The adrenergic innervation of the rat central amygdaloid nucleus: a light and electron microscopic immunocytochemical study using phenylethanolamine N-methyltransferase as a marker

Using immunocytochemistry of phenylethanolamine N-methyltransferase for light and electron microscopy, investigations were carried out to document the morphology of adrenergic afferents innervating the rat central amygdaloid nucleus and to analyse the manner in which contacts with neurons of the nucleus are formed. With the light microscope, dense terminal plexus of phenylethanolamine N-methyltransferase-immunoreactive axons with typical large boutons (diameter>1 m) were found in the medial central nucleus, extending into its ventral subdivision and the adjacent intra-amygdaloid portion of the bed nucleus of the stria terminalis. Electron microscopy of the medial central nucleus showed phenylethanolamine N-methyltransferase-immunoreaction product in the cytoplasm of intervaricose axons and boutons. Large adrenergic boutons contained numerous small clear vesicles and, occasionally, large dense-cored vesicles. In serial sections, most boutons formed synaptic contacts. Synapses of immunoreactive terminals were mainly of the asymmetric type and localized preferentially on medium sized to small dendrites and dendritic spines. Structures postsynaptic to adrenergic boutons were often additionally contacted by non-labelled terminals. The study gives evidence that adrenergic afferents exert a direct synaptic influence on medial central nucleus neurons. The peripheral localization of the majority of adrenergic synapses, their asymmetric configuration, and the presence of non-adrenergic synapsing terminals in their immediate vicinity indicate that the major component of the adrenergic input is of an excitatory nature, and is integrated with innervation from other sources.Abbreviations BNST _i Intra-amygdaloid portion of the bed nucleus of the stria terminalis - CL lateral central amygdaloid nucleus - CLc lateral capsular central amygdaloid nucleus - CM medial central amygdaloid nucleus - CN central amygdaloid nucleus - CV ventral central amygdaloid nucleus - DAB diaminobenzidine - EM electron microscopy - ir immunoreactive - LM light microscopy - NPY neuropeptide tyrosine - PBS phosphate buffered saline - PFA paraformaldehyde - PNMT phenylethanolamine N-methyltransferase     

Interaction between adrenergic fibers and intermediate cholinergic neurons in the rat spinal cord: a new double-immunostaining method for correlated light and electron microscopic observations

Relationships between cholinergic neurons and adrenergic fibers in the intermediate region of the rat thoracic spinal cord were examined using a new immunohistochemical double-staining method for light and electron microscopic observations. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase and stained bluish green by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products using beta-galactosidase as a marker. On the same sections, adrenergic fibers were labeled by a polyclonal antiserum to phenyl-ethanolamine-N-methyltransferase and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in the light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the four discrete areas of the intermediate region: the principal intermediolateral nucleus, the central autonomic nucleus, the intercalated nucleus and the funicular intermediolateral nucleus. These cell groups seemed to be connected to each other by their processes, and they showed a "ladder-like appearance" as a whole. Phenylethanolamine-N-methyltransferase-like immunoreactive fibers were present only along this "ladder-like structure" and were the most rich in the principal intermediolateral nucleus. In the electron microscope, some of the choline acetyltransferase-like immunoreactive neurons, which were identified by light micrographs, were found to receive synaptic inputs from phenylethanolamine-N-methyltransferase-like immunoreactive boutons in the principal intermediolateral nucleus. These findings suggest that the adrenergic axons in the principal intermediolateral nucleus directly affect the activity of the cholinergic preganglionic sympathetic neurons.     

Electron microscopic identification of reticulo-subthalamic axon terminals in the cat

Synaptic boutons emanating from axons of nucleus tegmenti pedunculopontinus origin were identified by electron microscopy in the neuropil of the subthalamic nucleus. Such boutons measure 1.5-3 microns, contain round synaptic vesicles and make asymmetrical axodendritic and axosomatic synaptic contacts with large subthalamic neurons. Very few contacts with vesicle-containing dendrites, and no contacts with the perikarya of the small neurons were observed. The present findings, in keeping with the relevant light microscopic and electrophysiologic data, furnish evidence for a substantial bilateral tegmenti pedunculopontinofugal projection that excites monosynaptically the relay subthalamic neurons.     

A GABA immunocytochemical study of rat motor thalamus: light and electron microscopic observations.

A light and electron microscopic study of GABA-immunoreactive neurons and profiles in the ventroanterior-ventrolateral and ventromedial nuclei of rat dorsal thalamus was conducted using antiserum raised against GABA. Less than 1% of the neurons in these motor-related nuclei exhibited GABA immunoreactivity, confirming previous reports that these nuclei are largely devoid of interneurons. Immunoreactive neurons in the ventral anterior-ventral lateral complex and ventromedial nucleus were bipolar or multipolar in shape, and tended to be smaller than non-immunoreactive neurons. GABA immunoreactivity in the neuropil consisted of labeled axon terminals and myelinated and unmyelinated axons, and was lower in the ventral anterior-ventral lateral complex and ventromedial nucleus than in neighboring thalamic nuclei. The density of neuropil immunolabeling was slightly higher in ventral anterior-ventral lateral complex than in ventromedial nucleus. GABA-immunoreactive axon terminals, collectively termed MP boutons for their medium size and pleomorphic vesicles (and corresponding to "F" profiles of some previous studies of thalamic ultrastructure), formed symmetric synapses and puncta adhaerentia contacts predominantly with large and medium-diameter (i.e. proximal) non-immunoreactive dendrites. Approximately 12 and 18% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, were GABA-immunopositive. Many of these immunoreactive profiles probably arose from GABAergic neurons in the thalamic reticular nucleus, substantia nigra pars reticulata and entopeduncular nucleus. Two types of non-immunoreactive axon terminals were distinguished based on differences in morphology and synaptic termination sites. Boutons with small ovoid profiles and round vesicles that formed prominent asymmetric synapses onto small-diameter dendrites were observed. Mitochondria were rarely observed within these boutons, which arose from thin unmyelinated axons. These boutons composed approximately 82 and 74% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, and were considered to arise predominantly from neurons in the cerebral cortex. In contrast, boutons with large terminals that contained round or plemorphic vesicles and formed multiple asymmetric synapses predominantly with large-diameter dendrites were also observed. Puncta adhaerentia contacts were also common. Mitochondria were numerous within large boutons with round vesicles, which arose from myelinated axons. Many of the large boutons were likely to have originated from neurons in the cerebellar nuclei. Approximately 6% of the boutons in the ventral anterior-ventral lateral complex and 8% in ventromedial nucleus were of the large type.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuronal and synaptic organization of the centromedian nucleus of the monkey thalamus: a quantitative ultrastructural study, with tract tracing and immunohistochemical observations

Summary The ultrastructure of the centromedian nucleus of the monkey thalamus was analysed qualitatively and quantitatively and projection neurons, local circuit neurons, and synaptic bouton populations identified. Projection neurons were mostly medium-sized, with oval-fusiform or polygonal perikarya, few primary dendrites, and frequent somatic spines; local circuit neurons were smaller. Four basic types of synaptic boutons were distinguished: (1) Small- to medium-sized boutons containing round vesicles (SR) and forming asymmetric contacts, identified as corticothalamic terminals. (2) Heterogeneous medium-sized boutons with asymmetric contacts and round vesicles, similar to the so-called large round (LR) boutons, which were in part of cortical origin. (3) Heterogeneous GAD-positive small- to medium-sized boutons, containing pleomorphic vesicles and forming symmetric contacts (F1 type), which included pallidothalamic terminals. (4) Presynaptic profiles represented by GAD-positive vesicle-containing dendrites of local circuit neurons. Complex synaptic arrangements, serial synapses and triads with LR and SR boutons engaging all parts of projection neuron dendrites and somata, were seen consistently, whereas classical glomeruli were infrequent. LR and SR boutons also established synapses on dendrites of local circuit neurons. F1 boutons established synapses on projection neuron somata, dendrites and initial axon segments. Compared to other previously studied motor-related thalamic nuclei, differences in synaptic coverage between proximal and distal projection neuron dendrites were less pronounced, and the density of synapses formed by local circuit dendrites on projection neuron dendrites was lower. Thus, compared to other thalamic nuclei, the overlap of different inputs was higher on monkey centromedian cells, and centromedian inhibitory circuits displayed a different organization.     

Ultrastructural features of non-commissural GABAergic neurons in the medial vestibular nucleus of the monkey.

The ultrastructural characteristics of non-degenerating GABAergic neurons in rostrolateral medial vestibular nucleus were identified in monkeys following midline transection of vestibular commissural fibers. In the previous papers, we reported that most degenerated cells and terminals in this tissue were located in rostrolateral medial vestibular nucleus, and that many of these neurons were GABA-immunoreactive. In the present study, we examined the ultrastructural features of the remaining neuronal elements in this medial vestibular nucleus region, in order to identify and characterize the GABAergic cells that are not directly involved in the vestibular commissural pathway related to the velocity storage mechanism. Such cells are primarily small, with centrally-placed nuclei. Axosomatic synapses are concentrated on polar regions of the somata. The proximal dendrites of GABAergic cells are surrounded by boutons, although distal dendrites receive only occasional synaptic contacts. Two types of non-degenerated GABAergic boutons are distinguished. Type A terminals are large, with very densely-packed spherical synaptic vesicles and clusters of large, irregularly-shaped mitochondria with wide matrix spaces. Such boutons form symmetric synapses, primarily with small GABAergic and non-GABAergic dendrites. Type B terminals are smaller and contain a moderate density of round/pleomorphic vesicles, numerous small round or tubular mitochondria, cisterns and vacuoles. These boutons serve both pre- and postsynaptic roles in symmetric contacts with non-GABAergic axon terminals. On the basis of ultrastructural observations of immunostained tissue, we conclude that at least two types of GABAergic neurons are present in the rostrolateral portion of the monkey medial vestibular nucleus: neurons related to the velocity storage pathway, and a class of vestibular interneurons. A multiplicity of GABAergic bouton types are also observed, and categorized on the basis of subcellular morphology. We hypothesize that "Type A" boutons correspond to Purkinje cell afferents in rostrolateral medial vestibular nucleus, "Type B" terminals represent the axons of GABAergic medial vestibular nucleus interneurons, and "Type C" boutons take origin from vestibular commissural neurons of the velocity storage pathway.     

Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphé nuclei and sympathetic preganglionic neurons in the rat spinal cord

Summary Electrophysiological and anatomical studies have suggested the existence of a pathway between the caudal raphé nuclei and regions of the spinal cord containing the sympathetic preganglionic neurons. However synaptic connections between cells in the raphé nuclei and identified sympathetic preganglionic neurons have not yet been shown. We have used a combination of anterograde tracing using Phaseolus vulgaris leucoagglutinin (PHA-L), retrograde tracing using a conjugate of cholera B chain and HRP and electron microscopy to look for such a pathway in rats. When PHA-L had been injected into the regions mainly restricted to the raphé pallidus and raphé magnus, synaptic contacts were found between PHA-L containing terminals and preganglionic neurons retrogradely labelled from the adrenal medulla. Out of the 43 synaptic contacts analysed, 26 were onto somata and 14 onto dendrites. 75% of the total appeared to have symmetric membrane specialisations, 20% asymmetric and the remainder could not be classified. Synaptic contacts were not seen in an animal in which the PHA-L injection site involved cells in the ventral raphé obscurus and surrounding gigantocellular reticular formation. These findings provide evidence of the existence of a direct monosynaptic pathway between cells in the raphé pallidus and/or caudal raphé magnus, and identified sympathetic preganglionic neurons and give further support for a role for the caudal raphé nuclei in sympathetic autonomic regulation.     

A GABAergic projection from the central nucleus of the amygdala to the parabrachial nucleus: an ultrastructural study of anterograde tracing in combination with post-embedding immunocytochemistry in the rat

To determine whether axonal terminals emanating from the central nucleus of amygdala (Ce) to the parabrachial nucleus (PBN) contain gamma-aminobutyric acid (GABA) as their neurotransmitter, an electron microscopic study was performed employing the combined techniques of WGA-HRP anterograde tracing and post-embedding immunocytochemistry for GABA. Our analysis distinguished a large population of GABA immunopositive axonal terminals from the Ce that exhibited symmetrical synaptic contacts with neurons in the lateral parabrachial nucleus. Additionally, most retrogradely labeled dendrites and perikarya received synaptic contacts from GABA immunoreactive terminals, with some of them originating from the Ce. The present study provides the first direct ultrastructural evidence for a monosynaptic, GABAergic link between Ce axons and neurons of the parabrachial nucleus via classical symmetrical synapses.     

Noradrenergic synaptic transmission in the superior cervical ganglion.

Morphology of dendrites of principal neurons in the rat superior cervical ganglion (SCG) was re-examined by an intracellular staining method followed by electron microscopic analysis. They exhibit a varying complexity in their morphology and arborization. Some dendrites show specializations such as a glomerular plexus, where extensively branched dendritic collaterals form synaptic connections comprising not only axodendritic synapses between preganglionic axons and principal cell dendrites, but also dendrodendritic synapses between principal cell dendrites. Most presynaptic elements of adrenergic synapses observed by conventional methods appear to represent these specialized dendritic collaterals of principal neurons. In denervated SCG, focal stimulation revoked an inhibitory postsynaptic potential in some principal neurons. The response was completely blocked by yohimbine, an alpha 2-adrenoceptor antagonist. The inhibitory synaptic connection between principal neurons via dendrodendritic synapses may be an important addition to the conventional scheme of intraganglionic synaptic transmission. Sympathetic ganglia may thus function as more than a simple relay station, with specialized neuronal circuitry that may be involved in the modulation of cholinergic transmission.     

Identification of an efferent projection from the paraventricular nucleus of the hypothalamus terminating close to spinally projecting rostral ventrolateral medullary neurons.

The paraventricular nucleus of the hypothalamus is increasingly being viewed as an important site for cardiovascular integration because of its connections to regions in the brain and spinal cord which are known to be important in cardiovascular control. Like the vasomotor neurons of the rostral ventrolateral medulla, descending axons from paraventricular neurons can be identified that form synapses on sympathetic preganglionic neurons in the thoracic spinal cord. The purpose of this study was to determine whether paraventricular axons project to the rostral ventrolateral medulla and whether they are closely apposed to reticulospinal neurons in this region. Descending paraventricular axons were labelled with biotin dextran amine, while rostral ventrolateral medullary neurons were retrogradely labelled from the spinal cord with wheatgerm agglutinin conjugated to horseradish peroxidase. This revealed, within the rostral ventrolateral medulla, paraventricular axon and terminal varicosities closely apposed to and apparently contiguous with retrogradely labelled spinally projecting neurons. Thus our study at the light microscopical level has shown the potential for the paraventricular nucleus to directly influence rostral ventrolateral reticulospinal neurons. We suggest these connections, if confirmed by electron microscopy, could be one means by which activation of paraventricular neurons elicits alterations in blood pressure.     

Adrenergic innervation of the rat nucleus locus coeruleus arises predominantly from the C1 adrenergic cell group in the rostral medulla.

Focal iontophoretic injections of the retrograde tracer Fluoro-Gold into the locus coeruleus were combined with immunocytochemistry for phenylethanolamine N-methyltransferase, the final enzyme in the synthesis of epinephrine. Retrograde labeling confirmed recent findings that the major afferents to the locus coeruleus are present in the ventrolateral (nucleus paragigantocellularis) and dorsomedial medulla (nucleus prepositus hypoglossi), areas containing the C1 and C3 adrenergic cell groups, respectively. The Fluoro-Gold label revealed morphologic details of locus coeruleus afferent cells. Labeled neurons in the prepositus hypoglossi region were typically round (10 microns diameter) or ellipsoidal and compressed against the ventricle wall (10 x 20 microns), while those in the paragigantocellularis were most often multipolar and ellipsoidal or triangular in shape (10 x 20-20 x 30 microns). Double labeling in the same tissue sections revealed that locus coeruleus afferent neurons are intercalated among phenylethanolamine N-methyltransferase-positive C1 and C3 neurons. Twenty-one per cent of locus coeruleus afferent neurons in paragigantocellularis stained for phenylethanolamine N-methyltransferase while only 4% of locus coeruleus afferents in the prepositus hypoglossi area exhibited phenylethanolamine N-methyltransferase immunoreactivity. In paragigantocellularis, doubly labeled neurons were usually the smaller locus coeruleus afferents, while in the prepositus hypoglossi phenylethanolamine N-methyltransferase labeling was found in all cell types that project to the locus coeruleus. Phenylethanolamine N-methyltransferase-positive fibers from the C1 and C3 cell groups form an adrenergic fiber bundle in the dorsomedial medulla; in the pons, these fibers appear to exit this bundle and innervate the locus coeruleus. Fibers from the neurons of the C3 cell group also appear to ascend on the dorsal surface of the medulla to innervate the locus coeruleus. The phenylethanolamine N-methyltransferase fiber innervation in the locus coeruleus was dense and highly varicose. Phenylethanolamine N-methyltransferase innervation in the dorsal pons was not restricted to the locus coeruleus but was also prominent in neighboring areas such as Barrington's nucleus and the lateral dorsal tegmental nucleus of Gudden.     

Differential synaptic inputs to the cell body and proximal dendrites of preganglionic parasympathetic neurons in the rat conus medullaris

Preganglionic parasympathetic neurons (PPNs) reside in the intermediolateral (IML) nucleus of the rat lumbosacral spinal cord and contribute to the autonomic control of visceral pelvic organs. PPNs provide the final common pathway for efferent parasympathetic information originating in the spinal cord. We examined the detailed ultrastructure of the type and organization of synaptic inputs to the cell body and proximal dendrites of PPNs in the rat conus medullaris. The PPNs were retrogradely labeled by a systemic administration of the B subunit of cholera toxin conjugated to horseradish peroxidase. We demonstrate four distinct types of synaptic boutons in apposition with PPN somata and proximal dendrites: S-type boutons show clear, spheroid vesicles; F-type boutons show flattened vesicles; dense-cored vesicle-type (DCV-type) boutons show a mixture of clear and dense-cored vesicles; L-type boutons were rare, but large, exhibited clear spheroid vesicles, and were only encountered in apposition with the PPN dendrites in our sample. The membrane surface covered by apposed boutons was markedly higher for the proximal dendrites of PPNs, compared with their somata. The inhibitory synaptic influence was markedly higher over the PPN somata compared with their proximal dendrites, as suggested by the higher proportion of putative inhibitory F-type boutons in apposition with the soma and a higher frequency of S-type boutons per membrane length for the proximal dendrites. Our studies suggest that the synaptic input to PPNs originates from multiple distinct sources and is differentially distributed and integrated over the cell membrane surface.     

Morphology of physiologically characterized medial lemniscal axons terminating in cat ventral posterior thalamic nucleus

1. Medial lemniscal axons were identified by extra- and intracellular recording in the thalamic ventral posterior lateral nucleus (VPL) of cats and injected intracellularly with horseradish peroxidase (HRP). 2. Axons were characterized in terms of their latencies of response to stimulation of the medial lemniscus in the medulla, their receptive fields, and the temporal patterns of their discharge in response to stimulation of the receptive field with natural, hand-held stimuli. One-hundred sixty-six axons were placed in five operational groups: hair transient (Ht) (n = 41); hair sustained (Hs) (n = 45); pressure transient (Pt) (n = 14); pressure sustained (Ps) (n = 27), and deep or joint (Jt) (n = 39). 3. There was a tendency for Jt axons to have their terminations in anterodorsal parts of VPL and for those in the four cutaneous categories to have theirs in more central parts of the nucleus. 4. Nineteen injected axons with receptive fields mainly on the distal forelimb were subjected to detailed morphological analysis in terms of extent of terminal field and number of boutons. All axons ended in localized terminal fields that were more extensive anteroposteriorly than in the other dimensions. All showed an overall similarity and similar ranges of variation. There was a tendency, however, for Jt axons to have the least extensive terminations with fewest boutons. Ps axons had the most extensive terminations and largest number of boutons; Hs axons had small terminations and few boutons but Ht axons had small-to-medium arborizations with many boutons; no Pt axons were sufficiently well stained to enable comparisons of them with the others. There were no marked differences in axon diameter or conduction velocity among the five types. 5. Boutons identified light microscopically tended to be clustered in linear chains along proximal dendrites of relay neurons and electron microscopy revealed that they were terminals making synaptic contacts on relay cell dendrites and on presynaptic dendrites of interneurons. 6. These results reveal more similarities than differences among lemniscal axon terminations in VPL. Further studies of a quantitative nature on stimulus-response coupling and on the geographic distribution of lemniscal synapses on relay neurons will be required to reveal how lemniscal input is translated into relay cell output in VPL.     

Parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat: localization, morphology, connectivity and ultrastructure

Summary We studied the distribution, morphology, ultrastructure and connectivity of parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat. Immunoreactive cell bodies were found in all layers of the entorhinal cortex except layer I. The highest numbers were observed in layers II and III of the dorsal division of the lateral entorhinal area whereas the lowest numbers occurred in the ventral division of the lateral entorhinal area, Most such neurons displayed multipolar configurations with smooth dendrites. We distinguished a type with long dendrites and a type with short dendrites. We also observed pyramidal immunoreactive neurons. A dense plexus of immunoreactive dendrites and axons was prominent in layers II and III of the dorsal division of the lateral entorhinal area and the medial entorhinal area. None of the parvalbuminimmunoreactive cells became retrogradely labelled after injection of horseradish peroxidase into the hippocampal formation. By electron microscopy, immunoreactivity was observed in cell bodies, dendrites, myelinated and unmyelinated axons and axon terminals. Immunoreactive dendrites and axons occurred in all cortical layers. We noted many myelinated immunoreactive axons. Immunoreactive axon terminals were medium sized, contained pleomorphic synaptic vesicles, and established symmetrical synapses. Both horseradish peroxidase labelled and unlabelled immunonegative cell bodies often received synapses from immunopositive axon terminals arranged in baskets. Synapses between immunoreactive axon terminals and unlabelled dendritic shafts and spines were abundant. Synapses with initial axon segments occurred less frequently. In addition, synaptic contacts were present between immunopositive axon terminals and cell bodies and dendrites. Thus, the several types of parvalbumin-containing neuron in the entorhinal cortex are interneurons, connected to one another and to immunonegative neurons through a network of synaptic contacts. Immunonegative cells projecting to the hippocampal formation receive axo-somatic basket synapses from immunopositive terminals. This connectivity may form the morphological substrate underlying the reported strong inhibition of cells in layers II and III of the entorhinal cortex projecting to the hippocampal formation.     

三叉神经本体觉中枢通路第二、三级神经元之间突触连接的发现及突触构筑学研究—溃变与HRP逆行追踪法相结合的电镜观察

将HRP注入丘脑腹后内侧核逆行追踪,将海人酸注入三叉神经脊束核吻侧亚核背内侧部造成顺行溃变,在电镜下对王百忍等发现的三叉神经本体觉中枢通路三级神经元所在地—”带状区”内的二、三级神经元的突触联系进行了探索。结果在“带状区”内发现了由溃变终末与HRP逆行标记的胞体、树突形成的轴-体、轴-树突触,从而在突触水平确证了这条通路的存在。另外还发现,同时存在着由正常轴突终末与HRP逆标神经元胞体、树突所形成的轴-体或轴-树突触;正常轴突终末与溃变终末所形成的轴-轴突触;溃变终末与未标记的神经元胞体、树突所形成的轴-体、轴-树突触。还见到少量的突触小球结构。本文也对”带状区”突触连接模式的特征及意义作了讨论。     

Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets

We investigated the effect on neurochemical phenotype of changing the targets innervated by sympathetic preganglionic neurons. In neonatal rats, the adrenal gland was transplanted into the neck, to replace the postganglionic neurons of the superior cervical ganglion. Transplanted adrenal glands survived, and contained noradrenergic and adrenergic chromaffin cells, and adrenal ganglion cells. Retrograde tracing from the transplants showed that they were innervated by preganglionic neurons that would normally have supplied postganglionic neurons of the superior cervical ganglion. The neurochemical phenotypes of preganglionic axons innervating transplanted chromaffin cells were compared with those innervating the normal adrenal medulla or superior cervical ganglion neurons. As in the normal adrenal gland, preganglionic nerve fibres apposing transplanted chromaffin cells were cholinergic. The peptide and calcium-binding protein content of preganglionic fibres was similar in normal and transplanted adrenal glands. In both cases, cholinergic fibres immunoreactive for enkephalin targeted adrenergic chromaffin cells, whilst cholinergic fibres with co-localised calretinin-immunoreactivity innervated noradrenergic chromaffin cells and adrenal ganglion cells. In contrast to the innervation of normal adrenal glands, these axons lacked immunoreactivity to nitric oxide synthase. In a set of control experiments, the superior cervical ganglion was subjected to preganglionic denervation in rat pups the same age as those that received adrenal transplants, and the ganglion was allowed to be re-innervated over the same time course as the adrenal transplants were studied. When the superior cervical ganglion was re-innervated by preganglionic nerve fibres, we observed that all aspects of chemical coding were restored, including cholinergic markers, nitric oxide synthase, enkephalin, calcitonin gene-related peptide and calcium binding proteins in predicted combinations, although the density of nerve fibres was always lower in re-innervated ganglia. These data show that the neurochemical phenotypes expressed by preganglionic neurons re-innervating adrenal chromaffin cells are selective and similar to those seen in the normal adrenal gland. Two explanations are advanced: either that contact of preganglionic axons with novel target cells has induced a switch in their neurochemical phenotypes, or that there has been target-selective reinnervation by pre-existing fibres of appropriate phenotype. Regardless of which of these alternatives is correct, the restoration of normal preganglionic codes to the superior cervical ganglion following denervation supports the idea that the target tissue influences the neurochemistry of innervating preganglionic neurons.     

The fine structure of nerve endings in the nucleus of the trapezoid body and the ventral cochlear nucleus

Large, calyciform axonal endings, as well as typical terminal boutons, have been previously described in the ventral cochlear nucleus and the nucleus of the trapezoid body by light microscopists. In the present study, these endings were examined with the electron microscope in chinchillas, rats, and a cat after perfusion fixation with osmium tetroxide. The calyces were found to consist of elongated processes arising from myelinated axons and making multiple synaptic contacts with perikarya and dendrites. This finding suggests that an important property of calyces is the large amount of synaptic activity that they can bring to bear on a single post-synaptic structure. Adjacent to the calyciform endings were variable numbers of boutons that made synaptic contacts with the same perikarya and dendrites. The majority of boutons contained smaller synaptic vesicles than those present in the calyces, implying both anatomical and functional differences between these two types of ending. It is suggested that many of these boutons in the ventral cochlear nucleus are endings of centrifugal, inhibitory fibers described by previous workers.     

The section-Golgi impregnation procedure. 2. Immunocytochemical demonstration of glutamate decarboxylase in Golgi-impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat

Sections of the cat's visual cortex were stained by an antiserum to glutamate decarboxylase using the peroxidase-antiperoxidase method; they were then impregnated by the section Golgi procedure and finally the Golgi deposit was replaced by gold. Neurons containing glutamate decarboxylase immunoreactivity were found in all layers of the visual cortex, without any obvious pattern of distribution. Fifteen immunoreactive neurons were also Golgi-impregnated and gold-toned, which enabled us to study the morphology and synaptic input of identified GABAergic neurons. These neurons were found to be heterogeneous both with respect to the sizes and shapes of their perikarya and the branching patterns of their dendrites. All the immunoreactive, Golgi-impregnated neurons had smooth dendrites, with only occasional protrusions. The synaptic input of glutamate decarboxylase-immunoreactive neurons was studied in the electron microscope. Immunoreactive neurons received immunoreactive boutons forming symmetrical synapses on their cell bodies. The Golgi-impregnation made it possible to study the input along the dendrites of immunoreactive neurons. One of the large neurons in layer III whose soma was immunoreactive was also Golgi-impregnated: it received numerous non-immunoreactive asymmetrical synaptic contacts along its dendrites and occasional ones on its soma. The same neuron also received a few boutons forming symmetrical synaptic contacts along its Golgi-impregnated dendrites; most of these boutons were immunoreactive for glutamate decarboxylase. Glutamate decarboxylase-immunoreactive boutons were also found in symmetrical synaptic contact with non-immunoreactive neurons that were Golgi-impregnated. A small pyramidal cell in layer III was shown to receive several such boutons along its somatic membrane. It is concluded that the combination of immunoperoxidase staining and Golgi impregnation is technically feasible and that it can provide new information. The present study has shown that there are many morphologically distinct kinds of aspiny GABAergic neurons in the visual cortex; that the predominant type of synaptic input to the dendrites of such neurons is from boutons forming asymmetrical synapses, but that some of the GABAergic neurons also receive a dense symmetrical synaptic input on their cell bodies, and occasional synapses along their dendrites, from the boutons of other GABAergic neurons. These findings provide a morphological basis, firstly, for a presumed powerful excitatory input to GABAergic interneurons and, secondly, for the disinhibition which has been postulated from electrophysiological studies to occur in the cat's visual cortex.     

大鼠外侧膝状体背核的主要突触构筑

外侧膝状体背核(DLG)的突触构成成份主要来自视网膜、视皮质、丘脑网状核的轴突终末和DLG内神经元的突起。视网膜和视皮质来的轴突终末对DLG投射神经元和中间神经元的树突形成非对称性突触,丘脑网状核的轴突终末则对这两类神经元构成对称性突触,而中间神经元树突具有轴突样作用,对投射神经元树突形成对称性突触。这些结果有助于我们了解视觉信息在DLG内的加工处理过程。