Topographically organized projections from the nucleus subceruleus to the hypoglossal nucleus in the rat: a light and electron microscopic study with complementary axonal transport techniques.

Projections from the nucleus subceruleus (nSC) to the hypoglossal nucleus (XII) were investigated with complementary retrograde and anterograde axonal transport techniques at the light and electron microscopic level in the rat. Injections of WGA-HRP into XII resulted in labeling of neurons in and around the nSC. Labeled nSC neurons were few in number (less than 4 per 40-60 microns sections) and variable in size and shape. Most labeled nSC neurons were medium-sized (mean = 16.89 microns), fusiform, triangular, or oval, with 3-4 dendrites typically oriented dorsomedially and ventrolaterally. These neurons were found throughout the rostrocaudal extent of the nSC but were most numerous medial, dorsomedial, and ventromedial to the motor trigeminal nucleus. Others were observed rostral to the motor trigeminal nucleus and ventral to the parabrachial nuclear complex. Confirmation of retrograde results was obtained following injections of tritiated amino acids or WGA-HRP into the nSC. This resulted in labeling throughout the rostrocaudal extent of XII mainly ipsilaterally. Labeled fibers descended the brainstem in the dorsolateral and, to a lesser extent, in the ventromedial component of Probst's tract. Fibers entered XII mainly rostrally along the lateral border of the nucleus. All regions of XII were recipients of nSC afferents, but the caudoventromedial quadrant contained the greatest density of terminal labeling. Electron microscopic evaluation confirmed that nSC afferents synapsed on motoneurons in XII. Axon terminals containing WGA-HRP reaction product were found contacting dendrites and somata, but primarily the former (81.3% versus 10.6%). Axodendritic terminals synapsed mainly on medium-to-small sized dendrites (less than 3 microns in diameter). The majority of labeled axodendritic terminals (90.1%) contained small, round, and clear synaptic vesicles (S-type: 20-50 nm) and were associated with an asymmetric (60.6%), symmetric (11.4%), or no (18%) postsynaptic specialization. By contrast, most axosomatic terminals contained flattened vesicles (F-type) and formed a symmetric or no postsynaptic specialization (75%). Large dense core vesicles (55-90 nm) were observed within a small proportion of all labeled axon terminals (1.3%). The results from this study demonstrate that the nSC projects to XII, preferentially targets a specific subgrouping of protrusor motoneurons, and synapses on both somata and dendrites, although mainly on the latter. The implications of these data are discussed relative to tongue control.     

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.     

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of tyrosine hydroxylase in rat nucleus accumbens

Immunocytochemical localization of tyrosine hydroxylase (TH) was used to determine the ultrastructural morphology and synaptic associations of catecholaminergic terminals in the nucleus accumbens of the rat. The brains were fixed by vascular perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde. Coronal sections cut with a vibrating microtome were incubated with rabbit antiserum to TH then immunocytochemically labeled by the peroxidase-antiperoxidase method. Immunoreactivity for the enzyme was found within unmyelinated axons and axon terminals. These terminals contained either all small clear or combined small clear and large dense core vesicles. Approximately 40% of the labeled terminals formed symmetric synapses with unlabeled proximal or distal dendritic shafts. The dendrites showed a spare distribution of spines. Axosomatic synapses and axonal associations of the TH-containing terminals also were detected. The recipient perikarya were usually 10-20 micrometers in diameter and contained an indented nucleus and abundant cytoplasm. The content of large dense vesicles and synaptic associations with somata and proximal dendrites suggest that a certain proportion of the TH-containing terminals within the nucleus accumbens are morphologically distinct from catecholaminergic terminals within the dorsal striatum. These differences are discussed in relation to neuropeptides and functions of the dopaminergic mesolimbic and nigrostriatal pathways.     

GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry

Previous light microscopic immunoperoxidase studies of glutamic acid decarboxylase (GAD)-immunoreactive neural elements in the rat basilar pontine nuclei revealed immunocytochemical reaction product in neuronal somata and axon terminals. In the present study, pre-embedding immunoperoxidase labeling of GAD or gamma-aminobutyric acid (GABA) and postembedding immunogold labeling of GABA allowed the ultrastructural visualization of these neural elements in the basilar pontine nuclei of colchicine-treated animals. At the electron microscopic level, immunolabeled neuronal somata exhibited smoothly contoured nuclei, whereas some dendrites also contained reaction product after immunocytochemical treatment and were postsynaptic to both immunoreactive and nonimmunoreactive axon terminals. Synaptic boutons immunoreactive for GAD or GABA exhibited cross-sectional areas that ranged from 0.1 to 3.8 microns 2 and generally appeared round or elongated in most sections. The majority (95%) of immunolabeled boutons contained pleomorphic synaptic vesicles and formed symmetric synapses at their postsynaptic loci; however, boutons exhibiting round vesicles and boutons forming asymmetric synapses (5%) were also immunopositive. Small (less than 1.5 microns 2) GAD- or GABA-labeled axon terminals formed synaptic contact mainly with small dendritic profiles, dendritic spines, and neuronal somata, whereas large labeled boutons (greater than 1.5 microns 2) formed synapses with all sizes of dendritic profiles. Occasionally, a single immunolabeled bouton formed synaptic contact with two separate postsynaptic dendrites. It is suggested that the immunolabeled neuronal somata and dendrites observed in the rat basilar pontine nuclei represent a population of pontine local circuit neurons; however, it is known that GABAergic cell groups extrinsic to the pontine gray provide afferent projections to the basilar pons, and therefore at least some immunoreactive axon terminals present in the pontine nuclei are derived from these extrinsic sources. The ultrastructural observation of GABAergic neural elements in the rat basilar pontine nuclei confirms previous light microscopic findings and provides an anatomical substrate through which GABAergic neurons, whether arising from an intrinsic or extrinsic source, might exert an inhibitory influence on target cells within the pontine nuclei.     

The lateral reticular nucleus of the opossum (Didelphis virginiana). I. Conformation,cytology and synaptology

When viewed in Nissl preparations, the lateral reticular nucleus (LRN) of the opossum can be divided into three subgroups: a medial internal portion, a lateral external portion and a rostral trigeminal division. Neurons within the internal division measure 13-45 μ in their greatest dimension whereas those within the external and trigeminal portions measure 11-32 μ and 14-27 μ respectively. Golgi impregnations reveal that many neurons in all three subdivisions display a radial dendritic pattern although some of the nerve cells within the external division have dendrites which orient mainly in a ventromedial to dorsolateral direction. The cell bodies of LRN neurons are relatively spine-free. However, a small percentage of neurons exhibit clusters of sessile spines on proximal and more distal dendritic segments. No locally ramifying axons or axon collaterals were found within the LRN. Synaptic terminals within the LRN were divided into four categories: (1) small terminals measuring 2.5 μ or less containing agranular spherical vesicles; (2) small terminals (2.5 μ or less) with agranular pleomorphic synaptic vesicles, i.e., a mixture of spherical and elliptical synaptic vesicles; (3) small terminals (2.5 μ or less) containing agranular spherical or pleomorphic vesicles with a variable number (4-27) of dense core vesicles; and (4) large terminals (greater than 2.5 μ) which contain agranular spherical synaptic vesicles and a variable number of dense core vesicles (1-17). Dendritic diameters were measured from Golgi impregnations and correlated with cross-sectioned profiles in electron micrographs to help determine the post-synaptic distribution of synaptic endings. Small terminals containing agranular spherical or pleomorphic synaptic vesicles contact the soma and entire dendritic tree in each portion of the nucleus, whereas the small terminals containing dense core vesicles are usually located on distal dendrites or spines. Some large terminals make multiple synaptic contacts with a cluster of spines, others contact groups of small (distal) dendrites. In order to identify two of the major afferent systems to the LRN, 15 adult opossums were subjected to either a cervical spinal cord hemisection or a stereotaxic lesion of the red nucleus. Two days subsequent to spinal hemisection, large terminals in the caudal part of the ipsilateral LRN exhibit either an electron dense or filamentous reaction. Their postsynaptic loci are spines and shafts of proximal dendrites or a number of distal dendrites and spines. In addition, small terminals containing spherical agranular synaptic vesicles undergo an electron dense reaction in the same areas. Their postsynaptic loci are proximal or distal dendrites. Two days subsequent to rubral lesions, small terminals containing agranular spherical synaptic vesicles undergo a dark reaction in rostral portions of the contralateral nucleus. They contact intermediate or distal dendrites and occasionally spines.     

