Fumio Kawakami

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection.

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.

Age-Related Changes in Growth Hormone Releasing Factor and Somatostatin in the Rat Hypothalamus

Distribution and staining intensities of growth hormone releasing factor (GR (GRF) and somatostatin (SRIF) were examined in young (3 months of age) and old (24 months of age) male rats of Sprague-Dawley strain, using the PAP immunocytochemical procedure. Some animals of each age group were intraventricularly injected with colchicine to demonstrate immunoreactive neuronal perikarya. GRF-immunoreactive intensities of old rats were markedly reduced in the median eminence as compared with those of young rats. No remarkable difference could be detected between SRIF immunoreactivities in the young and old animals, since intensive SRIF immunoreactivities were found in the external layer of the median eminence of both groups of animals. Between two age groups injected with colchicine, we also found no difference in the distribution and staining intensities of immunoreactive perikarya of GRF and SRIF in the hypothalamus and also detected no significant difference in total neuron numbers of each peptide. These findings suggest that the synthesis and/or release of GRF in GRF-containing neurons are decreased, though GRF-containing neurons themselves remain alive and have the capacity to synthesize GRF.

Loss of day-night differences in VIP mRNA levels in the suprachiasmatic nucleus of aged rats

Age-related decreases in circadian oscillating activity are speculated to be one of the causes of psychiatric symptoms. To explore the effects of aging on vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN), we investigated the changes in VIP mRNA levels in aged rats compared with young-adult rats under a light/dark cycle using in situ hybridization combined with microcomputer-based imaging analysis. In the young-adult rats, total signals of VIP mRNA in the light-phase showed a significant decrease compared with those on the dark-phase. The VIP signal level in the aged rats was markedly lower than that in young-adults in both light and dark phases. Moreover, in the aged rats, there were no significant differences in VIP mRNA level between the light and dark phases. These results suggest that gene expression of VIP neurons, a main component of the circadian oscillating system, becomes disturbed in the aged rat brain.

Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in the rat hypothalamus, particularly in the arcuate nucleus, the paraventricular nucleus and the median eminence, were investigated by light and electron microscopic immunocytochemistry. The main distributional areas of immunoreactive neuronal perikarya were found to be the arcuate nucleus, the periventricular nucleus and the paraventricular nucleus by light microscopic immunocytochemistry. Immunoreactive neuronal perikarya showed a characteristic distributional pattern in the arcuate nucleus. In the paraventricular nucleus they were distributed in both the magnocellular and parvocellular portions. A large number of immunoreactive terminals were observed throughout the external layer of the median eminence, particularly its lateral portion. A moderate number of immunoreactive terminals were also observed in the internal layer of the median eminence. By electron microscopic immunocytochemistry immunoreactive neuronal perikarya both in the arcuate and paraventricular nuclei showed generally well-developed cell organelles such as mitochondria, r-ER, and Golgi complex. In addition, immunoreactive dense granules were dispersed throughout the perikarya. A large number of immunoreactive terminals containing immunoreactive dense granules, clear vesicles and mitochondria were observed in the vicinity of pericapillary spaces of the external layer of the median eminence. This observation strongly suggests that neurotensin-like immunoreactive substance is released into the portal capillaries.

The influence of serotonergic inputs on peptide neurons in the rat suprachiasmatic nucleus: An immunocytochemical study

The influence of serotonin (5-hydroxytryptamine; 5-HT) innervation on peptide-containing neurons in the rat suprachiasmatic nucleus (SCN) was investigated by peroxidase-anti-peroxidase (PAP) immunocytochemistry. The 5-HT neuronal system was chemically severed by 5,6-dihydroxytryptamine (5,6-DHT) injection into the medial forebrain bundle bilaterally. After this treatment, a marked decrease of vasoactive intestinal peptide (VIP)-like immunoreactivity in neuronal perikarya occurred in the SCN corresponding to a decrease in number of 5-HT immunoreactive fibers and terminals. However, no alteration of arginine-vasopressin-like immunoreactivity was detected between 5,6-DHT-treated animals and the controls. It is speculated that VIP-like immunoreactive neurons play an important role in the SCN under the influence of strong 5-HT innervation.

