Immunohistochemical localization of the neural cannabinoid receptor in rat brain

A sample of UK consumers (N = 311) was interviewed in order to identify the attitudinal, cognitive and involvement characteristics of probable early adopters of polyunsaturated fatty acid (PUFA) fed fish. Attitude to fish significantly influenced PUFA fish, premium price PUFA fish, PUFA salmon, PUFA eel and PUFA sturgeon purchase. Involvement in healthy eating influenced PUFA fish, premium price PUFA fish and PUFA salmon purchase. Cognitive style did not influence PUFA fish and premium price PUFA fish purchase; nor, contrary to earlier research, did cognitive style and involvement interact to influence intended PUFA fish purchases.     

Immunohistochemical localization of UDP-glucuronosyltransferases in rat brain during early development

OBJECTIVE: Benign tumors account for less than 1% of testicular tumors and the incidence is even lower in children. A rare case of epidermoid cyst of the testis in a child is described. The differential diagnosis and treatment options are discussed. METHODS/RESULTS: A case of unilateral epidermoid cyst of the testis in an 11-year-old boy is presented. The clinical and diagnostic aspects are discussed. Definitive diagnosis could be made only after surgical excision. CONCLUSIONS: Pathological analysis of the entire testis is warranted to make the definitive diagnosis of epidermoid cyst. However, preservation of the testis can be considered, particularly in those cases with bilateral involvement, if supported by solid, consistent diagnostic evidence, including intraoperative biopsy.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

Diazepam alters caffeine-induced effects on beta-endorphin levels in specific rat brain regions

The present study was conducted to evaluate the effects of caffeine and the benzodiazepine agonist diazepam, and a combination of both on beta-endorphin (beta-EN) levels in specific rat brain regions. Male Sprague-Dawley rats (150-200 g) adapted to a 12-hour light: 12-hour dark illumination cycle were used in this study. Caffeine (10 mg/kg), diazepam (2 mg/kg) or a combination of caffeine (10 mg/kg) and diazepam (2 mg/kg) were administered intraperitoneally to rats at 11:00 hr. Control animals were injected with saline. Animals were sacrificed by decapitation 1 h after injection, the brains were immediately removed; the cortex, hippocampus, hypothalamus and midbrain were dissected and their B-EN levels measured by radioimmunoassay. Caffeine administration significantly increased B-EN levels in the cortex. Similarly, administration of diazepam alone resulted in a significant increase of B-EN levels in cortex. However, concurrent administration of diazepam and caffeine resulted in higher increase of B-EN levels in cortex. No significant changes in B-EN levels were detected in hippocampus and midbrain after administration of either caffeine or diazepam alone. On the other hand, when diazepam and caffeine were concurrently administered a significant increase of B-EN levels were observed in the midbrain. Moreover, administration of diazepam alone resulted in a significant increase of B-EN levels in hypothalamus. This increase was still observed following concurrent administration of diazepam and caffeine. These results clearly indicate that diazepam alters caffeine-induced effects on B-EN in specific rat brain regions.     

Immunohistochemical localization of enkephalin in peripheral sensory axons in the rat

Impaired energy metabolism plays an important role in neuronal cell death after brain ischemia, and apoptosis has been implicated in cell death induced by metabolic impairment. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. In order to clarify the involvement of poly(ADP-ribosyl)ation and apoptotic pathway in 3-NP induced cell death, we examined poly(ADP-ribosyl)ation and the apoptosis related gene protein expression after systemic administration of 3-NP by immunohistochemistry. Poly(ADP-ribosyl)ation was evidently detected in the striatal lesion but not in any other region. Immunoreactive ratio of Bcl-2 to Bax significantly increased both in the striatum and cortex. The data suggest that striatal cell death involves poly(ADP-ribosyl)ation and also apoptotic pathway in part following administration of 3-NP.     

The N-methyl-D-aspartate receptor pathway is involved in hypoxia-induced c-Fos protein expression in the rat nucleus of the solitary tract

In chloroplast photosystem II, the extrinsic polypeptide of 33 kDa is involved in the stabilization the Mn cluster in charge of water splitting and in the fulfilment of the Ca(2+)-cofactor requirement for oxygen evolution. The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures. The FTIR spectroscopic results showed that the isolated polypeptide is characterized by a major proportion beta-sheet conformation (36%) with 27% alpha-helix, 24% turn, and 13% beta-antiparallel structures.     

