Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka

Orexin/hypocretin is a neuropeptide that is involved in the regulation of feeding behavior and the sleep–wakefulness cycle in mammals. Melanin-concentrating hormone (MCH) is believed to be another candidate involved in food intake in teleost fish as well. Thus, it is interesting to examine whether neural connections exist between the neurons producing these two hormones. We first examined the localization of orexin-like immunoreactivity (orexin-LI) in the brain of the medaka Oryzias latipes by using immunohistochemistry. We further examined the interaction between the orexin and MCH neurons in the medaka brain by performing double-staining immunohistochemistry. Orexin-LI cell bodies were located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-LI fibers were detected not only in the hypothalamus but also extensively throughout the brain. Some orexin-LI fibers were in close contact with the MCH-immunoreactive (ir) cell bodies in the hypothalamus, as revealed by double-staining immunohistochemistry. Moreover, a few MCH-ir fibers were in close contact with the orexin-LI cell bodies. These results suggest that in the medaka brain, orexin performs various functions, including neuromodulation, and that neural connections exist between the orexin and MCH neurons.     

Immunocytochemical detection of the neurokinin B receptor (NK3) on melanin-concentrating hormone (MCH) neurons in rat brain

The presence of the neurokinin B receptor (NK3 receptor) in the rat lateral hypothalamus and the zona incerta was previously reported. The aim of the present study was to define its cellular localization in these areas. Investigations, coupling immunocytochemical and in situ hybridization techniques, focussed on two neuron populations: the melanin-concentrating hormone (MCH) neurons and a population of neurons recognized by an ovine prolactin antiserum (PRL-ir neurons). While PRL-ir neurons did not exhibit NK3 immunoreactivity, 57%±6% of MCH neurons were strongly stained by the NK3 antiserum. These results suggest that neurokinin B is involved in the regulation of MCH neuron activity via the NK3 receptor; they provide new bases for further investigations on MCH role in the control of food and water intake.     

Testosterone-driven seasonal regulation of vasopressin and galanin in the bed nucleus of the stria terminalis of the Djungarian hamster (Phodopus sungorus)

The sexually dimorphic vasopressin system of the bed nucleus of the stria terminalis (BNST) is the most sensitive neurotransmitter system regulated by sex steroids in rats and mice. In addition to vasopressin, the BNST neurons also express a second neuropeptide, galanin, whose expression also appears to be regulated by testosterone in laboratory rodents. Seasonal fluctuations of sex steroids in photoperiodic rodents feed back on the brain to regulate the expression of sex steroid sensitive genes. The seasonal rhythm of circulating sex steroids is generated by photoperiod-controlled melatonin secretion, resulting in a seasonal stimulation and involution of the gonads. We have studied the seasonal expression of vasopressin and galanin in BNST neurons and their target areas in the Djungarian hamster (Phodopus sungorus). Furthermore, we analyzed the effect of testosterone on vasopressin and galanin by testosterone supplementation in animals where reproduction was inhibited by exposure to a short photoperiod. Exposure to short photoperiod induced a major reduction in the expression of vasopressin in BNST neurons, as well as in their target areas, the lateral septum (LS) and the lateral habenula (LHb). Galanin expression in the BNST and its target areas was also strongly reduced, although this reduction did not result in an almost complete disappearance of the neuropeptide as observed for vasopressin. Testosterone was able to reverse this reduction for both vasopressin and galanin. However, while the mRNA expression in BNST neurons recovered within 2-4 days, recovery of the neuropeptide immunoreactivity in the target areas, LS and LHb, required more than 3 weeks. The photoperiod-driven testosterone rhythm thus appears to be a major regulator of extra-hypothalamic vasopressin and galanin in the Djungarian hamster. The long delay between mRNA recovery in the cell body and the neuropeptide recovery in the target areas may be due to progressive filling up of the axon terminals. Alternatively, this delay might be indicative of a seasonal structural plasticity.     

Co-expression patterns of cocaine- and amphetamine-regulated transcript (CART) with neuropeptides in dorsal root ganglia of the pig

In the present study the neuronal distribution of CART was evaluated immunohistochemically in porcine dorsal root ganglia (DRGs). In co-localization studies the co-expression patterns of CART with SP, CGRP, galanin, CALB and LENK were investigated by means of triple immunohistochemical stainings. In porcine DRGs, the expression of CART was found in approximately 5% of primary sensory neurons. The vast majority (ca. 95%) of CART-immunoreactive (IR) neurons were small and middle sized, and only 5% were categorized as large. CART-IR neurons additionally exhibiting the presence of SP/CGRP (ca. 12%), SP/CALB (ca. 12%), SP/LENK (ca. 5%) were found. The vast majority of CART-IR/CGRP-IR neurons did not display immunoreaction to SP (ca. 60%). Subclasses of CART-IR/LENK-IR/SP-negative (ca. 5%), as well as CART-IR/CALB-IR/SP-negative neurons (ca. 10%), were also visualized. In addition, CART-IR neurons with no immunoreactivities to any of the neuropeptides studied were also shown. In porcine DRGs none of the CART-IR neurons exhibited the presence of galanin. The results obtained in the study suggest that CART may functionally modulate the activity of the porcine primary sensory neurons. It is concluded that co-expression of CART with CGRP, SP, LENK and CALB in subsets of the pig L1-L6 DRGs neurons provide anatomical evidence for a CART role in pain processing.     

Vitamin D nuclear binding to neurons of the septal, substriatal and amygdaloid area in the Siberian hamster (Phodopus sungorus) brain.

Autoradiographic experiments were performed on brains of Siberian hamsters (Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alzheimer's disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.     

Coexistence of acetylcholinesterase-, human growth hormone-releasing factor(1–37)-, α-melanotropin- and melanin-concentrating hormone-like immunoreactivities in neurons of the rat hypothalamus: a light and electron microscope study

Using an antiserum (AS) raised against rat cerebral acetylcholinesterase (AChE), we revealed a neuron population in lateral and dorsal areas of the posterior rat hypothalamus. These neurons were previously described using antibodies to human growth hormone-releasing factor(1-37) (GRF-37), alpha-melanotropin (alpha-MSH) and melanin-concentrating hormone (MCH). Different intracytoplasmic distributions of the immunodeposits were observed depending on the used serum. Ultrastructural investigations demonstrated that AChE-AS labeled rough endoplasmic reticulum and nuclear envelope in control rats. MCH-AS stained Golgi apparatus in control animals and secretory granules in colchicine-injected rats. GRF-37-AS always revealed secretory granules, and alpha-MSH-AS gave the same staining only after colchicine injection.     

Enteroantigen (eAg)‐binding B lymphocytes in the mouse – phenotype,distribution, function and eAg‐specific antibody secretion

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co‐transplanted with Treg‐depleted (CD4+CD25?) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg‐presenting cells than unfractionated splenocytes or eAg? B cells in causing proliferation of eAg‐specific T cells. In comparison with eAg? B cells, eAg+ B cells secrete a significant amount of IL‐10 in vitro, suggesting an anti‐inflammatory potential. Compared with wild‐type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme‐specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19⁺ B cells by eAg and hybridization with hypoxanthine‐aminopterin‐thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg‐specific antibodies.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.