Light and electron microscopic immunocytochemistry of β-endorphin/β-LPH-like immunoreactive neurons in the arcuate nucleus and surrounding areas of the rat hypothalamus

beta-Endorphin/beta-LPH-like immunoreactive neurons in the hypothalamic arcuate nucleus and its surrounding areas were visualized by light and electron microscopic immunocytochemistry. Immunoreactive processes were found in the vicinity of the pia mater, in the lateral part of the external layer of the median eminence and near the lateral wall of the third ventricle. Neuronal perikarya contained immunoreactive dense granules as well as developed cell organellae. They received neuronal inputs from other neurons through axoplasmic and axodendritic synapses. Immunoreactive neuronal processes containing dense granules and mitochondria were found as preterminal elements on non-immunoreactive neuronal soma and dendrites. Immunoreactive processes also make intimate contact with capillaries in the arcuate nucleus near the median eminence.

Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment in constant darkness

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

Light and electron microscopic immunocytochemistry of GRF-like immunoreactive neurons and terminals in the rat hypothalamic arcuate nuclues and median eminence

Growth hormone-releasing factor (GRF) synthesizing neuronal perikarya and terminals were investigated by light and electron microscopic immunocytochemistry using rat hypothalamus. Immunoreactive neuronal perikarya were located mainly in the ventrolateral part of the arcuate nucleus. They contained well developed cell organella such as mitochondria and rough surfaced endoplasmic reticulum with some expansion. They also contained immunoreactive dense granules (80-120 nm in diameter). On the surface of the immunoreactive neuronal perikarya were frequently found non-immunoreactive axo-somatic synapses. Therefore, the GRF-like immunoreactive neurons were assumed to receive neuronal inputs from other neurons on their neuronal soma. In the external layer of the median eminence large numbers of immunoreactive terminals were distributed particularly around the capillaries of the portal vessel. Electron microscopic immunocytochemistry revealed large numbers of immunoreactive terminals containing immunoreactive dense granules, synaptic vesicles and mitochondria in the vicinity of the basement membrane of the pericapillary space of the portal vessel. Therefore, we concluded that GRF-like immunoreactive substances are released into the portal capillaries from the nerve terminals, which originate from the neuronal perikarya in the ventrolateral part of the arcuate nucleus, and act on growth hormone release in the anterior pituitary. We also suggest that GRF-like immunoreactive neurons have abundant terminal arborization in the external layer of the median eminence.

Morphological survey of neurotensin-like immunoreactive neurons in the hypothalamus ☆

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in in the rat hypothalamus were investigated by light and electron microscopic immunocytochemistry. Distributional density and pattern of these elements were clarified. Fine structure of immunoreactive neuronal perikarya with respect to development of cell organellae and immunoreactive dense granules was also elucidated. Features of immunoreactive processes, dendrites and preterminal axons were examined electron microscopically. In addition to the above findings by light and electron microscopic immunocytochemistry, we examined the coexistence of dopamine and neurotensin-like immunoreactive substances in these same neurons in the arcuate and periventricular nuclei. This was proved by the application of fluorescence histochemistry and immunocytochemistry on the same sections. Moreover, we speculated that the ascending noradrenergic neurons influence the neurotensin immunoreactive neurons in the paraventricular nucleus since a marked decrease in the number of neurotensin-like immunoreactive neuronal perikarya was observed after transection of ascending noradrenergic pathway.

Serotonin depletion by p-chlorophenylalanine decreases VIP mRNA in the suprachiasmatic nucleus.

The influence of serotonergic inputs on the levels of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN) was examined by in situ hybridization combined with morphometrical analysis. Depletion of serotonin by p-chlorophenylalanine methyl ester (300 mg/kg i.p. daily for 3 days) caused a marked decrease in VIP mRNA signal levels in the ventrolateral part of the SCN. This finding suggests that serotonergic input controls VIP production at the mRNA level.