Hidehiko Ochiai

GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining

A double immunocytochemical method combining the preembedding PAP technique and the postembedding immunogold technique was used to examine interactions between GABAergic and serotonergic neurons in the same tissue sections of the dorsal raphe nucleus of the rat. A large number of immunogold stained GABAergic axon terminals were found to be presynaptic to strongly PAP immunostained serotonergic perikarya and dendrites. The types of synapses were mostly symmetrical although a few asymmetrical ones were also found. No axo-axonic synapse between the GABAergic axon terminals and the serotonergic neuronal profiles was found. These results suggest that GABAergic neurons could modulate serotonergic neurons in the dorsal raphe nucleus through synaptic relations.

Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14).

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and its amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

SummaryThe correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after 3H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after 3H-DA, 3H-NA or 3H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of 3H-MA into TRH-like immunoreactive nerve terminals, where silver grains after 3H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after 3H-MA injection to total nerve terminals labeled after 3H-MA infusion silver grains were equally very low in 3H-DA, 3H-NA or 3H-5HT, amounting to less than 6.1%.

Cytological evidence for different types of cerebrospinal fluid-contacting subependymal cells in the preoptic and infundibular recesses of the frog

SummaryBlue-green fluorescent subependymal cells with intraventricular processes were shown by the fluorescent histochemical method to be distributed from the preoptic recess to the infundibular recess of the frog hypothalamus. Electron microscopy revealed at least two types of CSF-contacting subependymal cells, type 1 containing large dense granules (about 100–200 nm in diameter) and type 2 containing small dense core vesicles (about 60–100 nm in diameter). Subsequent to fixation in permanganate solution, the small dense core vesicles in type 2 cells reacted with the fixative and consistently showed a dense content. However, the large granules in type 1 cells were mostly pale or less dense after this fixation.Two hours after intraventricular injection of 3H-dopamine, a large number of silver grains appeared only in the cytoplasm of intraventricular processes possessing dense core vesicles (type 2 cells). A few grains were also found in the perikarya. It is concluded that type 2 cells are catecholamine-storing cells. It is suggested that type 1 cells in the infundibular recess are peptidergic neurons which may secrete some hypothalamic regulating hormones of the anterior pituitary. Most of these cells in the preoptic recess belong to the neurosecretory cells of the preoptic nucleus, while some cells probably function similarly to those in the infundibular recess.

Catecholamine-Peptide Interactions in the Hypothalamus

Besides the neurohypophyseal hormones oxytocin (OT) and vasopressin (VP), which belong to the classic neurosecretory systems, several other kinds of biologically active peptide have been detected in the mammalian hypothalamus. Of the latter peptides, the following are known to be present in the hypothalamus: 1. Hypophyseotropic hormones, or so-called hypothalamic hormones — thyrotropin-releasing hormone (TRH); luteinizing hormone-releasing hormone (LHRH); corticotropin-releasing hormone (CRH, CRF); somatostatin (somatotropin release-inhibiting hormone, SRIF); and growth hormone-releasing factor (GRF) 2. Anterior pituitary hormones — corticotropin (ACTH) and others 3. Opioid peptides — endorphins, enkephalins, and dynorphins 4. Gastrointestinal peptides — neurotensin (NT), substance P, 5. Peptides in a central angiotensin system.

Distribution of PNP 14 (β-synuclein) in neuroendocrine tissues: Localization in Sertoli cells

Phosphoneuroprotein (PNP 14) is abundant in the central nervous system and is localized at nerve endings but not in synaptic vesicles. In this study, we examined the presence of PNP 14 in various endocrine tissues of the rat. PNP 14 was not detected in the endocrine cells of the intestine, testes, or adrenal gland, but it was present in axon terminals in both the medulla of the adrenal gland and the anterior pituitary gland.

Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching

SummaryFreeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per μm2. The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.

Ultrastructural demonstration of the catecholaminergic innervation of vasopressin neurons in the paraventricular nucleus of the rat by double-labeling immunocytochemistry

The catecholaminergic innervation of vasopressin (VP) neurons in the paraventricular nucleus (PVN) of the rat was studied at the electron microscopic level by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase method with the postembedding immunogold staining method. Tyrosine hydroxylase-like immunoreactive nerve terminals were found to establish synapses with neurophysin II-like immunoreactive neuronal perikarya and their processes. This provides morphological evidence for catecholaminergic control of the release of VP, at the PVN level.

Electron-Microscopic Immunocytochemistry of Neuropeptide Y Immunoreactive Innervation of Vasopressin Neurons in the Paraventricular Nucleus of the Rat Hypothalamus

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.

Ultrastructural demonstration of dopamine-β-hydroxylase immunoreactive nerve terminals on vasopressin neurons in the paraventricular nucleus of the rat by double-labeling immunocytochemistry

The noradrenergic innervation of vasopressin (VP) neurons in the paraventricular nucleus (PVN) of the rat was studied ultrastructurally by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase method for dopamine-beta-hydroxylase (DBH) with the post-embedding immunogold staining method for neurophysin II, the carrier protein of VP. DBH-like immunoreactive nerve terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neuronal perikarya and their processes. This provides morphological evidence for noradrenergic control of the release of VP, at the PVN of the rat.