Research reportTumor necrosis factor α increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons
Introduction
Tumor necrosis factor α (TNFα), first identified as an anti-tumor agent [16], is a pro-inflammatory cytokine necessary for activating immune function [6], [22]. While TNFα knock-out mice are outwardly normal, they are susceptible to infection [44]. TNFα is also implicated in mediating brain damage caused by trauma [38], ischemia [45], inflammation [55], demyelinating diseases [30], multiple sclerosis [63], and Alzheimer’s [61] and Parkinson’s diseases [7]. In contrast, TNFα is thought to be neuroprotective in hypoxia [42], ischemia [11], demyelinating disease [41], trauma [58], Alzheimer’s disease [3], and excitotoxic insult, particularly that due to AMPA receptor activation [17], [40], [53]. Whether TNFα is damaging or protective appears to depend on many factors such as timing, acutely damaging but long-term protective [77]; the presence of specific receptors on target cells, TNF-R1 damaging versus TNF-R2 protective [24], [48], [80]; and the presence or absence of compounds that modify TNFα action [15], [60].
In addition to a role in the process of neurodegeneration/neuroprotection, TNFα also participates in mediating whole-organism responses to infection such as fever [56], decreased food intake [49], and increased sleep [33]. These effects are mediated, in part, by TNFα actions in the brain [43], [51], [64]. Cells such as microglia, astrocytes, and neurons express TNFα [9], [26], [57] and TNF receptors are expressed on neurons [7], [8], [17]. Further, TNFα mRNA expression and TNFα protein have a diurnal variation in the hypothalamus [10], [23]. By way of example, TNFα is implicated in physiological sleep regulation [34]. Thus, TNFα inhibitors, such as soluble TNF receptor, inhibit spontaneous sleep [71], and mice lacking the 55-kDa TNF receptor have reduced sleep [21]. Administration of TNFα intracerebroventricularly or microinjection into the preoptic area of the hypothalamus promotes sleep [35], [64]. TNFα and other cytokines are also implicated in the regulation of other physiological processes [46], [50], [76].
If TNFα has a function in physiological regulation, then it should affect normal neuronal activity and synaptic communication in a time frame consistent with observed TNFα behavioral actions (minutes to hours), rather than requiring extended exposures or manifested days after exposure. However, our knowledge of TNFα involvement in normal physiological processes is limited since most TNFα related research is oriented towards understanding its neuropathological actions.
AMPA receptor mediated neurotransmission is one of the most common forms of excitatory communication between neurons in the brain. Therefore we choose to test our hypothesis that TNFα effects normal synaptic communication by examining the effects of TNFα on AMPA-induced changes in cytosolic calcium. We further extended this beyond AMPA receptors to also include depolarization-induced calcium signals, since depolarization-induced calcium signaling is also a key regulator of neuronal activity. We found that TNFα induces an up-regulation of these responses within 3–6 h of treatment.
Section snippets
Hippocampal cultures
Neuronal cultures were prepared from day 19–21 rat fetuses. Isolated hippocampi were placed in ice-cold Hank’s balanced salt solution (HBSS), washed several times with HBSS, and then incubated in Hepes-buffered Dulbecco’s modified Eagle’s medium (HDMEM) for 10–15 min at 37 °C. After incubation, cells were mechanically dissociated by several gentle aspirations into a 20-ml syringe through first a 20-gauge needle, and then through a 22-gauge needle. Dissociated cells were then centrifuged and
Characterization of AMPA response
AMPA (10 μM) induced a prompt increase in cytosolic Ca2+ that was blocked by the presence of the AMPA receptor antagonist CNQX (Fig. 1A). A 10-μM amount of AMPA produced a just maximal response (Fig. 1B), thus this concentration of AMPA was used as the standard challenge in all subsequent studies.
AMPA opens AMPA-type ionotropic glutamate receptors and would be expected to depolarize neurons and thus open voltage-dependent calcium channels (VDCC), and thereby possibly activate synaptic activity.
Discussion
The AMPA-induced increase in cytosolic Ca2+ can be divided into roughly two components, a direct AMPA-induced influx, likely to be mediated by Ca2+ influx through AMPA-activated, calcium-permeable ionotropic glutamate receptors [12], and an indirect response that was dependent upon the activity of voltage-dependent Na+ and Ca2+ channels. The indirect response is likely to be due to AMPA-induced depolarization, although the magnitudes of AMPA-induced responses were typically smaller than the Ca2+
Acknowledgements
This work was supported, in part, by National Institutes of Health grants No. NS25378 and NS31453.
