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Tumor necrosis factor α increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons

Acute behavioral effects of tumor necrosis factor α (TNFα) have been previously reported, however the cellular basis for these actions are unknown. To address this issue we examined the effects of TNFα on AMPA- and depolarization-induced changes in cytosolic Ca2+ in cultured hippocampal neurons. Single cell Ca2+ levels were determined with the fluorescent calcium indicator fura-2. TNFα caused an up-regulation of AMPA (10 μM)- and depolarization (55 mM KCl)-induced Ca2+ responses. This effect occurred within a window of concentrations (1 and 10 ng/ml but not 0.1 or 100 ng/ml) and times (3 and 6 h but not 1 and 24 h). The effect was dependent upon protein synthesis (blocked by cycloheximide) and was prevented by the soluble TNF receptor and by a soluble TNF receptor fragment. Treatment with the soluble TNF receptor fragment also caused a decrease in the basal response. The TNFα treatment protocols did not appear to produce any toxicity to the neurons. Results are consistent with the hypothesis that TNFα regulates proteins known to be involved in neuronal communication (AMPA receptors) and cell regulation (voltage-dependent calcium channels) in a relatively rapid period of time (a few hours). These actions may be related to the behavioral effects produced by TNFα that occur within this time frame.

GHRH and IL1β increase cytoplasmic Ca2+ levels in cultured hypothalamic GABAergic neurons

GHRH and IL1beta regulate sleep via the hypothalamus. However, actions of these substances on neurons are poorly understood. In this study, we found both GHRH (100 nM) and IL1beta (1.2 pM) acutely increased cytosolic Ca(2+) in 7.6 and 4.0% of cultured hypothalamic neurons tested, respectively, and 1.2% of neurons responded to both. The neurons that responded were mostly GABAergic (96, 81, and 100% for GHRH, IL1beta, and dual-responsive neurons, respectively).

TNFα siRNA reduces brain TNF and EEG delta wave activity in rats

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFα were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFα siRNA treatment specifically reduced TNFα mRNA by 45% in vitro without affecting interleukin-6 or gluR1–4 mRNA levels. In vivo the TNFα siRNAα reduced TNFα mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFα-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFα siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFα siRNA attenuates TNFα mRNA and TNFα protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFα is involved in CNS physiology including sleep regulation.

Role of Transforming Growth Factor (TGF)-β Type I and TGF-β Type II Receptors in the TGF-β1-Regulated Gene Expression in Pituitary Prolactin-Secreting Lactotropes1

Transforming growth factor β1 (TGF-β1) inhibits pituitary lactotrope proliferation and secretion of PRL in an autocrine/paracrine manner. In this study, the role of TGF-β1 type I (TβR-I) and TGF-β type II (TβR-II) receptors in TGF-β1-regulated gene expression in lactotropes was determined using anterior pituitary cells known to be responsive to TGF-β1 growth inhibition and using a transformed PR1 cell line known to be nonresponsive to TGF-β1 growth inhibition. Treatment with TGF-β1 inhibited cell proliferation and decreased PRL mRNA levels in anterior pituitary cells, but in PR-1 cells, the treatment caused only decreased PRL mRNA levels. Affinity labeling of TGF-β binding proteins indicated that anterior pituitary cells contain several TGF-β-binding protein complexes, including the 65 kDa size TβR-I and 95 kDa size TβR-II. In the PR1 cells, the major complex found was similar to the 65 kDa size of TβR-I. Immunocytochemistry identified TβR-I and TβR-II receptor proteins in lactotropes but detected primari...

Whisker Stimulation increases expression of Nerve Growth Factor- and Interleukin-1β-immunoreactivity in the Rat Somatosensory Cortex

Activity-dependent changes in cortical protein expression may mediate long-term physiological processes such as sleep and neural connectivity. In this study we determined the number of nerve growth factor (NGF)- and interleukin-1beta (IL1beta)-immunoreactive (IR) cells in the somatosensory cortex (Sctx) in response to 2 h of mystacial whisker stimulation. Manual whisker stimulation for 2 h increased the number of NGF-IR cells within layers II-V in activated Sctx columns, identified by enhanced Fos-IR. IL1beta-IR neurons increased within layers II-III and V-VI in these activated columns and IL1beta-IR astrocytes increased in layers I, II-III and V as well as the external capsule beneath the activated columns. These whisker-stimulated increases in the Sctx did not occur in the auditory cortex. These data demonstrate that expression of NGF or IL1beta in Sctx neurons and IL1beta in Sctx astrocytes is, in part, afferent input-dependent.

Glutamate induces the expression and release of tumor necrosis factor-α in cultured hypothalamic cells

Tumor necrosis factor-alpha (TNFalpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFalpha involvement in physiological sleep regulation, e.g., TNFalpha induces sleep and brain levels of TNFalpha increase during prolonged wakefulness. The immediate cause of enhanced TNFalpha production in brain is unknown. We investigated whether glutamate could signal TNFalpha production because glutamate is a neurotransmitter associated with cell activation and wakefulness. We used primary cultures of fetal rat hypothalamic cells to examine the expression and release of TNFalpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFalpha was detected in both the media and cells under basal conditions. Stimulation of the cells with 1 mM glutamate for 2 h produced an increase in media content of TNFalpha, whereas cell content was elevated at earlier time points. Using trypan blue exclusion and MTT assays, there was no evidence of cell toxicity with this stimulation protocol. Immunocytochemical staining revealed that TNFalpha was expressed by approximately 25% of the neurons and approximately 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFalpha. We conclude that TNFalpha is constitutively expressed and released by healthy cultures of hypothalamic cells and that activation of the cells with a non-toxic challenge of glutamate increases TNFalpha production. These findings support the hypothesis that TNFalpha can participate in normal physiological regulation of sleep and feeding.