Ultrastructure of the mammillotegmental projections to the ventral tegmental nucleus of Gudden in the rat.

This study examines the termination pattern of axons from the medial mammillary nucleus within the ventral tegmental nucleus of Gudden (TV) in rats by using anterograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) and visualized with tetramethylbenzidine. The neuropil of TV contains three classes of axodendritic terminals, that is, terminals containing round, flat, and pleomorphic synaptic vesicles. These types make up 55.6%, 26.1%, and 18.3%, respectively, of all normal axodendritic terminals. Injection of WGA-HRP into the medial mammillary nucleus permits ultrastructural recognition of anterogradely labeled terminals within the TV. More than 80% of the labeled terminals contain round synaptic vesicles and form asymmetric synaptic contacts, whereas about 16% contain flat synaptic vesicles with symmetric synaptic contacts. There are a few labeled terminals with pleomorphic vesicles and only a few axosomatic terminals. Almost all labeled terminals are small, having diameters of less than 1.5 microns. Compared with the distributions of normal and labeled terminals with round vesicles, there is an increase of the percentage of labeled terminals with round vesicles on the intermediate dendrites (1-2 microns diameter) and a decrease on the distal dendrites (less than 1 micron diameter). Anterogradely labeled axon terminals often contact retrogradely labeled dendrites. These results suggest that the medial mammillary neurons send mainly excitatory as well as a few inhibitory inputs to the dendrites of TV and have direct reciprocal contacts with the TV neurons.     

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [³H]5-HT. The [³H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [³H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [³H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 μm in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [³H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.     

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla of the rat. I. Normal ultrastructure

The electron microscopic localization of the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was examined in the rostral ventrolateral medulla (RVL) of adult rats. The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were immunocytochemically labeled using a rabbit polyclonal antiserum to PNMT and the peroxidase-antiperoxidase method. A semi-quantitative ultrastructure analysis revealed that the perikarya constituted 9% of the total immunoreactive profiles observed in the RVL. The labeled somata were large (18-24 microns) and were characterized by an indented nucleus and abundant cytoplasm with numerous mitochondria. An average of 136.8 +/- 11.6 mitochondria were present per 100 microns2 cytoplasm, which is 38% greater than the numbers found for PNMT-immunoreactive neurons in the nucleus of the solitary tract. Moreover, the labeled somata were often found in direct apposition to the basal lamina of small capillaries and neighboring astrocytic processes. The remaining labeled profiles were neuronal processes of which 72% were dendrites. Both the PNMT-labeled somata and dendrites received primarily symmetric contacts from unlabeled axon terminals. Only a few axons and terminals containing immunoreactivity for PNMT were observed. The axons were both unmyelinated and myelinated. The PNMT-immunoreactive terminals were characterized by a mixed population of vesicles and by the formation of synaptic junctions with both unlabeled dendrites and PNMT-labeled perikarya and dendrites. The ultrastructural morphology and proximity to blood vessels and glia suggest a high metabolic activity and possibly a chemosensory function of PNMT neurons in the RVL. The existence of myelinated and unmyelinated axons could imply that PNMT-containing neurons have different conduction velocities in efferent pathways to the spinal cord or other brain regions. Furthermore, the multiple types of synaptic interactions between labeled and unlabeled axons and dendrites support the concept that adrenergic neurons modulate and are modulated by neurons containing the same or other putative transmitters in the RVL.     

