Effect of Sustained Physiological Hyperinsulinaemia on Hypothalamic Neuropeptide Y and NPY mRNA Levels in the Rat

Neuropeptide Y (NPY) synthesized in the arcuato-paraventricular projection in the rat hypothalamus is thought to play an important role in controlling energy homeostasis. The factors that regulate hypothalamic NPY are not known but, amongst others, insulin has been postulated as an inhibitory modulatory agent. To test this hypothesis, normal male rats were given either insulin (2 units/day) or saline via subcutaneous osmotic minipumps for 3 days. Euglycaemia was maintained by a concomitant glucose infusion in insulin-infused rats which had peripheral insulin levels 5–8 times higher than saline-infused controls. Hyperinsulinaernic rats ate 42% less than controls, but their total energy intake (food intake plus glucose infusion) was higher than that of controls, and they gained more weight than controls during the experimental period. Hyperinsulinaemia had no significant effect on hypothalamic NPY mRNA or NPY levels in the arcuate nucleus. NPY concentrations in the paraventricular nucleus were, however, significantly increased by 73% in hyperinsulinaemic rats, but were closely similar to controls in all other areas. Insulin may act as a satiety factor in that hyperinsulinaemic rats ate less, but the fact that these animals had increased total energy intake and gained excessive weight suggests that insulin may not function as an overall regulator of energy balance. In addition, physiological hyperinsulinaemia does not apparently inhibit NPY gene expression in the arcuate nucleus. Due to the lack of effect of hyperinsulinaemia on NPY synthesis in the arcuate nucleus, the elevated NPY concentrations in the paraventricular nucleus could result from a reduction of its release, which would be in keeping with the reduction in food intake.     

Protein kinase C epsilon contributes to basal and sensitizing responses of TRPV1 to capsaicin in rat dorsal root ganglion neurons

Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCepsilon) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCepsilon (DNPKCepsilon) as a strategy to demonstrate that PKCepsilon is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCepsilon to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in-vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS-treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCepsilon is essential for normal TRPV1 responses in vitro and in vivo.     

Neuroactive Steroid Levels in a Transgenic Rat Model of CMT1A Neuropathy

Charcot–Marie–Tooth type 1A (CMT1A) represents 80% of all the demyelinating hereditary motor and sensory neuropathies. As recently suggested, neuroactive steroids may have a role in a therapeutic strategy for peripheral neuropathies, including CMT1A. To this aim, an accurate qualitative and quantitative analysis of neuroactive steroid levels in this disease could be extremely important to define effective pharmacological strategies. We here analyzed by liquid chromatography-tandem mass spectrometry the levels of neuroactive steroids present in the sciatic nerve of male and female peripheral myelin protein 22 transgenic rats (PMP22_tg rats; i.e., an experimental model of CMT1A) and of the corresponding wild-type littermates. We observed that, both in PMP22_tg rats and in the wild types, the levels of neuroactive steroids, such as progesterone, tetrahydroprogesterone (THP), isopregnanolone (3β,5α-THP), testosterone, dihydrotestosterone, and 5α-androstane-3α, 17β-diol (3α-diol) are sexually dimorphic. It is interesting to note that the levels of 3β,5α-THP and of 3α-diol, which are exclusively detectable in sciatic nerve of female and male rats, respectively, are strongly decreased in PMP22_tg rats. 3β,5α-THP and 3α-diol are modulators of gamma-amino butyric acid A receptor. Thus, the present findings may be considered an interesting background for experiments aimed to evaluate the possible therapeutic effects of modulators of this neurotransmitter receptor in male and female PMP22_tg rats.     

BDN F Down-regulates Neurotrophin Responsiveness, TrkB Protein and TrkB mRNA Levels in Cultured Rat Hippocampal Neurons

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal TrkB protein does not appear to be just a developmental phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro , this leads to long-term functional desensitization to BDNF. NT-3 and NT-4/5. as well as down-regulation of trkB and trkC mRNA.     

Perinatal Oxygen Restriction Does Not Result in Reduced Rat Frontal Cortex Synaptophysin Protein Levels at Adulthood as Opposed to Postmortem Findings in Schizophrenia

Synaptophysin, a synaptic vesicle protein and a marker for synaptic density has been found to be reduced in postmortem prefrontal cortex of schizophrenia patients, consistent with evidence for synaptic deficits in schizophrenia. The contribution of both genetic and environmental factors to the etiology of schizophrenia is well established, and obstetric complications have been suggested as a non-genetic risk factor of schizophrenia. As there is only scarce evidence for a genetic linkage between synaptophysin’s chromosomal locus (Xp11.22) and schizophrenia, we hypothesized that early neonatal exposure of rat pups to oxygen restriction would result in reduced frontal cortex synaptophysin protein levels at adulthood. We studied the effects of anoxia or hypoxia on 7-day-old rats frontal cortex synaptophysin protein levels assessed by Western blotting 4 and 7 weeks following the exposure. In hypoxia- or anoxia-exposed rats, synaptophysin protein levels were elevated both 4 and 7 weeks after the exposure. Two-way ANOVA followed by post hoc LSD analysis showed that the effect was predominantly at 4 weeks after exposure and that only anoxia-exposed rats differed significantly from control rats (p = 0.019). These results are in contrast to postmortem findings in schizophrenia and suggest that reduced synaptophysin protein levels in schizophrenia patients’ postmortem brain do not result from perinatal oxygen deprivation.     

Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina

The present study aimed to analyze the gene and protein expression and the pattern of distribution of the vanilloid receptors TRPV1 and TRPV2 in the developing rat retina. During the early phases of development, TRPV1 was found mainly in the neuroblastic layer of the retina and in the pigmented epithelium. In the adult, TRPV1 was found in microglial cells, blood vessels, astrocytes and in neuronal structures, namely synaptic boutons of both retinal plexiform layers, as well as in cell bodies of the inner nuclear layer and the ganglion cell layer. The pattern of distribution of TRPV1 was mainly punctate, and there was higher TRPV1 labeling in the peripheral retina than in central regions. TRPV2 expression was quite distinct. Its expression was virtually undetectable by immunoblotting before P1, and that receptor was found by immunohistochemistry only by postnatal day 15 (P15). RNA and protein analysis showed that the adult levels are only reached by P60, which includes small processes in the retinal plexiform layers, and labeled cellular bodies in the inner nuclear layer and the ganglion cell layer. There was no overlapping between the signal observed for both receptors. In conclusion, our results showed that the patterns of distribution of TRPV1 and TRPV2 are different during the development of the rat retina, suggesting that they have specific roles in both visual processing and in providing specific cues to neural development.     

TRPV1在中枢神经系统中的双重作用及其参与精神分裂症的可能机制

瞬时受体电位香草酸亚型 1（TRPV1）在中枢神经系统广泛分布,并在氧化应激、神经炎 症、神经凋亡、突触损伤方面均发挥了双重作用。精神分裂症是一组以阳性症状、阴性症状、精神运动 性障碍及现实检验能力严重受损为特征表现的精神障碍,其发病机制尚未完全阐明。目前研究发现, TRPV1可能在精神分裂症中发挥潜在作用。本文对TRPV1在中枢神经系统中的双重作用进行归纳总结, 以期解释 TRPV1 参与精神分裂症的可能机制。     

Effect of Surgical and Chemical Sensory Denervation on Non-neural Expression of the Transient Receptor Potential Vanilloid 1 (TRPV1) Receptors in the Rat

Pretreatment with the ultrapotent capsaicin analog resiniferatoxin (RTX) has been applied as a selective pharmacological tool in inflammation and pain studies to desensitize transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerve endings. The discovery of TRPV1 receptor on non-neural cells challenges systemic RTX desensitization as a method acting exclusively on a population of sensory neurons, but not on non-neural cells. Systemic RTX desensitization was used for chemical denervation and transection of the sciatic and saphenous nerves for surgical denervation in rats. Quantitative real-time PCR and immunohistochemistry were applied to investigate the presence and alterations of the TRPV1 receptor mRNA and protein following chemical and surgical denervation. We provided the first evidence for non-neural TRPV1 immunopositivity and mRNA expression in the rat dorsal paw and plantar skin as well as the oral mucosa. Neither chemical nor surgical denervation influenced the level of TRPV1 receptor mRNA and protein expression in non-neural cells of either skin regions or mucosa. Therefore, RTX and consequently capsaicin remain to be considered as selective neurotoxins for a population of primary afferent neurons.     

颞叶癫痈大鼠海马内Rnd1 mRNA及蛋白表达变化

目的：动态观察小分子GTPase Rho家族的Rnd1 mRNA及其蛋白在氯化锂-毛果芸香碱（匹罗卡品）致痫大鼠模型海马中的表达变化，探讨其在颞叶癫痫发生发展中的作用。方法：在氯化锂-毛果芸香碱颞叶癫痫模型中应用逆转录聚合酶链反应（RT—PCR）检测癫痫持续发作（SE）后各时间点海马内Rnd1 mRNA的表达变化，并运用免疫组织化学染色法及Neo—Timm染色法分别检测齿状回门区、CA1区及CA3区中该蛋白在不同时间点的表达变化及苔藓纤维出芽（MFS）情况。结果：实验发现模型组于SE后8h内即出现Rnd1表达上调，SE后约1d达高峰，7d左右回复至对照组水平，此后其mRNA表达水平与对照组相似；而免疫组化染色发现Rnd1蛋白表达从SE后8h内即开始上调，约3d达高峰，至7d虽略有回落，但仍高于对照组水平，且这种情况可一直持续至慢性期。结论：急性期海马齿状回门区Rnd1表达上调可能通过促进MFS的发生参与了颞叶癫痫的发生。     

Histological Evidence of Protein Aggregation in Mutant SOD1 Transgenic Mice and in Amyotrophic Lateral Sclerosis Neural Tissues

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.     

