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Journal Article 2012; 33(4): 393-400 PubMed PMID: 22936265 Citation  Keywords:  Animals, Cattle, Cerebellar Nuclei:cytology, Female, Glutamic Acid:metabolism, Hypothalamus:physiology, Immunity, Humoral:physiology, Male, Neuroimmunomodulation:physiology, Neurons:immunology, Rats, Rats, Sprague-Dawley, Serum Albumin, Bovine:immunology,.   

OBJECTIVES: We explored effect of glutamatergic neurons in the fastigial nucleus (FN), one of three cerebellar nuclei, on humoral immunity and revealed that this effect was mediated by the hypothalamus via FN-hypothalamic glutamatergic transmission.

METHODS: Rats were immunized with bovine serum albumin (BSA). On the third day after the immunization, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase for glutamate synthesis, was microinjected in bilateral FN and D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on plasma membrane, was microinjected in both sides of lateral hypothalamic area (LHA). Glutamate content in the hypothalamus was examined by high-performance liquid chromatography (HPLC). Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to measure B lymphocyte percentage in mononuclear cells of peripheral blood and levels of anti-BSA IgM and IgG antibodies in the serum, respectively.

RESULTS: DON injection in bilateral FN reduced B lymphocyte percentage and anti-BSA IgM and IgG levels, and simultaneously decreased glutamate content in the hypothalamus. Combined treatment with DON in the FN and with THA in the LHA elevated B cell number and anti-BSA IgM and IgG levels and increased hypothalamic glutamate content compared with DON treatment alone. However, combined treatment with DON in the FN and with THA in the ventrolateral thalamic nuclei (VL) did not significantly alter DON-dependent changes in B cell number and antibody levels, although the co-treatment altered DON-dependent glutamate content in the thalamus.

CONCLUSION: Cerebellar FN glutamatergic neurons participate in modulation of humoral immunity and this effect is mediated by the hypothalamus via FN-hypothalamic glutamatergic transmission....

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Journal Article 2012; 33(2): 124-132 PubMed PMID: 22592192 Citation  Keywords:  Animals, Gene Expression:physiology, Hypothalamo-Hypophyseal System:physiology, Male, Myocardium:cytology, Pituitary-Adrenal System:physiology, Protein Transport, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Rats, Wistar, Receptors, Oxytocin:biosynthesis.   

OBJECTIVES: Cardiovascular system is regulated by a diverse array of hormones, neurotransmitters and neuropeptides. Oxytocin and its receptors (OTR) were also shown to regulate cardiovascular functions and this hormone was even called cardiovascular hormone. In recent publication, we demonstrated the expression of mRNA of OTR by real-time quantitative PCR (RT qPCR) in all rat heart compartments. The aim of this study was to investigate the effects of acute restraint stress on OTR mRNA expression in two rat strains with different activity of HPA axis.

METHODS: Adult male Sprague-Dawley and Lewis rats, the latter strain reported to have lower HPA activity, were used in RT qPCR studies and Wistar rats in immunofluorescent ones. Both acute restraint (IS) and this stress combined with the immersion of rats in water (ICS) lasted 60 min. Gene expression of OTR mRNA was estimated in all heart compartments after 1 or 3 hours after stress termination (IS1, IS3, ICS1, ICS3). The relative expression was calculated using 2(-ΔΔC)T method. In immunofluorescent studies we used commercial specific OTR antibodies.

RESULTS: In RT qPCR studies we found higher expression of OTR mRNA in atria than in ventricles and no statistical differences between Sprague-Dawley and Lewis rats under basal conditions. Relative expression of OTR mRNA after 60 min lasting stress exposure differed in dependence on the stress type and partly on the time interval after the stress termination. When compared to controls, in rat left atria both stressors caused inhibition of OTR mRNA expression in both rat strains. In rat ventricles, which have very low OTR mRNA expression, there was a significant difference in the effect of two stressors. In most groups ICS displayed the increase of OTR mRNA expression if compared to IS groups. Immunofluorescent studies revealed changes induced by acute restraint stress in all heart compartments. The immunofluorescent studies suggested that acute stress induces higher colocalization of OTR with the nuclei than it was observed in the controls.

CONCLUSIONS: The expression of OTR mRNA in all heart compartments of controls as well as after stress exposure in Sprague-Dawley and Lewis rats support the notion that OTR plays a regulatory role in the cardiovascular system and is also involved in the regulations in the heart after stress. The immunofluorescent observation that OTRs coexpress in areas of cell nuclei in certain heart compartments and after acute stress, compared to controls, requires further studies....

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Journal Article 2011; 32(6): 805-810 PubMed PMID: 22286791 Citation  Keywords:  Animals, Hypothalamo-Hypophyseal System:physiology, Male, Myocardium:cytology, Oxytocin:metabolism, Pituitary-Adrenal System:physiology, RNA, Messenger:metabolism, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Receptors, Oxytocin:genetics,.   

