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Query: EC:1.11.1.7 (peroxidase)
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The monosynaptic targets of different functional types of geniculocortical axons were compared in the primary visual cortex of monkeys. Single thalamocortical axons were recorded extracellularly in the white matter by using horseradish-peroxidase-filled pipettes. Their receptive fields were mapped and classified as corresponding to those of parvi- or magnocellular neurons in the lateral geniculate nucleus. The axons were then impaled and injected intraaxonally with horseradish peroxidase. Two magnocellular (MA) and two parvicellular (PA) axons were successfully recovered and reconstructed in three dimensions. The two MA axons arborised mainly in layer 4C alpha, as did the two PA axons in layer 4C beta. Few collaterals formed varicosities in layer 6. Both MA axons had two large, elongated clumps of bouton (approx. 300-500 x 600-1,200 microns each) and a small clump. One PA axon had two clumps (each with a core appr. 200 microns in diameter); the other had only one (appr. 150-200 microns in axon had 1,380; one MA axon had 3,200 boutons; and those of the more extensive MA axon were not counted. The distribution of postsynaptic targets as well as the number of synapses per bouton has been established for a sample of 150 PA boutons and 173 MA boutons from serial ultrathin sections. The MA axons made on average 2.1 synapses per bouton compared to 1.79 for one PA axon and 2.6 for the other. The sample of boutons taken from the two physiological types of axons contacted similar proportions of dendritic spines (52-68%), shafts (33-47%), and somata (0-3%). The postsynaptic elements were further characterized by immunostaining for GABA. All postsynaptic perikarya and some of the dendrites (4.5-9.5% of all targets) were positive for the amino acid. Near the thalamic synapse GABA-negative dendritic shafts frequently contained lamellar bodies, an organelle identical in structure to spine apparatus. Dendritic shafts and spines postsynaptic to the thalamocortical boutons frequently received an adjacent synapse from GABA-immunoreactive boutons. The similarity between the magno-and parvicellular axons in their targeting of postsynaptic elements, including the GABAergic neurons, suggests that the structural basis of the physiological differences between 4C alpha and 4C beta neurons should be sought in other aspects of the circuitry of layer 4C, such as local cortical circuits, or in the far greater horizontal extent of the thalamocortical and GABAergic axons in layer 4C alpha compared to those in the beta subdivision.
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PMID:Arborisation pattern and postsynaptic targets of physiologically identified thalamocortical afferents in striate cortex of the macaque monkey. 280 70

Type B neurons of the bullfrog sympathetic ganglia were examined to confirm the existence of axon collaterals and the distribution of synaptic contacts using the intracellular horseradish peroxidase (HRP) labeling method. The mean diameter of the perikarya was 60.8 (+/- 11.5 standard deviation; n = 36) X 43.8 (+/- 11.3) microns and the mean diameter of the initial segments of axons was 6.0 (+/- 1.8; n = 36) microns. Axon collaterals were found in 6 cells among 36 examined. They branched from axons at 61-167 microns from the perikaryon of origin. Short-axon collaterals containing vesicles (diameter: about 70 nm) were also observed to protrude from the stem axons. Spine-like processes were observed from the cell soma, axon hillock and the initial segment of the axon. They enclosed synaptic axon varicosities, or extended into the extracellular space without any synaptic contact. Serial sections revealed 171 axon varicosities in contact with a single ganglion cell; 32 (18.7%) varicosities were seen on the somata. 66 (38.6%) on the axon hillock and 73 (42.7%) on the initial segment of the axon which extended 100 microns from the perikaryon. Synaptic terminals were also found on the axon as far as 494 microns from the cell body of origin. These findings would provide a morphological basis for interaction between bullfrog sympathetic neurons at pre- or postsynaptic sites.
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PMID:Synaptic structure and axon collaterals of type B neurons in bullfrog sympathetic ganglia: intracellular horseradish peroxidase (HRP)-labeling study. 281 70

