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

2.1 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. 2A-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. 2B) 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). No significant differences in NGF- or Fos-IR cells were observed between the stimulated and unstimulated sides in the auditory cortex.

2.2 Whisker stimulation increased the number of IL1β-IR cells in the Sctx

The number of IL1β-IR cells increased in the stimulated columns (Fig. 2C) as evidenced by the larger number of Fos-IR cells in these columns in the adjacent section (comparing Fig. 2A with 2C from the same rat). A significant increase in the number of IL1β-IR pyramidal-shaped neurons occurred in layers II–III, V and VI in the stimulated columns compared to the adjacent unstimulated columns (Table 1). No significant differences in IL1β-IR cells were observed between the stimulated and unstimulated sides in the auditory cortex. Additionally, the number of IL1β-IR astrocytes in layers I, II–III and V as well as the underlying external capsule demonstrated significant increases in the stimulated columns (Table 2). However, no differences were observed between the two sides of the auditory cortex and its adjacent external capsule.

2.3 Morphology of NGF and IL1β-IR cells

NGF-IR cells in layers II–VI appeared to be neurons based on the size and the pyramidal morphology of cell bodies with apical dendrites (Fig. 3). Whisker stimulation increased the density and number of NGF-IR neurons in the stimulated columns (Fig. 3B, D, E & H). IL1β-IR cells in layers II–VI also appeared to be pyramidal-shaped neurons with apical dendrites or astrocytes (Fig. 4). The IL1β-IR in neurons appeared in the cytoplasm surrounding the nucleus and extended into the apical dendrites. Whisker stimulation increased the density and number of IL1β-IR neurons in the stimulated columns (Fig. 4B, D, E & H). Additionally, IL1β-IR astrocytes in the external capsule increased in density in the stimulated column relative to the unstimulated column (Fig. 5).

2.4. Colocalization of IL1β-IR with NeuN-, Fos- and NGF-IR cells

Double labeling immunohistochemistry demonstrated that IL1β– pyramidal-shaped cells colocalized with the neuronal nuclear marker, NeuN (Fig. 6A). The nucleus was brightly stained by NeuN. Also IL1β-IR stained the cytoplasm of cells containing Fos-IR in the nucleus (Fig. 6B). Also IL1β-IR cells colocalized with NGF-IR cells (Fig. 6C).

3.1 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 increases NGF mRNA levels (Schwarting et al., 1994) and the number of TNF-IR cells in the Sctx (Churchill et al., 2008). Further, natural whisker stimulation during sleep deprivation increases the number of NGF-IR cells in the Sctx, while a unilateral whisker trim decreases the number of these cells in the Sctx connected to this side (Brandt et al., 2001). These prior findings as well as the data reported herein, support the conclusion that localized neural activity regulates the level of SRSs.

In another group of rats that were killed at the same time as these rats (11 h after light onset) but without experimenter-induced whisker stimulation, the levels of Fos- and IL1β-IR cells were similar to that observed in this study (Hight et al., in review). In those rats, the number of Fos- and IL1β-IR cells were significantly greater when the rats were killed at 11 h after dark onset compared to when they were killed at the end of the light period, suggesting that the natural stimulation of the whiskers that occurs in the dark also enhances the levels of Fos and IL1β-IR cells.

A second finding, that sensory stimulation enhances the number of IL1β-IR astrocytes in the Sctx and the white matter layer below the Sctx, supports the hypotheses that astrocytes respond to neural activity and produce SRSs in response to neural activity. Whisker stimulation increases the calcium signaling in astrocytes within the Sctx (Wang et al., 2006) and visual stimulation increases calcium responses in specific columns of the Vctx (Schummers et al., 2008). In the current study, the increased number of IL1β-IR astrocytes in the Sctx layers and the external capsule below the stimulated Sctx suggests that enhanced neural activity results in an increased expression of the cytokine IL1β in astrocytes. These results are consistent with previous findings that the homeostatic response regulating ocular dominance in the visual cortex is mediated by astrocytic TNF (Kaneko et al., 2008) and together confirm the importance of astrocyte-neuron communication in neuronal networks (Fellin, 2009).