Comparison of the ultrastructure of cortical and retinal terminals in the rat dorsal lateral geniculate and lateral posterior nuclei

We compared the ultrastructure and synaptic targets of terminals of cortical or retinal origin in the rat dorsal lateral geniculate nucleus (LGN) and lateral posterior nucleus (LPN). Following injections of biotinylated dextran amine (BDA) into cortical area 17, two types of corticothalamic terminals were labeled by anterograde transport. Type I terminals, found throughout the LGN and LPN, were small, drumstick-shaped terminals that extended from thin axons. At the ultrastructural level in both the LGN and LPN, labeled type I corticothalamic terminals were observed to be small profiles that contained densely packed round vesicles (RS profiles) and contacted small-caliber dendrites. In tissue stained for gamma amino butyric acid (GABA) using postembedding immunocytochemical techniques, most dendrites postsynaptic to type I corticothalamic terminals did not contain GABA (97%). Type II corticothalamic terminals, found only in the LPN, were large terminals that sometimes formed clusters. At the ultrastructural level, type II terminals were large profiles that contained round vesicles (RL profiles) and contacted large-caliber dendrites, most of which did not contain GABA (98%). Retinogeniculate terminals, identified by their distinctive pale mitochondria, were similar to type II corticothalamic terminals except that 26% of their postsynaptic targets were vesicle-containing profiles that contained GABA (F2 profiles). Our results suggest that type I corticothalamic terminals are very similar across nuclei but that the postsynaptic targets of RL profiles vary. Comparison of the responses to retinal inputs in the LGN and to layer V cortical inputs in the LPN may provide a unique opportunity to determine the function of interneurons in the modulation of retinal signals and, in addition, may provide insight into the signals relayed by cortical layer V.     

Direct catecholaminergic innervation of spinal dorsal horn neurons with axons ascending the dorsal columns in cat

Previous ultrastructural studies have shown that catecholamine-containing nerve terminals in the spinal dorsal horn form synaptic junctions with dendrites and somata, but the identity of the neurons giving rise to these structures is largely unknown. In this study we have investigated the possibility that spinomedullary neurons, which project through the dorsal columns to the dorsal column nuclei, are synaptic targets for descending catecholaminergic axons. Neurons with axons ascending the dorsal columns were retrogradely labelled after uptake of horseradish peroxidase by their severed axons in the thoracic (T10–T12) or cervical (C2–C3) dorsal columns. After the retrogradely labelled neurons were visualized, the tissue was immunocytochemically stained with antisera raised against tyrosine hydroxylase or dopamine-β-hydroxylase. Three hundred forty-three retrogradely labelled neurons within laminae III–V of the lumbosacral dorsal horn were examined under high power with the light microscope. In Triton X-100 treated material, over 60% of cells were found to have dopamine-β-hydroxylase-immunoreactive varicosities closely apposed to their somata and proximal dendrites. The number of contacts per cell varied from 1 to 22, with a mean number of 4.5. Fewer cells (34%) received contacts from axons immunoreactive for tyrosine hydroxylase as a consequence of the weaker immunoreaction produced by this antiserum. Correlated light and electron microscopic analysis confirmed that many of these contacts were regions of synaptic specialization and that immunostained boutons contained pleomorphic (round to oval) agranular vesicles together with several dense core vesicles. These observations suggest that catecholamines regulate sensory transmission through this spinomedullary pathway by a direct postsynaptic action upon its cells of origin. Such an action would be predicted to suppress transmission generally through this pathway. © 1993 Wiley-Liss, Inc.     

