Introduction Northern analysis of human tissues [12], whereas p38g is

Brain Research 887 2000 350–358 www.elsevier.com locate bres Research report Immunolocalization of p38 MAP kinase in mouse brain a a , b c Masumi Maruyama , Tatsuhiko Sudo , Yoshitoshi Kasuya , Takashi Shiga , d a Bing-Ren Hu , Hiroyuki Osada a Antibiotics Laboratory , RIKEN, 2-1 Hirosawa Wako, Saitama 351-0198 Japan b School of Medicine , Chiba University, Chiba, Japan c Institute of Basic Medical Sciences , University of Tsukuba, Tsukuba, Japan d Queen ’s Medical Center, Hawaii, USA Accepted 3 October 2000 Abstract p38 has been implicated to play a critical role in regulating apoptosis in PC12 and cerebellar granule cells, and is inactivated in cultured fetal neurons in response to insulin. Though p38 is activated in microglia after ischemia, the physiological functions of p38 in the brain are not well understood. As a first step to elucidate the physiological functions of p38 in the central nervous system, we raised a polyclonal antibody against p38 and performed immunohistochemical examination to demonstrate the localization of p38 in mouse brain. Strong p38 immunoreactivity was apparent in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, intense p38 immunoreactivity was often observed in myelin sheath-like structures but not in axons. This is the first demonstration of the localization of p38 in the central nervous system and provides an anatomical basis for understanding physiological roles of p38.  2000 Elsevier Science B.V. All rights reserved. Theme : Neurotransmitters, modulators, transporters and receptors Topic : Signal transduction: gene expression Keywords : p38 MAP kinase; Immunohistochemistry; Oligodendrocyte; Central nervous system 1. Introduction Northern analysis of human tissues [12], whereas p38g is expressed only in skeletal muscle [16] and d has limited p38 was first identified as either an anti-inflammatory expression in kidney and lung [13]. drug CSAID binding protein [15], a lipopolysaccharide In vitro studies have suggested that p38 regulates gene LPS activated kinase [8] or a stress responsive kinase expression by phosphorylating transcription factors, CRE- [21]. p38 is a member of mitogen-activated protein kinases BP1 ATF2 [19], MEF2C [9], CHOP [25] and a transcrip- MAPK and is activated by dual phosphorylation of the tional regulator, Max [27]. Meanwhile p38 phosphorylates conserved TGY motif [8,15,21]. p38 has been implicated cellular kinases such as MAPKAPK2 3 [17,21], resulting to play roles in converting extracellular stresses to cellular in hyperphosphorylation of HSP27 to modulate actin responses. Thus far, four members of p38 have been dynamics [7]. Although the functional relevance and identified in the human. Among them, p38a and b have significance in physiological conditions are not well under- been shown to be expressed relatively ubiquitously, by stood, p38 may play a role in stress responses. More recently, p38 has been implicated to play a critical role in regulating apoptosis in PC12 [26] and cerebellar granule cells [14] by its activation. Conversely, p38 is inactivated in cultured fetal neurons in response to insulin Corresponding author. Tel.: 181-48-467-9542; fax: 181-48-462- [10]. Global forebrain ischemia results in apoptosis in 4669. E-mail address : sudopostman.riken.go.jp T. Sudo. hippocampal CA1 neurons and activation of p38 in 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 6 3 - 8 M . Maruyama et al. Brain Research 887 2000 350 –358 351 microglias [24]. Moreover analysis performed in ATF2 chased from CLEA Japan Inc. Under deep anesthesia with null mice has indicated that ATF2 plays a critical role in an intraperitoneal injection of sodium pentobarbital, mice the hippocampal CA3 [20]. These results suggest that p38 were perfused through the left cardiac ventricle with saline may play a critical part in maintenance of central nervous followed by ice-cold 4 paraformaldehyde in 0.1 M system CNS through the determination of survival and phosphate buffered saline PBS, pH 7.4. The brain was or death in response to extracellular stimuli. then removed and immersion fixed in the same fixative for Here we report the localization of p38 in the adult 12 h at 48C. For paraffin sections, the brain was dehydrated mouse brain as a first step to understanding its physiologi- in graded ethanol, embedded in paraffin, and sectioned at 4 cal functions in the CNS. mm thickness. For cryostat sections, the brain was im- mersed in the series of 5, 15 and 30 sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T.

2. Material and methods compound Miles Inc., and sectioned at 10 mm thickness.