3.2 Colocalization of activity dependent increases in NGF and IL1β

Our findings that IL1β-IR colocalizes with NGF-IR within cells in the Sctx layer V is consistent with the previous findings that NGF and IL1β colocalize in the frog nervous system during development (Jelaso and DeLong, 2005). The colocalization of NGF and IL1β in Sctx neurons may be due to their mutual dependence on nuclear factor kappa B (NF-κB); both are upregulated after NF-κB activation (Carter et al., 1996; Cao et al., 1999; Mamidipudi et al., 2002; Mémet, 2006; Krueger et al., 2007). Further, an NGF-dependent association of interleukin 1 receptor associated kinase with the p75 neurotrophin receptor could explain, in part, the interaction between IL1β and NGF in neurons and in immune cells (Mamidipudi et al., 2002; Friedman, 2005). Sleep deprivation-dependent increases in NF-κB translocation into the nucleus, the main step in initiating transcription of a number of sleep-regulatory genes, occur in the cortex (Chen et al., 1999) and hypothalamus (Brandt et al., 2004). These observations suggest that cortical activity increases the nuclear translocation of NF-κB and subsequent transcription of NGF and IL1β mRNAs in an activity-dependent manner.

3.3 Network circuitry transferring the activity dependent increases in IL1β and NGF to sleep regulatory centers

After application of IL1β or TNF onto the somatosensory cortex, Fos- and IL1β-IR neurons are induced in the somatic region of the reticular nucleus of the thalamus. Additionally, IL1β-IR astrocytes increase in the perireticular zone of the internal capsule (Churchill et al., 2005; Yasuda et al., 2007). Furthermore, cortical application of IL1β up-regulates Fos-IR cells in hypothalamic regions implicated in sleep regulation, such as the ventrolateral preoptic area and the median preoptic nucleus (Yasuda et al., 2007). These results as well as the increases in IL1β-IR astrocytes in the external capsule support the idea that the local state initiated in cortical columns by activity-induced SRSs is communicated via cortical connections to sleep regulatory circuits to coordinate whole organism sleep.

Previous studies in our lab suggest that unilateral cortical application of SRSs, such as IL1β and TNF, increase sleep intensity evidenced by enhanced EEG SWA on the ipsilateral side (Yoshida et al., 2004; Yasuda et al., 2005b). Thus the whisker stimulation-induced IL1β and NGF may provide an endogenous local source of SRSs that promote sleep within the stimulated cortical columns. Indeed, when rats are sleep deprived after unilateral whisker removal, the side ipsilateral to the cut whiskers receive disproportionate sensory stimulation and exhibit enhanced NGR-IR (Brandt et al 2001) and enhanced EEG delta power in subsequent sleep (Vyazovskiy et al., 2000; 2002). Multiple additional studies using measurements of EEG, or blood flow, or evoked response potentials, indicate that excessive afferent input to a region of the cortex is associated with deeper subsequent sleep (reviewed Krueger et al 2008). For instance, excessive right hand stimulation during wakefulness increases EEG SWA during subsequent sleep in the left cortex in humans (Kattler et al., 1994). Since rats increase their tactile and whisker activity and decrease their visual input during the dark, Sctx EEG SWA is higher during this time than that of the Vctx (Yasuda et al., 2005a). Conversely in the light, EEG SWA of the Vctx is higher than that of the Sctx. Further the probability of a cortical column entering a sleep-like state, as determined from higher amplitude evoked response potentials, is afterent activity-dependent (Rector et al., 2009).

3.4 Conclusion

In conclusion, the present study provides direct evidence that two SRSs, NGF and IL1β, are up-regulated in the Sctx by excessive afferent input. Our findings add new evidence to the hypotheses that sleep is an activity-dependent event regulated in part by astrocytic and neuronal SRSs in the Sctx.

4.1 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 approved by the Institution of Animal Care and Use Committee.