Cytokine mRNA induction by Interleukin-1β or Tumor Necrosis Factor α in vitro and in vivo

Hypothalamic and cortical mRNA levels for cytokines such as interleukin-1beta (IL1beta), tumor necrosis factor alpha (TNFalpha), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are impacted by systemic treatments of IL1beta and TNFalpha. To investigate the time course of the effects of IL1beta and TNFalpha on hypothalamic and cortical cytokine gene expression, we measured mRNA levels for IL1beta, TNFalpha, interleukin-6 (IL-6), interleukin-10 (IL-10), IL1 receptor 1, BDNF, NGF, and glutamate decarboxylase-67 in vitro using hypothalamic and cortical primary cultures. IL1beta and TNFalpha mRNA levels increased significantly in a dose-dependent fashion after exposure to either IL1beta or TNFalpha. IL1beta increased IL1beta mRNA in both the hypothalamic and cortical cultures after 2-6 h while TNFalpha mRNA increased significantly within 30 min and continued to rise up to 2-6 h. Most of the other mRNAs showed significant changes independent of dose in vitro. In vivo, intracerebroventricular (icv) injection of IL1beta or TNFalpha also significantly increased IL1beta, TNFalpha and IL6 mRNA levels in the hypothalamus and cortex. IL1beta icv, but not TNFalpha, increased NGF mRNA levels in both these areas. Results support the hypothesis that centrally active doses of IL1beta and TNFalpha enhance their own mRNA levels as well as affect mRNA levels for other neuronal growth factors.

Effect of ethanol on calcium regulation in rat fetal hypothalamic cells in culture.

The effects of acute exposure to ethanol on calcium regulation in primary cultures of rat fetal hypothalamic cells was studied with the use of the calcium indicator fura-2 and digital imaging techniques. We found that ethanol caused cytoplasmic calcium to increase in a dose-dependent and reversible manner, and these increases could be observed at pharmacologically relevant doses (34 mM). At 170 mM ethanol 65% of 1059 cells examined responded to ethanol with an increase in cytoplasmic calcium. Removing bath calcium eliminated the ethanol-induced calcium response in most cells (76% of 427 cells). In most cells exposure to thapsigargin (20 nM) had no significant effect on the ethanol-induced calcium increase (87% of 67 cells examined). The ethanol-induced calcium increase was reduced by 79+/-5% (n=110 cells) by the P/Q-type calcium channel blocker omega-agatoxin-TK (20 nM), by 51+/-10% (n=115 cells) by the N-type calcium channel blocker omega-conotoxin-GVIA (100 nM), and by 26+/-3% (n=90 cells) by the T-type calcium channel blocker flunarizine (1 microM). The L-type calcium channel blocker nifedipine (1 microM) had complex actions, sometimes inhibiting and sometimes increasing the calcium response. These results demonstrate that ethanol can directly modulate cytoplasmic calcium levels in hypothalamic cells mostly by a pathway that involves extracellular calcium and voltage-dependent calcium channels, and that this response may participate in the biological effects of acute ethanol exposure.

Colocalization of prolactin and proliferating cell nuclear antigen in the anterior pituitary during estrogen-induced pituitary tumors

Chronic estrogen treatment induces prolactin (PRL)-secreting pituitary tumors in laboratory animals. To determine earlier events of tumorigenesis, we studied cell proliferation in the pituitary following 7-30 days of estrogen administration in Fischer 344 rats. Immunohistochemical localization of proliferative cells by proliferating cell nuclear antigen (PCNA) staining and lactotropes by PRL staining revealed that estrogen treatment caused a time-dependent increase in the number of proliferative cells and lactotropes. Although the increase in lactotropic cell number paralleled the increase in PCNA-reactive cell number, only approximately 30% of lactotropes reacted simultaneously with the PCNA antibody. These results indicate that a subset population of lactotropes proliferates under the influence of estrogen during tumorigenesis.

Interleukin-1β stimulates growth hormone-releasing hormone receptor mRNA expression in the rat hypothalamus in vitro and in vivo

Changes in growth hormone‐releasing hormone (GHRH), GHRH‐receptor (R), somatostatin and interleukin (IL)‐1β mRNA levels were determined in fetal rat hypothalamic cultures after administration of IL‐1β (1, 10, 100 ng/ml, 2 h incubation), and in adult rat hypothalamus 5 h after intracerebroventricular injection of IL‐1β (2.5 and 25 ng). IL‐1β stimulated GHRH‐R mRNA expression both in vitro (10 and 100 ng/ml) and in vivo (2.5 and 25 ng). Somatostatin mRNA was significantly stimulated and GHRH mRNA slightly reduced in vitro, while these mRNA species were not altered in vivo in response to IL‐1β. IL‐1β stimulated its own expression both in vitro (10 and 100 ng/ml) and in vivo (25 ng). IL‐1β‐induced mRNA responses occurred 2 h after treatment in vitro (incubation times, 30 min to 6 h). IL‐1β also elicited slight GHRH releases in vitro. Up‐regulation of hypothalamic GHRH‐R by IL‐1β may explain previous findings suggesting that IL‐1β stimulates GHRH activity.