Quantitative immunoelectron microscopic colocalization of GABA and enkephalin in the ventrocaudal periaqueductal gray of the rat

In the present ultrastructural study in the ventrocaudal periaqueductal gray (PAG) of the rat, the relationship and the association between GABAergic and enkephalinergic neuronal elements were investigated using postembedding colocalization immunogold electron microscopic technique in order to establish the precise relationship between these two important neurotransmitters in this part of the brain stem. The GABA-like neuronal elements were immunoreacted with 20 nm gold particles and the enkephalin (ENK)-like immunoreactive neurons were labeled with 10 nm gold particles. Double labeling of sections with ENK and GABA produced colocalization in 23.3% and 1.2% of axon terminals and dendrites, respectively. Most of the double-labeled terminals contained more GABA-like than ENK-like immunolabeling. Approximately 19.4% of the labeled axon terminals and 8.5% of the labeled dendrites contained only GABA-like immunoreactivity, while 24% of the immunolabeled dendrites were immunoreactive with only ENK-like immunoreactivity. The synapses between the two kinds of immunolabeled neuronal profiles appear to be both asymmetrical and symmetrical. GABA-like immunolabeled terminals contained small, clear, pleomorphic or round vesicles and were found to make synapses with ENK-like immunolabeled and nonimmunolabeled dendrites, whereas most of the ENK-like immunolabeled axon terminals contained dense-cored vesicles. Approximately half of the axon terminals (51%) and dendrites (56%) in the ventrolateral PAG were not labeled for either GABA or for ENK immunoreactivity. The results are discussed in terms of GABAergic inhibition of antinociceptive mechanisms in the ventrolateral PAG and of the activation of these mechanisms by ENK neurotransmitter.     

Topography and synaptology of mamillary body projections to the mesencephalon and pons in the rat.

The anterograde and retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was used to study the anatomical organization of descending projections from the mamillary body (MB) to the mesencephalon and pons at light and electron microscopic levels. Injections of WGA-HRP into the medial mamillary nucleus resulted in dense anterograde and retrograde labeling in the ventral tegmental nucleus, while injections in the lateral mamillary nucleus resulted in dense anterograde labeling in the dorsal tegmental nucleus pars dorsalis and dense anterograde and retrograde labeling in the pars ventralis of the dorsal tegmental nucleus. Anterogradely labeled fibers in the mamillotegmental tract diverged from the principal mamillary tract in an extensive dorsocaudally oriented swath of axons which extended to the dorsal and ventral tegmental nuclei, and numerous axons turned sharply ventrally and rostrally to terminate topographically in the dorsomedial nucleus reticularis tegmenti pontis and rostromedial pontine nuclei. The anterograde labeling in these two precerebellar relay nuclei was distributed near the midline such that projections from the lateral mamillary nucleus terminated mainly dorsomedial to the terminal fields of projections from the medial mamillary nucleus. In the dorsal and ventral tegmental nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites and to a lesser extent with neuronal somata. A few labeled terminals contained pleomorphic vesicles and formed symmetric synaptic junctions with dendrites and neuronal somata. Labeled axon terminals were also frequently found in synaptic contact with retrogradely labeled dendrites and neuronal somata in the dorsal and ventral tegmental nuclei. These findings indicate that neurons in the dorsal and ventral tegmental nuclei are reciprocally connected with MB projection neurons. In the nucleus reticularis tegmenti pontis and medial pontine nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites. The present study demonstrates that projections from the medial and lateral nuclei of the MB are topographically organized in the mesencephalon and pons. The synaptic morphology of mamillotegmental projections suggests that they may have excitatory influences primarily on the distal dendrites of neurons in these brain regions.     

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.     

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.     