OBJECTIVES: Oxytocin (OT) is a neuropeptide acting both as a peripheral hormone and in the brain as neurotransmitter and neuromodulator. In addition to its well-known effects on milk-ejection and uterine contraction, OT was shown to exert neuroendocrine regulation of heart functions. The aim of this study was to investigate the expression of mRNA of OT receptors (OTR) in rat hearts by real-time quantitative PCR (qPCR). The study was performed in Sprague-Dawley (SD) and Lewis (LE) rat strains, the latter having lower activity of HPA axis.

METHODS: We used adult male SD and LE rats. OTR mRNA expression was detected in all heart chambers by comparing their threshold cycle values (CT) to CT of reference gene β-actin. The relative expression ratios were calculated using the 2-ΔΔCT method. The specificity of reaction of primary antibody with OTRs was tested by Western Blot and localization of OTR in the heart compartments was performed by immunofluorescence with commercial OTR specific antibodies.

RESULTS: We found expression of OTR mRNA in all heart compartments. The expression of OTR mRNA in both atria (LA, RA) was much higher than in the ventricles (RV, LV). By using two-way ANOVA we found no statistical differences between corresponding compartments of SD and LE rats. Immunohistochemical studies showed that OTR staining is not related to neuronal tissue and findings from left atrium indicate that prevalent localization of OTR is on cell membranes of cardiomyocytes.

CONCLUSIONS: The finding of expression of OTR mRNA by real-time qPCR and proof of OTR staining by immunohistochemistry in all heart compartments indicate that OT and its receptors may have function as a cardiovascular hormone. The differences in the HPA axis activity, as is exemplified in Sprague-Dawley and Lewis rat strain, do not project in the expression of OTR mRNA under basal condition. The effect of activity of HPA on OTR expression should be studied under stimulated conditions as it was performed in the behavioral studies....

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Journal Article 2011; 32(6): 811-815 PubMed PMID: 22286789 Citation  Keywords:  Animals, Behavior, Animal:physiology, Estradiol:pharmacology, Estrogens:pharmacology, Facial Pain:physiopathology, Female, Male, Ovariectomy, Pain Measurement, Pain Threshold:drug effects, RNA, Messenger:metabolism, Random Allocation, Rats, Rats, Sprague-.   

OBJECTIVES: P2X3 receptors are expressed in trigeminal ganglia (TG) and participate in the transduction of facial pain. However, the mechanisms underlying P2X receptor-mediated nociception at different estrogen levels has not been examined.

METHODS: In this study, female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), and estrogen-treated groups. In each group, the facial mechanical pain threshold was tested and the TG were harvested for a real-time PCR analysis of P2X3 receptor mRNA and western blot analysis of protein level.

RESULTS: In OVX rats we found that the mechanical pain threshold was significantly decreased compared with that in sham rats. Estrogen replacement reversed the decrease. The expression of P2X3 mRNA level in TG from OVX rats was significantly increased, consistent with the enhanced P2X3 receptor in protein level. Estrogen replacement could decrease the expression of P2X3 receptor in both mRNA and protein level.

CONCLUSION: These results indicate that estrogen might modulate the transduction of facial pain by inhibiting P2X3 receptor in TG....

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Journal Article 2011; 32(6): 763-768 PubMed PMID: 22286787 Citation  Keywords:  Animals, Dehydration:physiopathology, Glucose Transport Proteins, Facilitative:metabolism, Glutamic Acid:metabolism, Male, Paraventricular Hypothalamic Nucleus:metabolism, Rats, Rats, Sprague-Dawley, Sodium:metabolism, Synaptosomes:metabolism, Water Depri.   

OBJECTIVES: The purpose of this study was to identify and characterize Na+-dependent, high affinity glutamate transporter (GLUT) activity in the hypothalamic paraventricular nucleus (PVN) and to compare GLUT activity in PVN of euhydrated versus water-deprived rats.

METHODS: Sprague-Dawley rats were deprived of water for two days before sacrifice. Control rats received water ad libitum. After sacrifice, PVN and cerebrum were removed and synaptosomes were prepared using standard techniques. Glutamate uptake was measured using [3H]-glutamate as substrate, physiological buffer, approximately 100 µg of synaptosomal tissue per assay and a Brandel cell harvester.

RESULTS: Glutamate uptake was saturable in PVN synaptosomes from euhydrated, control rats with a Vmax of 541 ± 22 pmol/min-mg protein (SEM) and Km of 17.6 ± 3.8 µM (SEM). In contrast, Vmax of glutamate uptake was 808 ± 58 pmol/min-mg protein in PVN of rats deprived of water for 2 days. This was significantly higher than controls (p<0.001). Km was 21.2 ± 7.3 µM and not significantly different from controls (NS).

CONCLUSIONS: Our results suggest that water deprivation of rats results in significantly higher synaptosomal glutamate uptake in PVN. Although the exact mechanism is unknown, increased transcription of the GLUT gene and/or increased cell surface expression of GLUT may contribute to the observed increase of glutamate uptake in dehydrated rats. Increased glutamate uptake may serve to restrict dehydration-induced activation of PVN efferent pathways specifically involved in release of neurohypophysial hormones and activation of sympathetic outflow that operate to maintain body fluid balance and cardiovascular function....

