of the synaptic connectivity of serotoninergic neurons grafted to the 5,7-dihydroxytryptamine-lesioned rat spinal cord.

The connections between the host and 5-hydroxytryptamine-containing neurons grafted to the spinal cord have been analysed using electron microscopic immunohistochemistry. Adult rats with 5,7-dihydroxytryptamine lesions of the brain and spinal cord received implants of embryonic medullary raphé neurons at three sites in the spinal cord. Eight to 10 months after grafting, the transplanted 5-hydroxytryptamine-positive neurons had formed extensive and complex contacts with spines, dendrites, perikarya and vesicle-containing structures in both the dorsal and ventral horns. Reinnervation of laminae IV-VI was less rich. In the graft itself, connections were also made between non-immunoreactive varicosities and 5-hydroxytryptamine-containing dendrites, and somata, but the exact origin of the afferents was not determined. Outside the implant site, no obvious synaptic junctions onto grafted 5-hydroxytryptamine-immunoreactive boutons were obvious, although labelled and unlabelled varicosities were often in close apposition. Synaptic junctions in the dorsal horn were predominantly symmetric, with the presynaptic varicosity containing mostly small agranular vesicles. By contrast, in the ventral horn most junctions were asymmetric, while the presynaptic element contained both small agranular and large dense-core vesicles. The results demonstrate that the types of synaptic contacts formed between the grafted 5-hydroxytryptamine neurons and the host spinal cord are remarkably similar to those found in intact spinal cord. In addition, the division of morphological differences that exists between 5-hydroxytryptamine-containing boutons in the normal dorsal vs ventral horns is also apparent in the transplanted animals. Finally, there appear to be present several anatomical substrates for the regulation by the host of 5-hydroxytryptamine output from the grafted neurons.     

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

γ-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I–III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I–III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons.

At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals.

These results confirm data obtained withl-glutamate decar☐ylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.     

Distribution of thyrotropin-releasing hormone in the rat spinal cord with special reference to sympathetic nuclei: A light- and electron-microscopic immunocytochemical study

Summary This paper deals with the distribution of thyrotropin-releasing hormone-like immunoreactivity in the spinal cord of the rat, and particularly in the sympathetic nuclei, at light and electron microscopic levels. In the dorsal horn, the inner part of laminae II and III displayed thin thyrotropin-releasing hormone immunoreactive profiles. Electron microscopy revealed small immunoreactive varicosities which made synaptic contact with small dendrites or dendritic spines. Dense thyrotropin-releasing hormone-like immunoreactivity was observed in all sympathetic nuclei (nucleus intermediolateralis pars fascicularis and principalis, nucleus intercalatus and dorsal commissural nucleus) except the nucleus intercalatus pars ependymalis. Electron microscopy showed many immunoreactive varicosities which were often in synaptic contact with dendrites (proximal or distal), rarely with perikarya and never with axons. Sometimes, the same immunoreactive varicosity made axodendritic contacts with two dendrites and, conversely one dendrite was sometimes synaptically contacted by two or more immunoreactive varicosities. The ventral horn displayed a diffuse thyrotropin-releasing hormone-like immunoreactivity except for the cremaster nucleus (at lumbar level) which was densely outlined by immunoreactive profiles. Occasionally a large cell body in lamina IX (a putative motoneuron) was outlined by immunoreactive profiles but ultrastructural studies revealed very few immunoreactive axosomatic synapses, while immunoreactive symmetrical or asymmetrical axodendritic synapses were observed. The present study clearly confirms the existence of thyrotropin-releasing hormone immunoreactive synapses, thus substantiating the physiological role of this hormone in the spinal cord.     

