Research ReportWhisker stimulation increases expression of nerve growth factor- and interleukin-1β-immunoreactivity in the rat somatosensory cortex
Introduction
Neurotrophic growth factors and other cytokines are posited to play a role in activity-dependent cellular mechanisms (Levi-Montalcini, 1966, Cellerino and Maffei, 1966, Boulanger and Poo, 1999, Thoenen, 2000, Krueger et al., 2008). Nerve growth factor (NGF) is part of a larger neurotrophic family that includes brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT3) and neurotrophic factor 4 (NT4); most of which show activity-dependent expression (Ickes et al., 2000, Lu, 2004, Tabuchi, 2008). For instance, NGF mRNA levels upregulate in response to seizure activity (Gall and Isackson, 1989, Zafra et al., 1990, Ernfors et al., 1991), depolarizing stimuli (Lindholm et al., 1994, Lu, 2004) and afferent input from whiskers (Schwarting et al., 1994). NGF protein is released in response to neural activity (Blochl and Thoenen, 1995, Blochl and Thoenen, 1996). Sleep deprivation, a method associated with enhanced neural activity (Vyazovskiy et al., 2009), increases the number of NGF-immunoreactive (IR) cells in the primary somatosensory cortex (Sctx) (Brandt et al., 2001). Neuronal expression of interleukin-1 beta (IL1β) and tumor necrosis factor alpha (TNF) are also activity-dependent. For example, whisker stimulation increases the number of TNF-IR cells in Sctx neurons (Churchill et al., 2008). IL1β mRNA levels are upregulated by epileptiform activity in the hippocampus and cerebral cortex (Eriksson et al., 2000, Ravizza et al., 2006). Manipulations that mimic neuronal activity, such as depolarization, dramatically increase the levels of IL1β released in hippocampal samples (Tringali et al., 1999). Induction of hippocampal long-term-potentiation (LTP) increases IL1β mRNA levels (Schneider et al., 1998). Enhanced neuronal activation, as evidenced by Fos-IR in the reticular thalamus, is accompanied by an increase in IL1β-IR in this region after cortical IL1β application (Yasuda et al., 2007). Blocking olfactory input by closing the nares decreases levels of IL1β-IR in the periglomerular region of the olfactory bulb (Lim and Brunjes, 1999).
Our interest in the neurotrophins/cytokines derives from their involvement in sleep regulation. All of the neurotrophins/cytokines mentioned have the capacity to enhance non-rapid eye movement (NREM) sleep and there is substantial evidence that TNF, IL1β and NGF are involved in physiological sleep regulation (reviewed Krueger et al., 2008). Furthermore, several modern theories of sleep mechanisms and function emphasize that sleep is dependent upon prior neuronal activity and is a fundamental property of highly interconnected neuronal networks such as cortical columns (Krueger and Obal, 1993, Kavanau, 1994, Benington and Heller, 1995, Tononi and Cirelli, 2003, Krueger et al., 2008). One test of these theories is to determine whether expressions of sleep regulatory substances such as NGF and IL1β change in the Sctx under conditions that affect sleep. Electroencephalographic (EEG) slow wave power (SWA) during NREM sleep, a measure of sleep intensity, is altered by whisker stimulation (Vyazovskiy et al., 2000) or time of day (Yasuda et al., 2005a, Alfödi et al., 1991). Thus, demonstration of the use-dependent expression of somnogenic neurotrophins and cytokines would be consistent with the proposal that sleep is a use-dependent property of localized networks.
The glial network has also been implicated as a candidate for mediating sleep homeostasis (Krueger et al., 2008, Halassa et al., 2009). Whisker stimulation increases astrocytic cytosolic calcium in the Sctx (Wang et al., 2006, Schipke et al., 2008). Similarly, in the visual cortex (Vctx), astrocytic calcium levels respond to specific visual stimuli and these features are mapped in close register to the neuronal maps (Schummers et al., 2008). Astrocytes, like neurons, release ATP in response to cell activity (Pascual et al., 2005, Jourdain et al., 2007, Burnstock, 2007, Burnstock, 2009). ATP is posited to be the activity-indexed signal that initiates the process by which the brain keeps track of past activity (Krueger et al., 2008). The purine type 2 receptors that respond to ATP release, in turn, are involved in processing pro-IL1 and subsequent release of mature IL1β from astrocytes (Ferrari et al., 2006; reviewed Dinarello, 2009). The IL1 thus released is longer-lived than extracellular ATP and is involved in initiating sleepiness and sleep (reviewed Krueger et al., 2008). Therefore we investigated whether IL1β-IR astrocytes respond to whisker stimulation.
In the current study, we used a whisker stimulation model that is a well-characterized animal model for activity-induced gene expression (Mack and Mack, 1992, Melzer and Steiner, 1997, Filipkowski et al., 2000). For instance, unilateral stimulation of whiskers activates Fos expression in the corresponding Sctx columns on the opposite hemisphere (Melzer and Steiner, 1997, Filipkowski et al., 2000). We manually stimulated whiskers unilaterally for 2 h, then determined NGF- and IL1β-IR in the Sctx. We found that stimulation increased the number of NGF- and IL1β-positive neurons and IL1β-IR astrocytes and that NGF and IL1β often colocalized in Sctx neurons.
Section snippets
Whisker stimulation increased the number of NGF-IR cells in the activated columns of the Sctx as evidenced by the activity marker Fos
Fos-IR increased in a columnar pattern in the Sctx contralateral to the whisker stimulation (Fig. 1A activated column is marked by the bar). The increased number of Fos-IR cells in the stimulated column was evident in all of the Sctx layers (Table 1). The number of NGF-IR cells (Fig. 1B) increased in layers II-V in an adjacent section. In the activated columns, the number of NGF-IR cells increased significantly in layers II-V but not in layer VI compared to the non-activated column (Table 1).
Activity-dependent increases in NGF and IL1β-IR cells
The major finding reported here is that whisker stimulation enhances the number of NGF and IL1β-IR neurons in the Sctx. However, these changes were not observed in the auditory cortex, since each side of the cortex receives sensory inputs from both ears (Hutson et al., 2008). Previously it was shown that epileptic activity increases expression of NGF (Gall and Isackson, 1989, Zafra et al., 1990, Ernfors et al., 1991) and IL1β (Eriksson et al., 2000, Ravizza et al., 2006) and whisker stimulation
Animals
Male Sprague–Dawley rats weighing 250–400 g were obtained from Taconic Farm, Inc. (Germantown, NY). Rats were housed on a 12 h/12 h light/dark cycle at 24 ± 2 °C. Seven rats were adapted to unilateral whisker stimulation as previously described (Four of these rats were the same rats analyzed in Group B in Churchill et al., 2008). Water and food were available ad libitum throughout the experiment. The use of rats was in accordance with Washington State University and international guidelines and was
Acknowledgments
We would like to acknowledge the assistance of Xin Guan in the manual whisker stimulation studies, Marc Urza for Western blot analyses, Pari Sengupta for confocal microscopic assistance, Andrea Boucher for cell counting and Bryan Slinker for his advice on statistical analyses. This research was supported by the NIH grants to JM Krueger, NS25378 and NS31453.
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