4.2 Unilateral Whisker stimulation in a plexiform chamber

Rats were adapted to unilateral manual whisker stimulation outside of their home cages in a cage (40 cm by 40 cm) divided into two halves by plastic netting as previously described (Churchill et al., 2008). Briefly, the rats were adapted to the whicker stimulation over the course of 8 days starting with 15 min of stimulation and adding 15 min of stimulation time for each subsequent day. On the final day, the rats were stimulated for 2 h prior to the kill. Since the stimulation of Fos-IR in the barrel cortex after whisker stimulation was maximal after 1h and back to normal by 14 h (Bisler et al., 2002), we did not include a group that was adapted to whisker stimulation on all 8 days, but not stimulated on the day of the kill. Therefore, we didn’t think that the adaptation days would have a long-term impact on IL1 or NGF-IR. Each investigator was assigned two rats, one for each hand. Each investigator followed each rat about the enclosed area brushing by hand the longer, more caudal mystacial whiskers (B0, B1, CO, C1, D0, D1, E1) on one side only at a rate of about 30 times per minute. The rats were killed at 10 min intervals starting at 1 h prior to dark onset. Following 2 h of unilateral whisker stimulation, the rats were anesthetized with isoflurane and cardiac-perfused with warm saline followed by cold 4% paraformaldehyde. Brains were then post-fixed in this fixative for 2 h and sunk in 20% sucrose overnight. The brains were frozen in crushed dry ice and stored at −80°C until sectioning.

4.3 Immunohistochemistry

Brains were sectioned coronally with a sliding microtome at 30 μm thickness and collected at 360 μm intervals for each antigen. Immunohistochemistry was performed as described previously (Churchill et al., 2005, 2008), using a rabbit polyclonal anti-human Fos antibody (1:10,000; Calbiochem, Oncogene Research Products, San Diego, CA), goat anti-rat IL1β (0.5 μg/ml, R&D Systems, Inc., Minneapolis, MN), rabbit anti-mouse NGF that cross reacts with rat NGF (1:2500 or 1:5000; Chemicon, Temecula, CA). These sections were incubated for 3 days at 4°C with gentle rocking.

Negative controls for the secondary antibodies were performed by omitting the primary antibody. The specificity of the NGF and Fos antibodies were previously published (Zhang et al., 2007; Berghorn et al., 1994). The specificity of the IL1β antibody was tested by preincubation with an excess of IL1β protein for 24 h at 4° C prior to the immunohistochemistry with the coronal sections. No specific IR-labeling was observed in the sections incubated without primary antibodies or with the preincubated IL1β antibody (Fig. 1A) as compared with the specific rat Sctx labeling (Fig. 1B). This R & D antibody to rat IL1β failed to show specific IR staining in IL1β knockout mice (Fig. 1C relative to wild-type mice Fig. 1D) generously provided by Dr. M.K. O’Banion. Western blot analyses (Churchill et al., 2008) of extracts of lipopolysaccharide-(LPS) activated macrophages showed labeling of the 34 kD and 17kD forms of IL1β by the IL1β antibody; if cells were pretreated with IL1β siRNA, labeling was reduced (Fig. 1E).

4.4 Imaging and Quantitative analysis

The Sctx and the auditory cortex (as a control cortical region) were identified in the brain sections as demarcated in the rat brain atlas by Paxinos and Watson (2007). The digital photographs were prepared on a Leica DMLB microscope with a Spot camera for quantitative analysis. The digital images were adjusted using Adobe Photoshop, only in contrast and brightness. A rectangular box was prepared using a transparent micron ruler for calibration and superimposed over each Sctx layer in the digital images. NGF-stained sections were photographed with the 10× objective and two 0.5 mm × 0.2 mm boxes were superimposed onto each of the Sctx or auditory cortex layers. IL1β-stained sections were photographed with a 20× objective and one 0.5 mm × 0.225 mm box was superimposed onto each of the neuronal layers or three 0.23 mm by 0.08 mm boxes were superimposed onto the white matter layers. Fos-IR in an adjacent section was determined and used to identify stimulated columns on the side contralateral to the manual whisker stimulation. A stimulated column was identified where the number of Fos-IR cells in layer IV increased more than 25% compared with the adjacent columns. Columns of stimulated Fos expression were also observed on the ipsilateral side due to inadvertent whisker stimulation on the opposite side of the face; these columns were not used in the data analyses. The numbers of NGF-, IL1β- and Fos-IR cells were counted for each layer in the stimulated and unstimulated columns as well as on both sides of the auditory cortex.

4.5 Statistical Analyses

A paired Student’s t-test was used to evaluate the differences in the number of IR cells between the stimulated (Fos-IR evident) and unstimulated columns (little Fos-IR) on the stimulated side as well as between the stimulated and unstimulated sides of the auditory cortex.