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

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

Fine distribution of substance P-like immunoreactivity in the dorsal nucleus of the vagus nerve in cats

The ultrastructure of substance P (SP)-immunoreactive elements in the cat dorsal motor nucleus of the vagus nerve was examined using pre- and post-embedding immunocytochemical procedures. Substance P-like immunoreactivity was observed in axon terminals and axon fibres which were mostly unmyelinated. Quantitative data showed that at least 16% of axon terminals contained SP. Their mean diameter was larger than that of their non-immunoreactive counterparts. Most (83%) SP-containing terminals were seen to contact dendrites but some were observed adjoining soma or entirely embedded in the cytoplasm of vagal neurons (4.5%). Only 0.5% were observed to contact soma of internuerons. A few immunoreactive axon terminals (4%) were observed in contact with non-immunoreactive axon terminals. Round agranular vesicles and numerous dense core vesicles were visible in most SP-containing axon terminals (84.6%). The immunogold procedure showed the preferential subcellular location of SP to be dense core vesicles. In 32.4% of cases, SP-containing terminals were involved in synaptic contacts that were generally of the asymmetrical Gray type 1 and mainly apposed dendrites. The theoretical total of synaptic contacts was 74.5% and this suggests the existence of weak non-synaptic SP innervation involving approximately 25% of SP-containing axon terminals. No axo-axonic synapses were observed in the dorsal vagal nucleus. These results support the hypothesis that SP found in the dorsal vagal nucleus originates partly from vagal afferents and is involved in direct modulation of visceral functions mediated by vagal preganglionic neurons.     

Endomorphin-2 immunoreactivity in the cervical dorsal horn of the rat spinal cord at the electron microscopic level

Endomorphin-2 is a newly discovered endogenous opioid peptide with high affinity and selectivity for the micro-opioid receptor, and potent analgesic activity, particularly in the spinal cord. Using immunoelectron microscopy, we examined the ultrastructure of the endomorphin-2-like immunoreactive processes and their synaptic relationships in the spinal cord. Endomorphin-2-like immunopositive dense-cored vesicles were observed in many axon terminals, and, in a few cases, were observed together with immunonegative dense-cored vesicles. Immunopositive axons with or without myelination were also observed. The endomorphin-2-like immunoreactive axon terminals formed synapses with both immunopositive and immunonegative processes. Most synapses were asymmetrical, but symmetrical synapses were also found. Examples of axo-dendritic, axo-somatic and axo-axonic contacts were observed.This first demonstration of the ultrastructure and synaptic relationships of endomorphin-2-like immunoreactive axon terminals in the spinal cord dorsal horn provides morphological evidence that this peptide functions as a transmitter regulating pain processes.     

Ultrastructural immunocytochemical study of the dopaminergic innervation of the rat lateral septum with anti-dopamine antibodies

The dopaminergic innervation of the rat lateral septum has been investigated at ultrastructural level by immunocytochemistry using the unlabelled peroxidase-anti-peroxidase method with anti-dopamine antibodies. The specificity of the reaction has been carefully checked by immunological and histochemical controls. A strong immunoreaction was observed in fibres of the lateral septum as well as in their cells of origin in the ventral tegmental area. In the lateral septum, dopamine-immunoreactive fibres were localized in two distinct areas. A first area, located ventrally in the anterior part of the septum was characterized by a high density of immunoreactive varicosities with barely visible intervaricose segments. A more dorsal area, extending throughout the anteroposterior region of the septum, was characterized by immunoreactive fibres in pericellular arrangements. Electron microscopic observations revealed no difference in the ultrastructure of dopamine-immunoreactive profiles in the different areas. Reaction product was found in vesicles, linked to microtubules and in the cytoplasm. Three types of vesicles were seen: (i) small vesicles (30-50 nm) with varying intensity of immunoreaction, filling up the varicosities; (ii) rare large clear vesicles (50-80 nm) with no internal immunoreaction; (iii) very rare large dense vesicles (50-100 nm) with a strong dopamine immunoreactivity. Labelled profiles were observed in clearly defined asymmetrical synaptic contacts with somata and dendrites. Due to the lack of previous work dealing with the use of anti-dopamine antibodies for electron microscope immunocytochemistry, our observations are compared to previous data obtained by more indirect labelling techniques.     

