Akira Konishi

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

Since the horseradish peroxidase (HRP) method was first applied to the peripheral nervous system by Kristensson and Olsson 12 and subsequently to the central nervous system by LaVail and LaVai116, it has become one of the most common experimental methods for demonstrating neuronal connectivity in the nervous system (for review see refs. 10 and 15). Previous studies have shown that the light microscopical procedure for demonstrating HRP can also be used for electron microscopical visualization of the enzyme, not only of the enzyme transported retrogradely to neuronal somata and dendrites3,5,8,1a,14,17,19,26, 28, but also of that transported anterogradely to axon terminals2,4,s,9,17,22-za,z6, zS. In an attempt to increase the sensitivity of the HRP method and to improve the reliability of tracing neuronal connections using retrograde and anterograde transport of HRP in combination with electron microscopy, the cobalt method of Adams 1 and the coupled oxidation reaction utilized by Lundquist and Josefsson 18 for the determination of low levels of tissue peroxidase were applied to the histochemical visualization of HRP. The coupled oxidation method, utilizing a continuous supply of hydrogen peroxide formed in the glucose oxidase (GOD) reaction, has been shown to be at least I0 times more sensitive than other colorimetric peroxidase assays 18, and also to be applicable to electron microscopical demonstration of HRP 27. In the present study, the coupled oxidation method, when applied to the histochemical demonstration of HRP in combination with cobalt method 1, was found to give excellent electron microscopical pictures of the electron dense reaction product. The experiments were performed in more than 50 adult cats anesthetized with i.p. Nembutal (35 mg/kg). For observation of the retrograde transport of HRP, 0.1-0.5 /~1 of 2 5 ~ HRP (Toyobo Grade-I-C, RZ: 3.4) was injected, in different experiments, into the masticatory muscles 2z, perineal muscles H, or wall of the urinary bladder 25. For detection of HRP transported anterogradely to axon terminals, HRP was injected in a variety of brain areas such as the cerebral cortical areas 21 and the motor trigeminal nucleus23; a single injection of 0.01~3.1 ~1 of 50 % HRP dissolved in

Commissural interneurons for masticatory motoneurons: A light and electron microscope study using the horseradish peroxidase tracer technique

Abstract Commissural interneurons for masticatory motoneurons of the cat were investigated using the horseradish peroxidase tracing technique. After injection of horseradish peroxidase into the motor trigeminal nucleus, neurons labeled with the enzyme were seen contralaterally around the motor trigeminal nucleus, mainly in the supra- and intertrigeminal regions and the lateral tegmental regions close to the medial border of the motor trigeminal nucleus, and additionally within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei. After placing lesions in the supra- and intertrigeminal regions, degenerated axon terminals were found electron microscopically in the motor trigeminal nucleus contralateral to the lesion. In accordance with these findings, axon terminals in the motor trigeminal nucleus were labeled orthodromically with horseradish peroxidase injected contralaterally into the supra- and intertrigeminal regions; both terminals filled with round synaptic vesicles and those containing flattened vesicles were labeled with the enzyme. The existence of many commissural neurons around the motor trigeminal nucleus would be consistent with the intricacy of bilateral mechanisms of jaw movements.

The site of origin of cardiac preganglionic fibers of the vagus nerve: An HRP study in the cat

After injection of horseradish peroxidase (HRP) into the right cardiac branches of the vagus nerve in the cat, the majority of HRP-labeled neurons were located ipsilaterally in the reticular formation ventrolateral to the nucleus ambiguus. Additionally, HRP-labeled neurons were also observed within the nucleus ambiguus (Am) and the dorsal motor nucleus of the vagus nerve (DM).

Thalamic afferents to the rostral portions of the middle suprasylvian gyrus in the cat

Abstract It was demonstrated morphologically that nerve cells in the rostrodorsal areas of the complex of the anterior ventral and lateral ventral nuclei, the posterior lateral nucleus and the lateral central nucleus of the thalamus in the cat send axons to rostral portions of the middle suprasylvian gyrus. In the animals with lesions in the rostral portions of the middle suprasylvian gyrus, marked cell loss and gliosis were observed mainly in the rostrodorsal areas of the complex of the anterior ventral and lateral ventral nuclei as well as in the posterior lateral nucleus. The identification of thalamoparietal projection cells was also attempted by means of retrograde axonal transport of the horseradish peroxidase. In all animals in which the horseradish peroxidase was injected in the rostral areas of the middle suprasylvian gyrus, peroxidase-labeled neurons were found in the rostrodorsal areas of the complex of the anterior ventral and lateral ventral nuclei, the posterior lateral and the lateral central nuclei. Functional significance of the thalamoparietal projection cells in the rostrodorsal areas of the complex of the anterior ventral and lateral ventral nuclei is discussed in terms of the relay cells of the cerebello-thalamoparietal pathways.