Castration Reduces the Nocturnal Rise of Pineal Melatonin Levels in the Male Rat by Impairing its Noradrenergic Input

The effects of castration and testosterone treatment on pineal day-night rhythms were studied in male rats. Bilateral gonadectomy was performed at 21 days of age. Testosterone propionate was given subcutaneously to castrated animals in a dose of 10 μg/100 g body weight during two consecutive days before sacrifice. Animals were killed 40 days after gonadectomy at four different times of a 12:12 h light-dark cycle (1600, 2400, 0400 and 0800h). Tyrosine hydroxylase activity was measured in individual pineals by means of high-performance liquid chromatography determination of L-DOPA formed. Pineal levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindole acetic acid were determined by high-performance liquid chromatography with amperometric detection, while pineal melatonin content was measured by radioimmunoassay. Castration abolished the day-night rhythms of pineal tyrosine hydroxylase activity and norepinephrine content, both by elevating their daytime levels and by blocking their nocturnal rise. In addition, gonadectomy drastically modified pineal indoleamine metabolism by increasing daytime levels of both 5-hydroxytryptamine and 5-hydroxyindole acetic acid, and by reducing the nocturnal elevation of pineal melatonin content. Testosterone treatment was unable to prevent the effect of orchidectomy on pineal rhythms of tyrosine hydroxylase activity, 5-hydroxytryptamine or 5-hydroxyindole acetic acid content, however it partially restored the day-night pineal rhythms of both norepinephrine and melatonin content. These results are indicative of a possible participation of reproductive hormones in the control of pineal rhythmic activity in the male rat. Apparently, since gonadectomy abolished the nocturnal rise of both pineal tyrosine hydroxylase activity and norepinephrine content, the primary site of action of reproductive hormones could be at the level of the superior cervical ganglion.     

Possible Relation of Hemin-Induced HO-1 Expression to the Upregulation of VEGF and BDNF mRNA Levels in Rat C6 Glioma Cells

Glial cells are generally considered to contribute to retaining the integrity of neural function through the protection of neuronal cells against neurodegenerative insults and also expected to play a potential role in the protection of cerebrovascular systems from various toxic insults of hemorrhaged blood, thus proposing a possible implication of glial cells in the recovery of brain function from the damage caused by cerebral hemorrhage. Based on this hypothetical idea, the direct effect of hemin on the expression of genes encoding heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) in glial cells was examined using rat C6 glioma cells as an in vitro model system. Hemin elevated both HO-1 and VEGF mRNA levels in the glioma cells at the concentration causing no critical damage to the cells, and the elevation of BDNF mRNA levels was also observed by exposing the cells to hemin under the same conditions. Furthermore, the elevation of VEGF and BDNF mRNA levels induced by hemin was blocked by pretreatment of the cells with the agents inhibiting not only HO-1 gene expression but also its enzymatic activity. These pharmacological studies indicate that hemin can induce the enhancement of VEGF and BDNF gene expression probably through the mechanism mediated by HO-1 activity in the glioma cells, proposing the possibility that glial cells are capable of contributing to the recovery of brain function from the damage caused by cerebral hemorrhage through the production of neurogenic and angiogenic factors.     

3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1-/- mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.     

Effect of Peripheral Axotomy on Gene Expression of NIDD in Rat Neural Tissues

Peripheral nerve lesion-induced production of neuronal nitric oxide synthase (nNOS) was implicated to influence a range of postaxotomy processes necessary for neuronal survival and nerve regeneration (Zochodne et al., Neuroscience, 91:1515–1527, 1999; Keilhoff et al., Journal of Chemical Neuroanatomy, 24:181–187, 2002, Nitric Oxide, 10:101–111, 2004). Protein–protein interactions represent an important mechanism in the control of NOS spatial distribution or activity (Alderton et al., Biochemical Journal, 357:593–615, 2001; Dedio et al., FASEB Journal, 15:79–89, 2001; Zimmermann et al., Proceedings of the National Academy of Sciences, 99:17167–17172, 2002). As one of the nNOS-binding proteins, nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) has recently been identified to increase nNOS enzyme activity by targeting nNOS to the synaptic plasma membrane in a postsynaptic density protein 95/discs-large/zona occlusens-1 domain dependent manner (Saitoh et al., Journal of Biological Chemistry, 279:29461–29468, 2004). In this paper, we established a rat model with peripheral axotomy to investigate the gene expression patterns of NIDD in neural tissues using TaqMan quantitative real-time polymerase chain reaction and in situ hybridization combined with immunofluorescence. It revealed that NIDD mRNA was upregulated after sciatic nerve transection with the similar expressing styles as that of the nNOS in the injured nerves, corresponding dorsal root ganglia, and lumbar spinal cord. These findings imply that NIDD may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival, and even pain process, possibly via regulating the enzyme nNOS activity. Chun Cheng and Mengling Chen both contributed equally to this work.     