This is a study of the chemoanatomical organization of the projection from the raphe nuclei to the main olfactory bulb in the rat. A heavy projection from the dorsal and median raphe nuclei to the main olfactory bulb was shown by both retrograde and anterograde tracing techniques. Following injections of 1% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the main olfactory bulb, up to 1300 neurons were retrogradely labeled in the dorsal and median raphe nuclei, a much larger number than has been suggested by previous studies. By combining 5-HT immunofluorescence with True blue retrograde fluorescent labeling, it was determined that the vast majority of raphe neurons that project to the olfactory bulb contain serotonin. Injections of WGA-HRP into dorsal and/or median raphe produced dense anterograde labeling in the glomeruli, while fewer labeled fibers were observed in the external plexiform layer, internal plexiform layer, and granule cell layer. In contrast to the number of other centrifugal afferents to the bulb, a significant contingent of fibers from the raphe nuclei enters the main olfactory bulb (MOB) from outside in, i.e., via the olfactory nerve layer. Serotonergic fibers in MOB were visualized by immunocytochemistry, and the distribution of specific 5-HT fibers closely matched the distribution of anterograde terminal label resulting from injections of WGA-HRP in the raphe nuclei. The serotonergic fibers have a specific laminar distribution and morphology in MOB. Thus, the density of the serotonergic innervation to the glomerular layer is 2-3 times that of any other layer in MOB. In addition to their preferential innervation of the glomeruli, glomerular and infraglomerular serotonergic fibers are morphologically different. Serotonergic fibers located in the glomerular layer are generally thick (0.25-0.60 micron) compared to the thinner (0.25-0.35 micron) fibers that predominate in infraglomerular layers. Another difference is that the glomerular fibers often contain varicosities that are greater than 1 micron in diameter, while varicosities along infraglomerular fibers are usually barely larger than the axonal diameter. Finally, glomerular fibers are much more intensely stained than infraglomerular fibers. Electrolytic lesions of the dorsal and median raphe caused a total depletion of serotonin fiber staining in the bulb, demonstrating that the raphe nuclei are the sole source of the serotonergic input to the main olfactory bulb. Thus, it has been demonstrated that serotonergic neurons in dorsal and median raphe project very heavily to glomeruli and less heavily to other layers in the main olfactory bulb.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat. 282 62

1. The developmental process of the monosynaptic reflex pathway was investigated morphologically and electrophysiologically in isolated lumbar spinal cords of new-born and fetal rats. 2. Dorsal root fibres were stained with horseradish peroxidase in the fourth lumbar (L4) segment at different ages ranging from embryonic day (E) 15.5 to post-natal day (P) 0. At E15.5, several collaterals issued from axons in the dorsal funiculus and reached the dorsal part of the dorsal horn. At E16.5, the number of collaterals entering the grey matter increased. Also, a group of collaterals extended ventralwards forming a bundle, and reached the intermediate region. At E17.5, a small number of collaterals reached the motor nuclei. The number of collaterals entering the motor nuclei increased almost linearly with age: 0 at E15.5 and at E16.5, 27 at E17.5, 184 at E18.5, 432 at E19.5 and 746 at P0. 3. The tips of collaterals and their branches had growth cones, boutons (round or oval varicosities) or other varicosities. The mean number of branches with these structures per collateral in the motor nuclei was 1.2 at E17.5, 2.5 at E18.5, 3.6 at E19.5 and 5.8 at E20.5. 4. The percentage of collaterals having growth cones in the motor nuclei was 75% at E17.5, 70% at E18.5, 38% at E19.5 and 15% at E20.5. 5. The mean number of boutons per collateral in the motor nuclei was 0.6 at E17.5, 3.2 at E18.5, 4.9 at E19.5 and 10.7 at E20.5. This increase with age was caused by both branching of collaterals and the increase in the number of boutons of the en passant type. The estimated total number of boutons in the motor nuclei of the L4 segment steeply increased after E17.5: 16 at E17.5, ca. 600 at E18.5, ca. 2000 at E19.5 and ca. 8000 at E20.5-P0. 6. Stimulation of the L4 dorsal root evoked a reflex response in the L4 ventral root, recorded as the ventral root potential, in two out of nine preparations at E15.5 and in all preparations at and after E16.5. The onset of the ventral root potential indicated the onset of excitatory post-synaptic potentials in motoneurones. The segmental latency of the ventral root potentials was markedly shortened between E17.5 and E18.5 (from 12.5 to 7.2 ms) and essentially unchanged at the later stages. 7. The magnitude of monosynaptic reflex responses in the L4 segment gradually increased with age during prenatal stages, becoming maximal at P2-3 and then decreased at the following stages (P4-P8).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Morphological and physiological studies of development of the monosynaptic reflex pathway in the rat lumbar spinal cord. 282 63