Localization of motoneurons innervating the tensor tympani muscles: An horseradish peroxidase study in the guinea pig and cat

Motoneurons innervating the tensor tympani muscle were identified in the adult guinea pig and cat by the horseradish peroxidase (HRP) method. After HRP injection into the tensor tympani muscle, HRP-labeled neurons were seen in the regions outside the cytoarchitectonically-defined confines of the trigeminal motor nucleus; in the regions rostral to the rostral pole of the nucleus, as well as in the regions ventral and ventrolateral to the nucleus at the levels of the rostral half (guinea pig) or the rostral two-thirds (cat) of the nucleus. The tensor tympani motoneurons were generally smaller than the masticatory motoneurons.

Multipolar neurons and axodendritic synapses in the mesencephalic trigeminal nucleus of the cat

In the mesencephalic trigeminal nucleus (MTN) of the adult cat, multipolar neurons with 1-9 smooth dendritic processes were labeled with horseradish peroxidase which was applied to central cut ends of the masseter, deep temporal, medial pterygoid, superior alveolar and inferior alveolar nerves. These constituted 40% of the total population of MTN neurons; most of them were jaw-closing muscle afferent neurons and located mainly at the levels of the superior colliculus. Axon terminals were found electron microscopically to make synaptic contacts upon horseradish peroxidase-labeled dendritic profiles of MTN neurons.

A quantitative electron microscope study of cerebellar axon terminals on the magnocellular red nucleus neurons in the cat.

In the red nucleus (RN) of the cat, the bouton covering ratio (BCR: the ratio of whole somatic surface length and that covered with axon terminals) and the density of axon terminals in contact with somatic profiles (DAST: the number of axosomatic terminals per micron of somatic surface membrane) were calculated in each neuronal somatic profile over 60 micron in diameter. The mean BCR was 61.4 +/- 1.43 (S.E. M.)%. The mean DAST of axosomatic terminals filled with spherical synaptic vesicles (S-terminals) was 27.7 +/- 0.95, and that of terminals with pleomorphic and/or flattened vesicles (F-terminals) was 10.3 +/- 1.12. Subsequently, sequential changes of the BCR and DAST of intact terminals were examined in the RN deafferented from the cerebellorubral fibers. The mean BCR and DAST were decreased most markedly during the survival period of 4-7 days; thus decrease was chiefly due to degeneration of S-terminals (BCR: 16.7 +/- 1.17 %, DAST of S-terminals: 7.1 +/- 1.12, DAST of F-terminals: 5.8 +/- 1.22). In the RN 11-63 days after the operation, both the BCR and DAST tended to re-increase slightly and the majority of the re-increased terminals appeared to be F-terminals. Possible meanings of this re-increase of axosomatic terminals are discussed.

Leucine-enkephalin-like immunoreactive afferent fibers to pudendal motoneurons in the cat

Leucine-enkephalin-like immunoreactive (ENK-LI) afferent fibers to the feline homologue of the Onufs nucleus (pudendal motoneurons) originate mainly from lamina X of the sacral cord. They make synaptic contacts chiefly upon dendrites of pudendal motoneurons; the synaptic terminals most often contain pleomorphic synaptic vesicles and occasionally round synaptic vesicles. Large-cored vesicles in the axon terminals within the Onufs nucleus often showed ENK-LI.

Cholinergic neurons in the nucleus tegmenti pedunculopontinus pars compacta and the caudoputamen of the rat: a light and electron microscopic immunohistochemical study using a monoclonal antibody to choline acetyltransferase

Large neurons in the nucleus tegmenti pedunculopontinus pars compacta (TPC) of the rat were shown to have choline acetyltransferase (ChAT) immunoreactivity by light and electron microscopic immunohistochemistry using a monoclonal antibody to ChAT. The ChAT-positive TPC neurons had triangular or multipolar cell bodies with diameters of 20-50 micron. The ultrastructural features of the ChAT-positive TPC neurons were similar to those of ChAT-positive neurons in the caudoputamen; each neuron had a distinct, pale nucleus with an indented nuclear envelope and large, cytoplasm-containing, well-developed endoplasmic reticulum and pale mitochondria.

A morphological evidence of direct connections from the cochlear nuclei to tensor tympani motoneurons in the cat: a possible afferent limb of the acoustic middle ear reflex pathways.

The central circuitry of the acoustic middle ear reflex activating the tensor tympani muscle was studied in the cat by tracer methods using horseradish peroxidase (HRP), wheat germ agglutinin (WGA) or HRP conjugated with WGA (WGA-HRP). The results indicate that the dorsal and ventral cochlear nuclei send fibers to motoneurons innervating the tensor tympani muscle, bilaterally with an ipsilateral dominance.