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Journal Article 2011; 32(4): 487-490 PubMed PMID: 21876485 Citation  Keywords:  Animals, Behavior, Animal:drug effects, Disease Models, Animal, Electroencephalography, Epilepsy, Absence:chemically induced, Grooming:drug effects, Male, Mastication:drug effects, Motor Activity:physiology, Rats, Rats, Sprague-Dawley, Veratridine:toxicit.   

OBJECTIVES: Veratridine was characterized previously as an experimental model of epilepsy in vitro. The aim of this preliminary investigation is to identify the pattern of seizure induced by this model in vivo.

MATERIAL AND METHODS: Veratridine (200 μg/kg) was administered intraperitoneally to male Sprague-Dawley rats and the electrical activity of the brain was recorded as surface electroencephalogram (EEG).

RESULTS: The animals developed behavioral effects manifested as grooming, masticatory movements, facial automatism and wet dog shakes (WDSs). There were episodes of complete quiescent periods for 2-5 minutes before the animals presumed activity which were repeated every 15-20 minutes. The seizure activity during this silent activity showed fast frequency signals in the surface EEG correlating with absence seizure. The WDS behaviour was associated with electrical spikes on the EEG. When the rats were pre-treated with 200mg/kg ethosuximide (ETX), EEG recordings did not display the same fast frequency signal as that observed in animals receiving veratridine only. The number and duration of WDSs were not altered by ETX (200-400 mg/kg).

CONCLUSION: Veratridine produced an absence like-seizure activity in the surface EEG, sensitive to ETX and correlates with its behavioural effects....

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Journal Article 2011; 32(2): 187-192 PubMed PMID: 21552198 Citation  Keywords:  Aging:metabolism, Animals, Animals, Newborn, Atrial Natriuretic Factor:metabolism, Cells, Cultured, Cochlea:cytology, Ear, Inner:physiology, Models, Animal, Rats, Rats, Sprague-Dawley, Receptors, Atrial Natriuretic Factor:metabolism, Stria Vascularis:cyto.   

OBJECTIVE: Atrial natriuretic peptide (ANP) regulates the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system. To assess the possible physiological role in the inner ear, we investigated the expression of ANP in primary culture of marginal cells, and then we detected the expression of ANP and its receptor (NPR-A) in cochlear tissues derived from neonatal and adult rats of various ages.

METHODS: Marginal cells were isolated from cochlear stria vascularis of the neonatal rats. The cultured cells were subsequently characterized by morphology, immunocytochemistry, and scanning electron microscope (SEM) analysis. In order to examine the expression of ANP in marginal cells as well as in the developing rats' cochleae, immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) were applied respectively.

RESULTS: The present study demonstrated typical characteristics of marginal cells, including cobblestone, polygonal monolayer, pleomorphic growth pattern and the expression of cell type-specific marker, and SEM analysis. Subsequently, immunoreactive product of ANP were found in cultured marginal cells. The mRNAs encoding ANP and NPR-A receptor was expressed in rats' cochleae from postembryonic stage to early postnatal period. During the maturation stage, ANP expression was gradually down-regulated, while the expression of NPR-A receptor was relatively stable.

CONCLUSION: ANP might be synthesized and secreted by marginal cells of stria vascularis, and could play an important role in modulating the microenvironment of the inner ear. In addition, ANP might contribute to development and growth process of cochlea....

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Journal Article 2010; 31(6): 761-765 PubMed PMID: 21196915 Citation  Keywords:  Animals, Circadian Rhythm:physiology, Male, Melatonin:secretion, Microscopy, Electron, Pineal Gland:physiology, Rats, Rats, Sprague-Dawley, Synapses:ultrastructure,.   

OBJECTIVE: The pineal gland is part of the circadian clock system and is under the predominant influence of the endogenous oscillator located in the suprachiasmatic nucleus. A polysynaptic pathway involving hypothalamus, spinal cord and sympathetic system regulates the so far best-studied aspect of its neuroendocrine output, i.e., the synthesis and secretion of melatonin. This parameter increases dramatically at night upon sympathetic activation in rats and many other mammals including man. In addition, parasympathetic, trigeminal, diencephalic and other sites or mechanisms connect the gland, mainly via its stalk, to the nervous system. However, their function for pineal metabolic or morphological features are hardly known. An interesting ultrastructural attribute of the pineal gland are ribbon synapses. These presynaptic structures in pinealocytes are composed of a ribbon and vesicles. They are thought to regulate and facilitate multivesicular release, and display a circadian rhythm with higher levels at night paralleling melatonin synthesis but regulated differently.

METHODS: To gain more insight into the roles of both, the non-sympathetic ("central") innervation and the regulation of pineal ribbon synapses, a surgical transection of the pineal stalk was conducted in rats and the number of synaptic ribbons (SR) were determined by electron microscopy from experimental, sham-operated and control animals.

RESULTS: The transection resulted in normal daytime levels but diminished the nocturnal increase of SR numbers when compared to controls or sham-operated rats.

CONCLUSION: These data provide first evidence that the central innervation of this neuroendocrine organ plays an important role in SR (up)regulation, and that this pathway is antagonistic to the sympathetic innervation....

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