Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species.

A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species.     

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.     

Localization of nerve growth factor-like immunoreactivity in rat nervous tissue

The use of immunofluorescence with affinity-purified antibodies enabled cytological localization of nerve growth factor-like material in the rat. Immunoreactivity was observed along various nerve tracts of the foetal rat brain and spinal cord at day 15 of gestation. Longitudinal pathways in ventral and dorsal spinal cord, ventral lower brain stem, posterior commissure, retroflex fascicle and in the olfactory bulb were all positive. A weaker and more widely spread immunostaining was visible in many areas in the central nervous system. Cranial nerves were strongly immunoreactive. Neuronal perikarya in the retina and the olfactory mucosa as well as filae olfactoriae and the olfactory nerve all the way to the olfactory bulb were also positive. In sensory ganglia and peripheral nerves most immunoreactivity was confined to supporting tissues, probably including Schwann cells. In irides, the pattern of immunoreactivity was similar to that of the sensory and autonomic innervation. More intensively fluorescent material was found in regrowing nerve fibres in iris transplants. Our histochemical results suggest that nerve growth factor and/or a related protein is present in large amounts along nerve pathways in supportive tissues of the peripheral nervous system as well as in the central nervous system during early development.     

Serotonin-like immunoreactivity in the central and peripheral nervous systems of the interstitial acochlidean Asperspina sp. (Opisthobranchia)

Species of Acochlidea are common members of the marine interstitial environment and defined in part by their minuscule size and highly divergent morphology relative to other benthic opisthobranchs. Despite these differences, acochlideans such as species of Asperspina display many plesiomorphic characteristics, including an unfused condition of their neural ganglia. To gain insight into the distribution of specific neural subsets within acochlidean ganglia, a species of Asperspina was studied by using anti-serotonin immunohistochemistry and epifluorescence and confocal laser scanning microscopy. Results reveal similarities between Asperspina and larger opisthobranchs in the general distribution of serotonergic perikarya in the central nervous system. Specifically, the arrangement of perikarya into regional clusters within the cerebral and pedal ganglia and the absence of immunoreactive perikarya in the pleural ganglia are similar to the model species of Aplysia californica, Pleurobranchaea californica, and Tritonia diomedea. Moreover, serotonergic innervation of the rhinophores in all opisthobranchs, including Asperspina sp., originates from the cerebral ganglion instead of directly from the rhinophoral ganglion. Serotonergic innervation of the body wall, including the epithelium, muscles, and pedal sole, appears to arise exclusively from pedal and accessory ganglia. These observations indicate a general conservation of serotonin-like immunoreactivity in the central and peripheral nervous systems of acochlidean and other benthic opisthobranchs.     

Distribution of PACAP-like immunoreactivity in the nervous system of oligochaeta

The marked similarity between the primary structures of human, other vertebrate, and the invertebrate tunicate PACAP suggests that PACAP is one of the most highly conserved peptides during the phylogeny of the metazoans. We investigated the distribution of PACAP-like immunoreactivity in the nervous system of three oligochaete (Annelida) worms with immunocytochemistry. The distribution pattern of immunoreactivity was similar in all three species (Lumbricus terrestris, Eisenia fetida, and Lumbricus polyphemus). The cerebral ganglion contains numerous immunoreactive cells and fibers. A few cells and fibers were found in the medial and lateral parts of the subesophageal and ventral cord ganglia. In the peripheral nervous system, immunoreactivity was found in the enteric nervous system, in epidermal sensory cells, and in the clitellum.     

Developmental expression of hexokinase 1 and 3 in rats

 Mammalian hexokinase types one and three (HK1 and HK3) are 100 kDa isozymes that phosphorylate glucose to glucose-6-phosphate. HK1 is present in most tissues but is especially prominent in brain and kidney. HK3 is less well studied, but may be most prominent in the spleen and lymphocytes. In this study, we determined the ontogeny of the expression of these isoforms in the rat. Using immunohistochemistry, we identified HK1 and HK3 immunoreactivity in the brain, heart, kidney, liver, skeletal muscle and spleen from gestational day 14 (E14) to 45 days after birth (P45). With the exception of the liver and spleen, we observed a similar age- and cell-dependent staining pattern for both isoforms in all organs studied. The brain and spleen were analyzed in more detail to identify specific regions of immunoreactivity during maturation. A transient expression of HK1 and HK3 was noted in the cell bodies of mature neurons, including layers V and VI of the cerebral cortex and the cerebellar Purkinje cells followed by localization to the white matter of the cerebrum and cerebellum. In the spleen, HK3 immunoreactivity was detected postnatally and appeared to track with the infiltration of B cells. Our demonstration of changing patterns of immunoreactivity for HK1 and HK3 in fetal and postnatal organs suggests that these HK isoforms are involved the process of development. We speculate that HK1 and HK3 share a complex interaction during development of these organs and regulate glucose metabolism at multiple levels during development. Accepted: 16 May 1997     

Select 3′,5′-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain

3′,5′-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26 months) versus young (5 months) Sprague–Dawley rats, with select experiments replicated in old (9 months) versus young (2 months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.     

