Tumor necrosis factor alpha: activity dependent expression and promotion of cortical column sleep in rats

Abstract

Cortical surface evoked potentials (SEPs) are larger during sleep and characterize a sleep-like state in cortical columns. Since tumor necrosis factor alpha (TNF) may be involved in sleep regulation and is produced as a consequence of waking activity, we tested the hypothesis that direct application of TNF to the cortex will induce a sleep-like state within cortical columns and enhance SEP amplitudes. We found that microinjection of TNF onto the surface of the rat somatosensory cortex enhanced whisker stimulation-induced SEP amplitude relative to a control heat-inactivated TNF microinjection. We also determined if whisker stimulation enhanced endogenous TNF expression. TNF immunoreactivity (IR) was visualized after 2 h of deflection of a single whisker on each side. The number of TNF-IR cells increased in layers II-IV of the activated somatosensory barrel column. In two separate studies, unilateral deflection of multiple whiskers for 2 h increased the number of TNF-IR cells in layers II-V in columns that also exhibited enhanced cellular ongogene (Fos-IR). TNF-IR also colocalized with NeuN-IR suggesting that TNF expression was in neurons. Collectively these data are consistent with the hypotheses that TNF is produced in response to neural activity and in turn enhances the probability of a local sleep-like state as determined by increases in SEP amplitudes.

Figure 1

Goat anti-rat TNF antibody made by R&D Systems showed one band in the Western blot analyses of the rat recombinant TNF (rTNF) at 17 kD (A and B-first lane) and one band in the sleep deprived Sctx at 26 kD (second lane). The 26 kD protein is the membrane bound form of TNF. Preabsorption with rat rTNF (B) blocked the antibody from labeling both recombinant rat TNF (first lane) and the 26 kD band in the Sctx (second lane).

Figure 2

Examples of time-triggered average traces to whisker twitches during NREMS after either TNF or heat-inactivated TNF (hiTNF) microinjection onto the surface of the Sctx. The SEP amplitude is greater after the TNF microinjection than after the heat-inactivated TNF. The increases in SEP amplitudes occurred whether the animal was awake or asleep.

Figure 3

A. Preabsorption of the primary antibody to rat TNF with rat recombinant TNF (3-fold greater concentration) completely blocked the darkly stained TNF-IR cells evidenced in B. B. Whisker deflection of Group A rats increased the number of TNF-IR cells in layer II–III & V underlying the electrode microarray on the side injected with the control heat-inactivated TNF relative to the adjacent unstimulated column. Bar = 0.7 mm. Higher magnification in the stimulated (C) and unstimulated columns (D) illustrates the neuronal-like shape of the TNF-IR cells with the greatest immunoreactivity surrounding the nuclear region and into the apical dendrite. Bar = 0.05 mm.

Figure 4

Manual whisker brushing for 2 h in Group B rats increased the number of TNF-IR cells in the Sctx of a stimulated (horizontal bar marks the stimulated column) but not in an adjacent unstimulated cortical columns (A). In this case a large increase in the number of TNF-IR cells was evident in the upper layers of the Sctx. For comparison, whisker stimulation increased the number of Fos-IR cells in an equivalent column of the Sctx in an adjacent section (B). Bar = 0.6 mm.

Figure 5

Confocal laser scanning microscopic images of the stimulated (B) and unstimulated (A) columns in the Sctx after double labeling with fluorescent probes for TNF (red)-IR and NeuN (green)-IR. Whisker stimulation increases the number of TNF-IR nuclei (B relative to A), but did not alter the number of NeuN-IR cells in the Sctx. Bar = 0.01 mm.

Figure 6

Confocal laser scanning microscopic images of the stimulated (B) and unstimulated (A) columns in the Sctx after double labeling with fluorescent probes for TNF (red)-IR and Fos (green)-IR. Whisker stimulation increases the number of TNF- and Fos-IR nuclei (B relative to A). Fos-IR was evident within nuclei surrounded by TNF-IR in the surrounding cytoplasm. Bar = 0.01 mm.