Antiserum raised against GABA coupled with glutaraldehyde to bovine serum albumin was applied to the guinea pig cochlea. Immunoreactivity was visualized as horseradish peroxidase reaction product in surface preparations of the organ of Corti using immunocytochemical techniques. Bright-field, differential interference contrast and video-enhanced contrast light microscopy were used. GABA-like immunoreactivity was found in axons and endings of efferent neurons in all turns of the cochlear spiral, but predominantly in the third turn and first half of the fourth turn. In these apical turns, immunoreactivity was seen in the efferent components: inner spiral bundle, tunnel spiral bundle, tunnel-crossing fibers, large nerve endings synapsing on outer hair cell bases, nerve endings high up on outer hair cells, nerve endings or varicosities close to outer hair cells, and outer spiral fibers. Some immunoreactive large nerve endings at outer hair cells were found in the apical half of the fourth turn. This study shows that axons and endings of efferent neurons in the organ of Corti of guinea pig contain GABA-like immunoreactivity with a distribution similar to that of GAD-like immunoreactivity as shown in a previous study. In both studies, many efferent nerve axons and endings were unstained, even in regions of maximal density of immunoreactivity in the apical turns. The evidence indicates that a subpopulation of efferent neurons projecting to the organ of Corti is GABAergic and very likely different from the lateral and the medial olivocochlear efferent systems.
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PMID:GABA visualized by immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons. 287 Jul 61

The distribution of serotonin (5-HT) and tyrosine hydroxylase (TH) was examined in the hypothalamus of juvenile baboons, 24 h after infundibular stalk section. Simultaneous immunostaining for 5-HT with peroxidase-antiperoxidase (PAP) and TH with 15 nm colloidal gold (IGS) was performed on Vibratome sections from 3 operated and 1 control female. Light microscopy revealed fine 5-HT immunopositive (5-HT+) fibers, presumably axons, in the suprachiasmatic nuclei and ventromedial hypothalamus (VMH) after stalk section. In addition, focal accumulations of swollen and heavily stained 5-HT+ fibers occurred on the side of the surgical approach. Enlarged fibers were densest in the medial preoptic area, lateral and VMH areas, and the median eminence. TH immunoreactivity (TH+) in VMH cell bodies and axons was only slightly increased over that in controls. Electron microscopy of areas of 5-HT+ and TH+ overlap (medial VMH and adjacent periventricular zone) showed that 5-HT+ profiles were mostly unmyelinated axons and irregular varicosities. A few myelinated 5-HT+ axons were also observed. TH+ perikarya, dendrites, axons and terminals showed gold labeling characteristic for this enzyme. However, colocalization of 5-HT (PAP) and TH (IGS) was present in a number of fiber varicosities in experimental animals only. Both single- and double-labeled profiles occurred in individual thin sections, thus arguing against antibody cross-reactivity. These results indicate that: hypothalamic 5-HT+ fibers project to the median eminence in primates; 5-HT fibers become more obvious after stalk section due to accumulation of transmitter; focal 5-HT+ immunoreactivity in the hypothalamus can increase dramatically after distant and mild surgical trauma, and coexistence of 5-HT and TH in single neurons can appear after acute stalk section and/or trauma in experimental animals. These findings might represent uptake of exogenous 5-HT or amplified expression of endogenous neurotransmitter, suggesting that plasticity of transmitter phenotype might follow acute surgical and/or endocrine intervention in mature primate brain. Neuroendocrine studies employing the stalk-sectioned primate might thus be radically affected.
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PMID:Immunostaining reveals accumulation of serotonin and coexistence with tyrosine hydroxylase in hypothalamic neurons of acutely stalk-sectioned baboons. 288 96

Synaptic organization of the intermediolateral nucleus of the guinea pig thoracic spinal cord was examined with particular focus on monoamine- and peptide-containing nerve terminals. Axon varicosities having flat synaptic vesicles constituted 17% of all axons in the nucleus and formed exclusively symmetric synapses. Enkephalin-, substance P-, somatostatin-, 5-hydroxytryptamine-, and catecholamine-immunoreactive nerve terminals were densely distributed, while neurotensin, vasoactive intestinal polypeptide-, oxytocin-, and cholecystokinin-8-immunoreactive nerves were sparse in the nucleus. Coexistence of 5-hydroxytryptamine and enkephalin was demonstrated, and coexistence of somatostatin and enkephalin as well as somatostatin and 5-hydroxytryptamine in the same axons was also shown by serial semithin sections. Catecholamine axons labelled by 5-hydroxydopamine formed axodendritic and axosomatic synapses and made direct synaptic contacts on the preganglionic sympathetic neurons identified by retrograde transport of horseradish peroxidase. Direct synaptic contacts from enkephalin- and substance P-immunoreactive axons to preganglionic sympathetic neurons were also revealed. Enkephalin-, substance P-, and 5-hydroxytryptamine-immunoreactive axons formed axodendritic and axosomatic synapses. Catecholamine axon varicosities constituted 19% of all axon varicosities in the nucleus and 30% of them showed synaptic specializations in a sectional plane. Axon varicosities immunoreactive to enkephalin, 5-hydroxytryptamine, and substance P constituted approximately 35, 19, and 13% of all axon varicosities, respectively, while those with synaptic contacts made up 27, 30, and 26%, respectively, in a sectional plane. Enkephalin-, 5-hydroxytryptamine-, and noradrenaline-immunoreactive axons showed mainly symmetric synaptic contacts.
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PMID:Synaptic structure of the monoamine and peptide nerve terminals in the intermediolateral nucleus of the guinea pig thoracic spinal cord. 288 97

