Elsevier

Brain Research

Volume 981, Issues 1–2, 15 August 2003, Pages 133-142
Brain Research

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

https://doi.org/10.1016/S0006-8993(03)02997-4Get rights and content

Abstract

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.

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.

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      TNF has a well-established role in synaptic scaling [18]. TNF potentiates AMPA-induced potentials at the post-synaptic membrane [43], and also acts to increase Ca++ conductance via AMPA-induced [44] and voltage-dependent mechanisms [45,46]. Additionally, TNF modulates glutamatergic transmission [16,44,45].

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