Morphological change of substance P receptor immunoreactive dendrites of preganglionic sympathetic neurons

Substance P (SP)-containing terminals and SP receptors (SPRs) are found on the dendrites of preganglionic sympathetic neurons (PSNs) in the intermedio-lateral nucleus (IML) of the spinal cord. The SP-containing fibers were thought to be of supraspinal origin. However, the primary sensory nerve fibers terminated around PSNs, and some of them directly on PSNs. We observed approximately 150 SPR-immunoreactive (ir) varicosities on the dendrites of PSNs in slices of the first thoracic segment (T1) in control rats. The number of varicosities decreased to 41% 14 days after hemisection of the spinal cord at the fourth cervical segment (C4), and to 55% 14 days after sectioning the spinal dorsal roots at the C8 and T1 levels. The number of varicosities decreased by 33% in 8-week-old rats which had been administered capsaicin subcutaneously within 24 hours after birth to eradicate unmyelinated sensory fibers. However, varicosities increased by 15% 15 minutes after injection of capsaicin into the plantar surface of the front paw to stimulate somatosensory nerve fibers in adult rats. The results demonstrate that SPR-ir varicosities on the dendrites of PSNs were modulated not only by the supraspinal nervous system but also directly by the primary sensory nerve terminals.     

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.     

Immunocytochemical evidence for calcitonin gene-related peptide-like neurons in the dorsal horn and lateral spinal nucleus of the rat cervical spinal cord

Calcitonin gene-related peptide (CGRP) in the dorsal horn of the rat spinal cord was assumed until now to be principally of primary afferent origin. It is shown here, on the basis of both light and electron microscopic immunocytochemical evidence, that some cell bodies of the dorsal horn and lateral spinal nucleus (LSn) of the rat cervical spinal cord contain a CGRP-like immunoreactivity. At the light microscopic level, immunoreactive cell bodies were observed in animals pretreated with colchicine injected intraventricularly, CGRP-like cell bodies were morphologically heterogeneous and distributed in the three superficial layers of the dorsal horn. They were very rare in lamina I and more numerous in laminae II and III. A group of immunoreactive cell bodies was also observed in the LSn. Using electron microscopic techniques, a few immunoreactive cell bodies were observed even in control animals. In addition, relatively numerous immunoreactive dendrites were observed in lamina II. The specificity of the reaction and the physiological implications of the results are discussed.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.     

Identification of dorsal root synaptic terminals on monkey ventral horn cells by electron microscopic autoradiography

Summary The projection of dorsal root fibres to the motor nucleus of the macaque monkey spinal cord has been examined utilizing light and electron microscopic autoradiography. Light microscopy demonstrates a very sparse labelling of primary afferent fibres in the ventral horn. Silver grains overlying radioactive sources are frequently clustered into small groups, often adjacent to dendritic profiles. Under the electron microscope, myelinated axons and a few large synaptic profiles containing rounded synaptic vesicles were overlain by numerous silver grains. These labelled profiles made synaptic contact with dendrites 1–3 m in diameter. The labelled profiles did not contact cell bodies or large proximal dendrites of ventral horn neurons. Frequently, small synaptic profiles containing flattened vesicles were presynaptic to the large labelled terminals and it is suggested that these axoaxonal synapses may mediate presynaptic inhibition of the primary afferent fibres. The relationship of the present findings to previously published physiological and anatomical studies is discussed.     

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

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

大鼠孤束核内5－羟色胺能轴突终末的突触联系

用顺行演变与免疫组织化学结合的双标技术，电镜下观察大鼠孤束核内５－羟色胺样免疫反应（５－ＨＴ－ＬＩ）轴突终末的突触联系，特别是与迷走神经初级传入（溃变）终末的关系。发现：１．５－ＨＴ－ＬＩ轴突终末和迷走神经初级传入终末终止于同一个树突形成轴－树突触；２．５－ＨＴ－ＬＩ轴突终末与树突形成轴－树突触；３．５－ＨＴ－ＬＩ轴突终末与迷走神经初级传入终末或非标记的轴突终未形成轴一轴相贴（ａｘｏ－ａｘｏｎｉｃ－ｃｏｎｔａｃｔ）。以上结果提示，孤束核内的５－ＨＴ能神经成分可能通过突触后、突触前机制调控经迷走神经传入的信息。     

Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey

Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18-25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus.