Localizing spinal-cord-projecting neurons in adult albino rats

Cytochrome oxidase activity was examined in the striate cortex (area 17) of squirrel monkeys at both the light and ultrastructural levels. Two prominent bands of reactivity were found in 4A and 4C with intermittent puffs of cytochrome oxidase reactivity in laminae 2 and 3. These puffs, spaced 0.5 mm apart, were in register with intermittent concentrations of activity in laminae 4B, 5, and 6. A thin band of reactivity was observed in lamina 1. The upper portion of 4C beta was less reactive than 4C alpha or the lower portion of 4C beta. Reactive neurons included stellate cells in all laminae and pyramidal cells in laminae 2 through 4B, 5, and 6. A row of large reactive pyramidal cells was observed in upper lamina 6. More reactive neurons were found in the puffs (laminae 2 and 3) than were observed in interpuff regions, and the reactive neurons were significantly larger than the nonreactive neurons. Reactive neurons contained two to three times as many reactive mitochondria as did the nonreactive neurons and often had indented nuclei. Based on the number of darkly or highly reactive, moderately reactive and lightly reactive mitochondria, puff regions were significantly different from nonpuff regions; there were approximately two times as many darkly reactive mitochondria in puff regions as compared to a similar nonpuff area. The majority of mitochondria (32% in puff; 44% in nonpuff) were found to reside in the dendritic profiles, which also contained the majority of highly reactive mitochondria. In a separate analysis, the total area of highly reactive mitochondria within puff regions was found to be twice the total area of highly reactive mitochondria in a comparable nonpuff region. An analysis of synapses showed that there were more asymmetrical synapses in both puff and nonpuff regions (55% and 54%, respectively) than symmetrical ones (45% in puff and 46% in nonpuff). There was an increase in mitochondrial reactivity in both asymmetrical and symmetrical synapses in the puff areas; however, the increased reactivity within asymmetrical terminals was significantly greater than that within symmetrical ones. Several somatodendritic synapses were observed and they were all of the symmetrical variety. Axospinous contacts were primarily of the asymmetrical type; however, symmetrical axospinous synapses were observed and were typically seen in association with an asymmetrical synapse. It was concluded that cytochrome oxidase activity is localized primarily within the dendritic profiles in both puff and nonpuff regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential glutamatergic innervation in cytochrome oxidase-rich and -poor regions of the macaque striate cortex: Quantitative EM analysis of neurons and neuropil

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase (CO)-rich puffs and CO-poor interpuffs in its supragranular layers. However, the neurochemical basis for their differences in metabolic activity and physiological properties is not well understood. The goals of the present study were to determine whether CO levels in postsynaptic neuronal compartments were correlated with the proportion of excitatory glutamate-immunoreactive (Glu-IR) synapses they received and if Glu-IR terminals and synapses in puffs differed from those in interpuffs. By combining CO histochemistry and postembedding Glu immunocytochemistry on the same ultrathin sections, the simultaneous distribution of the two markers in individual neuronal profiles was quantitatively analyzed. As a comparison, adjacent sections were identically processed for the double labeling of CO and GABA, an inhibitory neurotransmitter. In both puffs and interpuffs, most axon terminals forming asymmetric synapses (84%)—but not symmetric ones, which were GABA-IR—were intensely immunoreactive for Glu. GABA-IR neurons received mainly Glu-IR synapses on their cell bodies, and they had three times as many mitochondria darkly reactive for CO than Glu-rich neurons, which received only GABA-IR axosomatic synapses. In puffs, GABA-IR neurons received a significantly higher ratio of Glu-IR to GABA-IR axosomatic synapses and contained about twice as many darkly CO-reactive mitochondria than those in interpuffs. There were significantly more Glu-IR synapses and a higher ratio of Glu- to GABA-IR synapses in the neuropil of puffs than of interpuffs. Moreover, Glu-IR axon terminals in puffs contained approximately three times more darkly CO-reactive mitochondria than those in interpuffs, suggesting that the former may be synaptically more active. Thus, the present results are consistent with our hypothesis that the levels of oxidative metabolism in postsynaptic neurons and neuropil are positively correlated with the proportion of excitatory synapses they receive. Our findings also suggest that excitatory synaptic activity may be more prominent in puffs than in interpuffs, and that the neurochemical and synaptic differences may constitute one of the bases for physiological and functional diversities between the two regions. © 1996 Wiley-Liss, Inc.     

Adrenergic fibers in the spinal cord of the monkey: light and electron microscopic study

The adrenergic innervation of the monkey (Macaca fascicularis) thoracic spinal cord was examined by means of peroxidase-antiperoxidase immunohistochemical method using antisera directed phenylethanolamine N-methyl transferase (PNMT). At light microscopic level the PNMT-positive profiles are seen as brown granules, presumably axon terminals, or varicose fibers. They are localized in the intermediolateral nucleus, central gray and the intermediate gray which connects the two. Occasional fibers are seen in ventral and dorsal horns. The descending adrenergic fiber tract is found in the lateral margin of the lateral funiculus. At electron microscopic level, the PNMT-positive presynaptic profiles exhibit densely packed small clear vesicles, a few large dense core vesicles and numerous mitochondria. They make synaptic contact with dendritic profiles (97%) and somatic profiles (3%) and demonstrate either symmetric or asymmetric synaptic specialization. The descending adrenergic fiber tract consists mainly of unmyelinated fibers and is located in the ventral half of the lateral funiculus.     

Patterns of cytochrome oxidase activity in the frontal agranular cortex of the macaque monkey

The laminar pattern of cytochrome oxidase activity was studied in the agranular frontal cortex (area 4-6 complex) of the macaque monkey. The cortex, stained with this method, showed 6 stripes of different enzymatic activity. On the basis of their characteristics and of the presence of highly active cells, the agranular frontal cortex could be parcellated in 5 areas (F1-F5). F1 very likely corresponds to area FA of von Bonin and Bailey. Rostral to F1 two large regions could be distinguished, one located medial to the spur of the arcuate sulcus and its imaginary caudal extension, the other laterally. The superior region was formed by areas F2 and F3. The first was located on the dorsomedial cortical surface, the other on the mesial surface. F3 possibly corresponds to the supplementary motor area. The inferior region was formed by areas F4 and F5. The rostral area (F5) showed transition characteristics that rendered it somehow similar to the prefrontal areas. It may correspond to the cytoarchitectonic area FCBm. The cytocrome oxidase technique is a useful means of parcellating the agranular frontal cortex and may greatly help in physiological and behavioral experiments.     

Quantitative and qualitative characteristics of dentate and fastigial afferents identified by electron microscopic autoradiography in the cat thalamus

Dentato- and fastigiothalamic afferents were identified in the VM and medial VA and VL using electron microscopic (EM)-autoradiography. Synaptic vesicles in labeled dentate and fastigial boutons differed significantly in both their size and shape, which allows these two types of terminals to be distinguished in the normal neuropil. Differences in the mode of terminations of cerebellar afferents upon the neurons in the thalamic nuclei studied are also discussed.     

Immunoreactive substance P in the substantia nigra of the monkey: light and electron microscopic localization

The microscopic localization of immunoreactive substance P in the monkey substantia nigra was investigated by the peroxidase antiperoxidase method. Immunoreactive substance P was identified in the substantia nigra pars compacta and pars reticulata in numerous myelinated and unmyelinated fibers, and within axon terminals which contained intensely labeled granular vesicles and formed predominantly axodendritic synapses. These morphologic results are consistent with known excitatory effects of SP on nigral neurons.     

Thiamine-like molecules in the cerebellar cortex of the rat: light and electron microscopic immunocytochemical investigation

Immunocytochemical methods were used for studying the location of thiamine-like molecules in the cerebellar cortex of the rat. A positive reaction was observed in several synapses and in several dendrites. This reaction was associated with tubular formations and synaptic vesicles. A positive reaction was also found in several mitochondria and in glial cells. The possible role of thiamine-like molecules in cerebellar cortex is discussed.     

Efferent connections of the centromedian and parafascicular thalamic nuclei in the squirrel monkey: a light and electron microscopic study of the thalamostriatal projection in relation to striatal heterogeneity.

The organization of the thalamostriatal projections arising from the centromedian (CM) and parafascicular (Pf) thalamic nuclei in the squirrel monkey (Saimiri sciureus) was studied at both light and electron microscopic levels. Following selective injections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the CM or Pf, patterns of terminal arborization within the striatum were compared to the biochemical heterogeneity of the striatum as revealed by immunohistochemical staining for the calcium-binding protein calbindin D-28k (CaBP), and histochemical staining for the enzymes acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase). The PHA-L-labeled axon terminals within the striatum were further analyzed at the ultrastructural level to characterize their pattern of synaptic organization. Dense and heterogeneous terminal fields occur in the "sensorimotor" territory of the striatum after CM injections, or in the "associative" striatal territory following Pf injections. In the associative territory labeled axons arborize in a diffuse manner predominantly within areas enriched with CaBP, AChE, or NADPH-diaphorase, representing the matrix compartment, and tend to avoid areas poor in these substances, corresponding to the patch/striosome compartment. In the sensorimotor territory labeled axons form bands that occupy a subregion of the NADPH-diaphorase-rich zone in the putamen. The terminal pattern of the CM-striatal projection suggests the existence of a more complex mosaic organization within the sensorimotor territory. Ultrastructural analysis of PHA-L-labeled elements within the striatum reveals that both CM and Pf projections form asymmetric synapses upon dendrites and spines of striatal cells. A total of 339 PHA-L-labeled boutons were examined after CM injections and compared to 293 boutons following Pf injections. After CM injections, 29% of PHA-L-labeled terminals form synapses on dendritic spines and 66% on dendritic shafts, whereas after Pf injections only 12% of synapses occur on dendritic spines compared to 81% on dendritic shafts. Labeled terminals forming axosomatic or axoaxonic synapses were not seen within the striatum following either CM or Pf injections. It is concluded that in the squirrel monkey: 1) Pf-striatal fibers profusely arborize within the matrix compartment of the associative territory, 2) CM-striatal fibers form bands that occupy a subregion of the NADPH-diaphorase-rich zone within the sensorimotor territory, and 3) that both Pf- and CM-striatal projections establish asymmetric synapses with dendrites and spines of medium-sized spiny cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Projections from the striate cortex to the diencephalon in the squirrel monkey (Saimiri sciureus). A light microscopic radioautographic study following intracortical injection of H3 leucine

Tritiated leucine was injected into three different regions of the striate cortex in nine squirrel monkeys and the axoplasmic transport of labelled protein in the neurons was used to identify terminal fields of projection in the thalamus. The regions of injection corresponded to those representing about 3°, 10°, and 30° eccentricity in the contralateral visual field. The periods of post-injection survival were 9–19 hours, 8 days, and 23 days. The exposure was about three weeks in all cases. In the thalamus terminal fields of projection were identified in the lateral geniculate nucleus, the posterior nucleus of the thalamus, the inferior and lateral pulvinar nucleus, the reticular nucleus of the thalamus and the pregeniculate nucleus. In most of the projections the location of the terminal fields varied systematically with the location of the injection site indicating a retinotopic organization. In eight of the nine experiments all of the thalamic projections were observed. In the cases with postinjection survival times of 8 and 23 days pronounced axonal labelling was seen, and the corticothalamic pathway could be traced from the site of injection to the thalamus. In one experiment in which only the superficial layers of the cortex were labelled by the injection no thalamic projection was found.     

Immunohistochemical localization of calcium-binding proteins, parvalbumin and calbindin-D 28k, in the adult and developing visual cortex of cats: a light and electron microscopic study

In the cat primary visual cortex, we investigated with immunohistochemical techniques the developmental changes in the cellular and subcellular localization of the Ca2+-binding proteins parvalbumin (PV) and calbindin-D 28K (CBP), in order to determine whether there is a correlation between the expression of Ca2+-dependent processes and the time course of the critical period for use-dependent plasticity. On the 54th day of gestation and at 1 week postnatally, both calcium-binding proteins were present only in a subpopulation of neurons in layers V and VI. During subsequent maturation, the number of PV(+) and CBP(+) neurons increased significantly and labeled cells were detected in more superficial layers. Moreover, the homogeneous labeling of some CBP(+) neurons in layers IV to VI decreased and changed to a punctate pattern. In adult cats PV(+) neurons were evenly distributed throughout layers II to VI, whereas CBP(+) neurons were concentrated in layers II/III. Only a few immunoreactive cells had morphological features characteristic of pyramidal cells; the large majority were nonpyramidal. Electron microscopy confirmed the presence of PV- and CBP-reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. Immunoreactive neurons received immunonegative asymmetric synapses on their dendritic shafts and made symmetric synaptic contacts with labeled and unlabeled somata and with unlabeled dendritic shafts. The large number and widespread distribution of immunoreactive neurons implies that PV and CBP play an important role in the regulation of calcium-dependent processes in the visual cortex. Furthermore, the developmental redistribution of PV and CBP points to changes in the organization of Ca2+-dependent processes during maturation.     

GABAergic elements in the neuronal circuits of the monkey neostriatum: a light and electron microscopic immunocytochemical study

An antibody raised in rabbits against a GABA-BSA conjugate was used together with the PAP technique to label elements in the neostriatum of three Old World monkeys. Light microscopy revealed numerous immunoreactive medium-size neurons of various staining intensities, some of which had indented nuclei, as well as an occasional large cell. The neuropil showed a plexus of fine processes with frequent puncta. Ultrastructurally, the medium-size GABA-positive neurons were of two types: one with smooth nuclei and scanty cytoplasm, similar to spiny I cells, the other with invaginated nuclear envelopes and more abundant perikaryon, resembling the aspiny type. Correspondingly, labeled dendrites were either spiny or varicose. Some stained axons were myelinated, and the boutons had either large and ovoid, or small and pleomorphic vesicles. All of these boutons formed symmetric synapses, the former type with GABA-positive dendritic shafts but also with unlabeled dendrites; the latter type usually with GABA-negative elements, either dendrites, dendritic spines, or somata. Synapses were also observed between unreactive boutons and immunostained dendrites. Terminals with densely packed, small round vesicles that established asymmetric synapses with spines were always GABA-negative. Glial elements were consistently unlabeled, save for some astroglial endfeet. These findings provide positive evidence for the existence of two classes of GABAergic striatal neurons corresponding to a long-axoned spiny I type and an aspiny interneuron. Furthermore, the simultaneous labeling of GABA-immunoreactive presynaptic and postsynaptic profiles offers possible morphologic bases for the various kinds of intrastriatal inhibitory processes, including the feedforward, feedback, and "autaptic" types.     

A light and electron microscopic study of GAT-1 positive cells in the monkey brainstem and spinal cord

The distribution of the GABA transporter GAT-1 was studied in the monkey brainstem and spinal cord, using an affinity purified polyclonal antibody against a synthetic peptide corresponding to the C terminus of GAT-1. Very dense staining was observed in the interpeduncular nucleus, the inferior olivary nucleus and the substantia gelatinosa of the spinal cord, whilst dense labelling was observed in the substantia nigra, cochlear nuclei, vestibular nuclei, the spinal nucleus of V, the area postrema and the ventral horn of the spinal cord. Electron microscopy showed that the labelled profiles consisted of axon terminals that formed symmetrical synapses, consistent with GABAergic terminals. Many of the nuclei that were densely labelled for GAT-1 were those that received primary auditory, vestibular, or somatosensory inputs and the high density of GAT-1 in these nuclei suggests that GAT-1 plays an important role in terminating the inhibitory effects of GABA, at these nuclei.     

Laminar origin of direct projection from cortex area V1 to V4 in the rhesus monkey

Direct projection from cortex area V1 to V4 in the rhesus monkey was demonstrated by means of retrograde axonal transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). This projection originated at the layers 2 and 3 of the only representation area of central visual field (0-6 degrees) in V1 and terminated at the central representation of V4, whereas there was no projection between the extracentral representations of V1 and V4. Correlation of the present finding with the previous findings suggests that this projection is predominantly involved in the system of color information processing.     

Distribution of cytochrome oxidase and parvalbumin in the primary visual cortex of the adult and neonate monkey,Callithrix jacchus

The anatomical distributions of the mitochondrial enzyme cytochrome oxidase (CO) and of the calcium binding protein parvalbumin (PV) were studied in the striate cortex of adult and neonate New World monkeys (Callithrix jacchus). In the adult marmoset, both proteins were found in laminar arrangements similar to those described for the macaque monkey, with prominent bands of PV-like immunoreactive (PV-LI) puncta in layers IV and IIIb, and fairly evenly distributed PV-LI nonpyramidal neurons. Furthermore, the pattern of CO activity in area 17 of the neonate marmoset was almost identical to the CO pattern described in neonate macaque and squirrel monkeys. It came, therefore, as a surprise to find that the adult pattern of PV-like immunoreactivity (PV-LI) in the marmoset striate cortex arises from a neonatal pattern strikingly different from that seen in any developmental stage of the macaque, or in any other mammal studied so far. In the deep layers IV through VI of the neonate marmoset, a large number of PV-LI neurons was stained in bandlike patterns, their number in layers IV and V exceeding the number of PV-LI neurons present in these layers of the adult marmoset area 17. Staining of layers IV and VI was restricted to area 17 and involved nonpyramidal cells and their exceeding the number of PV-LI neurons present in these layers of the adult marmoset area 17. Staining of layers IV and VI was restricted to area 17 and involved nonpyramidal cells and their processes. The stained band of layer V, in contrast, continued throughout most of the neocortex. In area 17, an estimated 10 to 20% of the stained cells in layer V exhibited pyramidal shapes. The findings show that the expression of PV by visual cortical cells occurs before birth and suggest that the comparatively early onset of PV expression is not dependent on the onset of textured vision. The exuberant number of stained cells in some layers, and particularly the staining of pyramidal cells, in the neonate marmoset, suggest that a considerable number of cells possesses the stainability for PV-LI only transiently, i.e., in the marmoset, these cells have a specific demand for parvalbumin during this phase of their development. © 1994 Wiley-Liss, Inc.     

Calretinin immunoreactivity in the cerebral cortex of the lizard Psammodromus algirus: A light and electron microscopic study

The present study describes the distribution and structural features of calretinin-immunoreactive neurons and fiber plexuses in the cerebral cortex of a lacertid lizard, at the light and electron microscopic levels, and also examines the colocalization of calretinin with parvalbumin and gamma-aminobutyric acid (GABA) in certain cortical regions. Calretinin-immunoreactive neurons are present throughout the cerebral cortex of Psammodromus and can be classified according to morphological and neurochemical criteria. Neurons in the medial cortex are small, spine-free and lack parvalbumin, whereas in the lateral cortex, calretinin-immunoreactive neurons display sparsely spiny dendrites and also lack parvalbumin. The dorsomedial and dorsal cortices contain most of the calretinin cortical neurons, which were located almost exclusively in the deep plexiform layer. These neurons are large, with an extensive spine-free dendritic tree. Most of the calretinin-immunoreactive neurons of dorsomedial and dorsal cortices are GABAergic and contain parvalbumin. Calretinin-immunoreactive fibers form two main afferent systems in the cortical areas. One probably intrinsic inhibitory system, arising from the calretinin and parvalbumin GABAergic neurons in the dorsomedial and dorsal cortices, makes symmetrical synapses on the soma and proximal dendrites of neurons located in the cell layers of the same cortical areas. The other system is formed by extremely thin axons running within the superficial plexiform layers of the medial, dorsomedial and dorsal cortices. These axons make asymmetrical synapses on dendrites or dendritic spines. We suggest that this system, probably extrinsic excitatory, arises from neurons located in the basal forebrain. J. Comp. Neurol. 382:382-393, 1997. © 1997 Wiley-Liss Inc.     

The distribution of somatostatin-immunoreactive neurons and fibers in the rat cerebral cortex: light and electron microscopic studies

The distribution of somatostatin (SOM)-immunoreactive neurons in rat motor and somatosensory cortices was studied by immunohistochemistry at both light and electron microscopic levels. Three types of SOM-positive neurons were described. Type A cells are large to medium in size, multipolar in shape and have 3-5 long primary cell processes. Type B cells are medium to small, fusiform in shape and have two primary processes. These two subtypes show abundant subcellular organelles and are heavily stained throughout their cytoplasm. Type C cells are small, ovoid or fusiform in shape and are lightly stained. These previously undescribed cells are the largest SOM-immunoreactive population in the cortex. Ultrastructurally they have few subcellular organelles and only a patchy immunostaining in the cytoplasm. SOM-immunoreactive neurons occur in all cortical layers except I, and are most numerous in layer V of the somatosensory and motor cortex. In SOM-positive dendrites, electron-dense immunoreactive/peroxidase end product is primarily associated with microtubules. Fine deposits also occur deep to the postsynaptic membrane asymmetric synapses making contact with such dendrites.     

Quantitative light and electron microscopic studies on the lateral hypothalamus in rat. Cell and synaptic densities

Cell and synaptic packing densities and the synapse/cell ratios were determined for the lateral hypothalamus of the rat. A quarter of a million neurons were found in this area each having approximately 16000 synapses. Of the approximately 3700 million synapses of the lateral hypothalamus 82% is axodendritic and 16% axosomatic.     

Noradrenergic innervation of the developing and mature visual and motor cortex of the rat brain: a light and electron microscopic immunocytochemical analysis

The noradrenergic (NA) innervation of the developing and adult visual and motor cortex of the rat was examined with light and electron microscopic immunocytochemistry by using antibodies against dopamine-beta-hydroxylase. At birth, NA fibers were present in both cortical areas, appearing as two tangential streams, one above and the other below the cortical plate. During the subsequent weeks, these two streams arborized gradually innervating all cortical layers. The adult pattern of distribution was attained by postnatal day 14, but the density of innervation, which was higher in the motor than in the visual cortex, appeared similar to the adult by the end of the third postnatal week. Electron microscopic analysis revealed that a low proportion of NA varicosities (the highest value was 12% in the adult motor cortex in single sections) were engaged in synaptic contact, throughout development, in both areas examined. The overwhelming majority of these synapses were symmetrical, involving predominantly small or medium dendrites. This evidence suggests that transmission by diffusion is the major mode of NA action in the developing and adult cerebral cortex. Noradrenaline released in the rare synaptic junctions may act mainly to reduce the activity of its cortical targets. The results altogether provide morphologic evidence for an involvement of noradrenaline in the development of the neocortex and, along with earlier data on the serotonergic system, indicate that the monoaminergic systems are endowed with a specific anatomic organization in various areas of the brain.     

Innervation of the zona fasciculata of the adult human adrenal cortex: a light and electron microscopic study

Summary Autonomic innervation of the adrenal cortex has been demonstrated in several species. Detailed ultrastructural studies on the innervation of the zona fasciculata of the normal human adrenal cortex are lacking. We report herein our observations on the pattern of innervation of the cells of the zona fasciculata of the normal adult human cortex at both the light and electron microscope levels. Postganglionic unmyelinated fibers were observed to descend from a dense capsular meshwork and to be distributed as delicate branches among the columns of endocrine cells. Immunoperoxidase staining confirmed the presence of nerve fibers in the zona fasciculata in a distribution similar to that observed after staining with silver impregnation methods. Ultrastructural findings lent further support to these observations by the demonstration of bundles of unmyelinated fibers with focal enlargements containing terminal boutons with both clear and dense core vesicles in close approximation with the endocrine cells.