We have used the indirect antibody peroxidase-antiperoxidase technique to analyze the course of serotonin (5-hydroxytryptamine; 5HT) fibers to the deep cerebellar nuclei; the distribution of serotonin within the nuclei; the continued course of 5HT fibers to the cerebellar cortex; and the lobular and laminar distribution of this indoleamine in the cerebellar cortex. Only rarely are fibers found in either the restiform body or the brachium pontis. However, a distinct bundle of serotoninergic axons is present in the medial aspect of the brachium conjunctivum. Axons arise from this bundle and course dorsally into the neuropil of the deep cerebellar nuclei. The densest immunostaining is present in posterior and ventral regions of all four cerebellar nuclei. Within the nuclei large (24% of total) and small (76% of total) varicosities are present. The average distance between varicosities on individual axons is 3.85 micron (S.D. = 1.2). The innervation of the cerebellar cortex is derived primarily from fibers that course through the deep nuclei. At levels caudal to the deep nuclei a single midsagittal band courses into lobules VIII and IX. In the cerebellar cortex, serotoninergic axons and varicosities are present in all lobules; however, the fiber density is not uniform. The densest distribution is present in vermal lobule VIII and the dorsal folia of lobule IX. Within the granule cell layer of lobules VIII and IX, immunoreactive elements form a midsagittal band, and to a lesser degree, two parasagittal bands. Beaded serotoninergic fibers course through the deep and middle portion of the granule cell layer and give rise to a plexus at the border between the Purkinje cell and granule cell layers. Within this plexus axons extend long distances in the transverse and sagittal planes. Long beaded axons oriented in the transverse plane of the folia are also present in the deep molecular layer. A few radial serotoninergic fibers ascend to the pial surface and give rise to very short tangential branches. In all three cortical layers, both large (19% of total) and small (81% of total) varicosities are present. The average distance between varicosities on individual fibers is 5.3 micron (S.D. = 2.2).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Localization of serotonin immunoreactivity in the opossum cerebellum. 298 15

In the leech Hirudo medicinalis inhibitory motor neurons to longitudinal muscles make central inhibitory connections with excitatory motor neurons to the same muscles. We have used a variety of physiological and morphological methods to characterize these inhibitory connections. The efficacy of the transmission between the inhibitors and the excitors was measured by using three intracellular electrodes, two in the inhibitor (one for injecting current and one for measuring voltage) and a third electrode in the excitor for measuring the resultant voltage changes. We have determined that delta Vpre/delta Vpost, or what we have called the transmission coefficient, is X = 0.51, as measured in the somata of the two cells. Evidence which we have obtained leads us to propose that these inhibitory connections between motor neurons are probably monosynaptic. The synaptic latency is consistent with a monosynaptic connection. In addition, a double-labeling technique, whereby one neuron was filled with Lucifer Yellow and the other with horseradish peroxidase (HRP), was used to determine the anatomical relationship between inhibitors and excitors in whole mounts. This revealed varicosities on the processes of inhibitor motor neurons which appear to make contact with processes of excitor motor neurons. A second double-labeling technique, whereby one neuron was filled with HRP and the other with an electron-dense particulate marker, revealed adjacent processes between an inhibitor and an excitor in electron microscopic thin sections which could be the sites of synaptic contact between the neurons. The connections appear to be mediated largely by graded transmitter release from the inhibitory motor neurons. Three different methods were used to demonstrate that synaptic transmission remained in the absence of impulses in the inhibitory motor neurons. These included eliminating the impulse-supporting portion of the motor neuron by pinching off its axon, abolishing impulses by replacing Na+ with Tris in the medium bathing the nerve cord, and increasing the threshold for impulse production by raising the Mg2+ and Ca2+ concentrations in the medium bathing the nerve cord.
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PMID:Physiological and morphological analysis of synaptic transmission between leech motor neurons. 299 80

Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18-25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus.
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PMID:Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey. 299 42


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