Projections from auditory cortex to the cochlear nucleus in rats: Synapses on granule cell dendrites

Previous work has demonstrated that layer V pyramidal cells of primary auditory cortex project directly to the cochlear nucleus. The postsynaptic targets of these centrifugal projections, however, are not known. For the present study, biotinylated dextran amine, an anterograde tracer, was injected into the auditory cortex of rats, and labeled terminals were examined with light and electron microscopy. Labeled corticobulbar axons and terminals in the cochlear nucleus are found almost exclusively in the granule cell domain, and the terminals appear as boutons (1–2 μm in diameter) or as small mossy fiber endings (2–5 μm in diameter). These cortical endings contain round synaptic vesicles and form asymmetric synapses on hairy dendritic profiles, from which thin (0.1 μm in diameter), nonsynaptic “hairs” protrude deep into the labeled endings. These postsynaptic dendrites, which are typical of granule cells, surround and receive synapses from large, unlabeled mossy fiber endings containing round synaptic vesicles and are also postsynaptic to unlabeled axon terminals containing pleomorphic synaptic vesicles. No labeled fibers were observed synapsing on profiles that did not fit the characteristics of granule cell dendrites. We describe a circuit in the auditory system by which ascending information in the cochlear nucleus can be modified directly by descending cortical influences. © 1996 Wiley-Liss, Inc.     

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla. II. Synaptic relationships with GABAergic terminals

The ultrastructural morphology of terminals synthesizing gamma-aminobutyric acid (GABA), as indicated by peroxidase immunoreactivity for its synthetic enzyme L-glutamate decarboxylase (GAD), was examined in the rostral ventrolateral medulla (RVL) of the adult rat brain. The objective of the study was to determine the types of synaptic associations between the GABAergic terminals and other neurons in the RVL, particularly the C1-adrenergic neurons containing phenylethanolamine N-methyltransferase (PNMT). The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were singly labeled with a sheep antiserum to GAD using the peroxidase-antiperoxidase (PAP) method. Additional sections were dually labeled using the PAP technique for the GAD antiserum and immunogold labeling for a rabbit antiserum against PNMT. Ultrastructural analysis revealed that peroxidase labeling for GAD was localized primarily to axons and axon terminals in both single and dual labeled material. The axons were small and unmyelinated. The GAD-labeled terminals were 0.5-2.0 microns in diameter and contained a large population of small clear vesicles usually associated with a few mitochondria. These terminals formed synapses with many dendrites, a few nerve cell bodies and axon terminals. The junctions were all symmetric and the postsynaptic structures failed to exhibit immunoreactivity when processed only for GAD labeling. In sections incubated with both GAD and PNMT antisera, the peroxidase-labeled GABAergic terminals formed symmetric synapses with nerve cell bodies and dendrites showing immunogold labeling for PNMT. In addition, the GAD-labeled terminals were presynaptic to other dendrites which appeared to have equal access to the antisera and gold markers, but failed to exhibit detectable immunoreactivity for PNMT. Both the PNMT-labeled and unlabeled somata and dendrites also received symmetric and asymmetric contacts from terminals containing neither GAD nor PNMT-immunoreactivity. We conclude that GABA is at least one of the inhibitory transmitters regulating adrenergic as well as non-adrenergic outflow from the RVL.     

Synaptic connections of a periodontal primary afferent neuron within the subnucleus oralis of the cat

The central axon of a primary afferent neuron that responded to light mechanical stimulation of the lower premolar teeth in a fast adapting fashion was intra-axonally injected with horseradish peroxidase in the cat. The labeled terminals within the rostrodorsomedial (Vo.r) and dorsomedial (Vo.dm) parts of subnucleus oralis were examined electron microscopically. The labeled ending had pale axoplasm, contained clear spherical synaptic vesicles, and formed multiple synapses with dendrites and/or unlabeled axonal endings with pleomorphic vesicles (P-endings). In these synaptic contacts, the labeled primary ending was presynaptic to dendrites and postsynaptic to P-endings. Labeled endings simultaneously synapsing with both dendrites and P-endings were more frequent in Vo.dm (28%) than in Vo.r (8.3%).     

Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.     

Dual ultrastructural immunocytochemical labeling of μ and δ opioid receptors in the superficial layers of the rat cervical spinal cord

The δ opioid receptor (DOR) and μ opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2–C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and <1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.