References (80)
- et al.
Immunocytochemical analysis of tumor necrosis factor and its receptors in Parkinson’s disease
Neurosci. Lett.
(1994) - et al.
Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit
Neuron
(1992) Effects of TNFα on immature and mature oligodendrocytes and their progenitors in vitro
Brain Res.
(2000)- et al.
Interferon-α,β and tumor necrosis factor-α enhance the frequency of miniature end-plate potentials at rat neuromuscular junction
Neurosci. Lett.
(1994) - et al.
Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis
Neuron
(1994) - et al.
Interleukin-1 β (IL1-β) and tumour necrosis factor (TNF) inhibit long-term potentiation in the rat dentate gyrus in vitro
Neurosci. Lett.
(1996) Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level
FEBS Lett.
(1991)- et al.
TNF-α increases the frequency of spontaneous miniature synaptic currents in cultured rat hippocampal neurons
Brain Res.
(1994) - et al.
Neuronal-associated tumor necrosis factor (TNFα): its role in noradrenergic functioning and modification of its expression following antidepressant drug administration
J. Neuroimmunol.
(1997) - et al.
TNF-α receptor fusion protein prevents experimental auto-immune encephalomyelitis and demyelination in Lewis rats: an overview
J. Neuroimmunol.
(1997)
TNFα reduces glutamate induced intracellular Ca2+ increase in cultured cortical astrocytes
Brain Res.
Intrapreoptic microinjection of TNF-α enhances non-REM sleep in rats
Brain Res.
Role of tumor necrosis factor-α in neuronal and glial apoptosis after spinal cord injury
Exp. Neurol.
Identification of the binding site of 55-kDa tumor necrosis factor receptor by synthetic peptides
Biochem. Biophys. Res. Commun.
Tumor necrosis factor-α attenuates N-methyl-d-aspartate-mediated neurotoxicity in neonatal rat hippocampus
Brain Res.
Central nervous system mechanisms contributing to the cachexia-anorexia syndrome
Nutrition
Tumor necrosis factor and interleukin-1 β: suppression of food intake by direct action in the central nervous system
Brain Res.
TNF-α receptors simultaneously activate Ca2+ mobilisation and stress kinases in cultured sensory neurons
Neuropharmacology
Marked diversity in the action of growth factors on N-methyl-d-aspartate-induced neuronal degeneration
Eur. J. Pharmacol.
TNF-α transgenic and knockout models of CNS inflammation and degeneration
J. Neuroimmunol.
Production of tumor necrosis factor-α by microglia and astrocytes in culture
Brain Res.
The divergent role of tumor necrosis factor receptors in infectious diseases
Microbes Infect.
Effect of ethanol on calcium regulation in rat fetal hypothalamic cells in culture
Brain Res.
Cellular basis for the acute inhibitory effects of IL-6 and TNF-α on excitation–contraction coupling
J. Mol. Cell. Cardiol.
Tumor necrosis factor alters synaptic transmission in rat hippocampal slices
Neurosci. Lett.
Evidence for the involvement of TNF and NF-κB in hippocampal synaptic plasticity
Synapse
Human mast cells release metaloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNFα
J. Immunol.
Tumor necrosis factors α and β protect neurons against amyloid β-peptide toxicity: evidence for involvement of a kB-binding factor and attenuation of peroxide and Ca2+ accumulation
Proc. Natl. Acad. Sci. USA
Model of thalamocortical slow-wave sleep oscillations and transitions to activated states
J. Neurosci.
Control of synaptic strength by glial TNFα
Science
TNF, immunity and inflammatory disease: lessons of the past decade
J. Invest. Med.
Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia
Mol. Med.
Distribution and characterization of tumor necrosis factor-α-like immunoreactivity in the murine central nervous system
J. Comp. Neurol.
Diurnal variations of tumor necrosis factor α mRNA and α-tubulin mRNA in rat brain
Neuroimmunomodulation
Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors
Nat. Med.
Nicotine blocks TNF-α-mediated neuroprotection to NMDA by an α-bungarotoxin-sensitive pathway
J. Neurobiol.
An endotoxin-induced serum factor that causes necrosis of tumors
Proc. Natl. Acad. Sci. USA
Calcium responses to growth hormone releasing hormone and interleukin-1β in cultured hypothalamic neurons is developmentally regulated
Sleep
Rapid colorometric assay for cell growth and survival
J. Immunol. Methods
Mice lacking the TNF 55-kDa receptor fail to sleep after TNFalpha treatment
J. Neurosci.
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