Differential regional expression and ultrastructural localization of alpha-actinin-2, a putative NMDA receptor-anchoring protein, in rat brain

Fast chemical neurotransmission is dependent on ionotropic receptors that are concentrated and immobilized at specific postsynaptic sites. The mechanisms of receptor clustering and anchoring in neuronal synapses are poorly understood but presumably involve molecular linkage of membrane receptor proteins to the postsynaptic cytoskeleton. Recently the actin-binding protein alpha-actinin-2 was shown to bind directly to the NMDA receptor subunits NR1 and NR2B (), suggesting that alpha-actinin-2 may function to attach NMDA receptors to the actin cytoskeleton. Here we show that alpha-actinin-2 is localized specifically in glutamatergic synapses in cultured hippocampal neurons. By immunogold electron microscopy, alpha-actinin-2 is concentrated over the postsynaptic density (PSD) of numerous asymmetric synapses where it colocalizes with NR1 immunoreactivity. Thus alpha-actinin-2 is appropriately positioned at the ultrastructural level to function as a postsynaptic-anchoring protein for NMDA receptors. alpha-Actinin-2 is not, however, exclusively found at the PSD; immunogold labeling was also associated with filaments and the spine apparatus of dendritic spines and with microtubules in dendritic shafts. alpha-Actinin-2 showed marked differential regional expression in rat brain. For instance, the protein is expressed at much higher levels in dentate gyrus than in area CA1 of the hippocampus. This differential regional expression implies that glutamatergic synapses in various parts of the brain differ with respect to their alpha-actinin-2 content and thus, potentially, the extent of possible interaction between alpha-actinin-2 and the NMDA receptor.     

Immunohistochemical localization of peroxisomal enzymes in developing rat kidney tissues

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, l-alpha-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.     

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas

Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.     

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.     

Immunohistochemical localization of cytosolic sialidase in the epithelium of rat cornea and conjunctiva

OBJECTIVE: To compare the hemodynamic change, course of recovery and adverse reaction in desflurane, sevoflurane and enflurane inhalation under low flow for patients undergoing selective abdominal surgery. METHODS: Following thiopental induction, 42 patients were divided into three groups: the first group received desflurane, the second sevoflurane and the third enflurane. During surgery, one of the agents around 1 minimum alveolar concentration (MAC) was used for maintenance, with fresh gas flow of 0.3-0.5 L/min for either desflurane or enflurane, and (0.8-1.0) L/min for sevoflurane. Heart rate (HR), blood pressure and end-tidal anesthetic concentration were monitored continuously. Time intervals from cutting off anesthetic to patient opening eyes, following commands, stating the time and location and recalling date of birth were all recorded. In addition, postoperative nausea or vomiting was traced. RESULTS: Desflurane caused the least cardiovascular depression. with mean arterial pressure (MAP) maintained significantly better at 10, 30 and 60 minutes of surgery and with HR stabilized right after incision as well. Its emergence was 2 times faster than sevoflurane, and 5-6 times quicker than enflurane. However, nausea or vomiting was found the lowest in patients receiving sevoflurane, though no distinct difference was shown between desflurane and enflurane. Nevertheless, patients under desflurane suffered less. CONCLUSIONS: Desflurane offers significant advantages for clinical anesthesia maintenance over sevoflurane and enflurane. It provides minimal cardiovascular depression, much quicker recovery, yet still causes some nausea during emergence.     

Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.     

c-Fos expression in brain in response to hypotension and hypertension in conscious rats

Hypotension- and hypertension-evoked expression of the protein product, Fos, of the immediate early gene c-fos was assessed throughout the rat brain as an approach for describing the neuronal populations that respond to alterations in arterial blood pressure. Conscious, chronically catheterized rats were treated with the vasoconstricting drug phenylephrine or the vasodilatating drug hydralazine to increase or decrease, respectively, arterial pressure by approx. 40 mm Hg for 90 min. Rats were then anesthetized, fixed by vascular perfusion, and sections representing the entire brain were processed for the immunocytochemical localization of Fos. In control rats treated with isotonic saline, few Fos-positive neurons were observed. In contrast, phenylephrine and hydralazine treatments resulted in different, yet reproducible, patterns of Fos expression in the brain, with hydralazine evoking Fos expression in more brain regions than phenylephrine. Brain regions containing Fos-positive neurons in rats treated with hydralazine included nucleus tractus solitarius, area postrema, caudal ventrolateral medulla, rostral ventrolateral medulla, bed nucleus of the stria terminalis, amygdala, paraventricular nucleus, supraoptic nucleus, subfornical organ and the Islands of Calleja. The nucleus tractus solitarius, paraventricular nucleus and the amygdala also contained Fos-positive neurons in phenylephrine-treated rats, although the number of Fos-positive neurons was always less than that noted in the hydralazine-treated rats and the location of Fos-positive neurons within these regions tended to differ between treatments. These results generally fit within an emerging understanding of brain circuitry underlying cardiovascular regulation.     

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.