Parvalbumin- and Calbindin D-28k-Immunoreactive Innervation of Orofacial Tissues in the Rat

Parvalbumin- and calbindin D-28k-immunoreactive (-ir) innervation was examined in orofacial tissues of the rat. Labial and facial skins were devoid of the calcium-binding protein (CaBP)-ir nerve endings, while the infraorbital and mental nerves contained numerous parvalbumin-ir axons. Labial and gingival mucosae were also devoid of the CaBP-ir nerve endings. The buccal mucosa and incisive papilla contained both encapsulated and unencapsulated endings, while the hard palate mucosa excluding the incisive papilla contained only unencapsulated endings. Encapsulated endings were found just beneath the epithelium or attached to the cartilaginous core of the incisive papilla. Unencapsulated endings in the lamina propria were subdivided into two types: simple (unramified) and complex (ramified). Neurites of simple endings were straight, curved, or coiled, while those of complex endings exhibited a bush-like appearance due to the ramification. In addition, palatal rugae contained intraepithelial endings. The unencapsulated complex endings in palatal rugae coexpressed parvalbumin- and calbindin D-28k-irs, whereas other endings were immunoreactive for parvalbumin alone. The pterygopalatine ganglion contained calbindin D-28k-ir pericellular fibers but not the ir cell bodies. A subpopulation of trigeminal ganglion neurons coexpressed both CaBPs. CaBP-ir encapsulated and unencapsulated endings in the oral mucosa probably include low-threshold mechanoreceptors, while parvalbumin-ir intraepithelial endings in the palatal mucosa may be involved in nociception.     

Rapid Alterations of BDNF Protein Levels in the Rat Brain after Focal Ischemia: Evidence for Increased Synthesis and Anterograde Axonal Transport

Cellular localization and tissue levels of BDNF protein were studied using immunocytochemistry and enzyme immunoassay, respectively, in the cortex and striatum at different reperfusion times (0–24 h) after 2 h of unilateral middle cerebral artery occlusion (MCAO) in rats. The distribution of neuronal injury was analyzed in NeuN-, cresyl violet-, and Fluoro-Jade-stained sections. At 2 h postischemia, but not at later time points, there was a several-fold increase of the number of BDNF-immunoreactive (-ir) cells in the ipsilateral cingulate and frontal cortices outside the damaged area. Animals with cortical injury showed loss of BDNF-ir fibers in the striatum at 2–24 h, whereas rats with cell damage confined to the striatum exhibited no such change. At 2–16 h, strongly BDNF-ir fibers were observed along the myelinated fascicles medially in the striatum, in the anterior commissure, and in the corpus callosum ipsilateral to the MCAO. BDNF protein levels were increased (by 133–213%) at 2 h in the cingulate and frontal cortices and decreased (by 40%) at 24 h in the striatum. These findings show that the increased expression of BDNF mRNA in cortical neurons previously demonstrated after transient focal ischemia is accompanied by elevated levels of BDNF protein. The rapid decline of BDNF protein levels suggests a pronounced release or anterograde axonal transport in the postischemic phase. The reduction of BDNF protein in the striatum of animals with cortical damage provides further evidence for anterograde transport, which is also supported by the accumulation of BDNF protein in several preterminal fiber systems. The changes of BDNF protein after focal ischemia could play a role for survival and plasticity of cortical and striatal neurons.     

Role of TRPV1 in susceptibility to PTZ-induced seizure following repeated hyperthermia challenges in neonatal mice

This study was designed to investigate the role of experimental febrile seizures in the induction of generalized clonic seizures and the involvement of heat-sensitive channel TRPV1. Pentylenetetrazol-induced clonic seizure was used as the seizure model, and Trpv1 gene knock-out and wild-type C57/BL6 mice were used as experimental subjects. Electroencephalograph and seizure behavior were recorded for the evaluation of the severity of seizures.Increased frequency of the experimental febrile seizures facilitated PTZ-induced generalized clonic seizures. Trpv1 gene deficiency decreased the properties of generalized clonic seizure. The intensity of experimental febrile seizures reduced the threshold to generalized clonic seizure, and Trpv1 gene deficiency decreased the susceptibility to PTZ-induced seizures following early-life hyperthermia challenges in mice.