Immunocytochemical localization of glycogen phosphorylase in primary sensory ganglia of the peripheral nervous system of the rat

Neuronal localization was investigated of glycogen phosphorylase (GP) in ganglia of the peripheral nervous system of the rat. Immunofluorescence and immunoenzymatic procedures were applied with a monoclonal anti-bovine brain GP antibody on paraformaldehyde-fixed, paraffin-embedded tissues. Immunoreactivity was only present in the somatic neurons of the mesencephalic trigeminal nucleus in the brain stem and in dorsal root ganglia (DRG), but not in the autonomic neurons of the superior cervical ganglia or in the sensory nuclei of the spinal cord. GP immunoreactivity was present as early as day 1 after birth. In the adult rat, staining was present in neurons of different sizes, and to varying intensities. No relationship was apparent between the staining intensities and morphologically distinguishable types of neurons. In DRG, the type of reactivity was the same from cervical to sacral ganglia. The selected occurrence of GP in specific neurons of the peripheral nervous system in contrast to the ubiquitous occurrence in all astrocytes of the central nervous system may indicate a different role of neuronal glycogen compared to astrocytic glycogen.     

Taurine in the insect central nervous system

1. Taurine is one of the most abundant free amino acids found in the tissues of insect nervous systems. A brief survey of its immunocytochemical distribution is provided for the brain of worker honeybees.2. The protocerebral mushroom bodies are prominent neuropiles of the insect brain. Immunoreactivity for taurine was compared in the mushroom body intrinsic Kenyon cells of Apis, Drosophila, and Locusta.3. In all three species Kenyon cells expressed immunoreactivity.4. The intensity of the immunoreactivity was, however, graded, depending on the species.5. Recent technical advances in the primary culture of the Kenyon cells of honeybees in a defined taurine-free medium provide the opportunity to investigate the action of taurine in a controlled environment.6. Taurine-like immunoreactivity has been described in the photoreceptor cells of insect and mammalian visual systems. Physiological evidence for similar functions of taurine in mammalian and insect nervous systems is reviewed.     

In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.)

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.     

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH₂. To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons. This work was supported by grants from the Ministry of Education, Sports, and Culture, Japan.     

Double-labelling data on somatostatin-like and tyrosine-hydroxylase-like immunoreactivities in chick embryo brain stem. I. Pons and mesencephalon.

With the aim of investigating some factors and mechanisms of the chicken brain development, the same thick sections of brain stems from twelve E13-to-E21-aged chick embryos were sequentially tested with a rabbit anti-Somatostatin antiserum, using a PAP-DAB technique, and with anti-tyrosine hydroxylase (-TH) monoclonal antibodies, using an indirect immuno-fluorescence technique. As regards the pons and mesencephalon, the following main results were obtained. At E21 almost the same distribution of the TH-like immunoreactivity (ir) as at E13 was observed. Neuroblasts in a central, relatively wide region of mesencephalic tegmentum and in the central portion of the pons showed TH-like ir. A co-localization of the 2 immunoreactivities was detected only at E18, within some neuroblasts of the mesencephalic and pontine regions with TH-like ir. It is possible that this transitory co-localization plays a role in the development of the pons and mesencephalon of this species.     

Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact. (J Histochem Cytochem 57:763–774, 2009)     

P2X(3) receptor expression at early stage of mouse embryogenesis

In this study we examined the expression of P2X(3) receptor in mouse embryos from E9.5 to E14.5 using immunohistochemistry. We found a uniform labeling in the developing trigeminal and dorsal root ganglia (DRG), while adult DRG and trigeminal ganglia expressed P2X(3) only in small-diameter neurons. In the brainstem, the mesencephalic trigeminal and facial motor nuclei were immunoreactive for P2X(3). P2X(3) was also transiently expressed in the developing brain, and precursors of spinal motor neurons. We also detected immunolabeling in the paravertebral sympathetic chain ganglia, in the sympathoadrenal cells and in non-neural tissues including testis, epidermis, wall of the aorta, as well as in subepidermal structures and mesenchymal tissues of limbs, branchial arches and tail.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.