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Expression analysis of neuroglobin mRNA in rodent tissues
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Neuroscience Vol. 115, No. 3, pp. 645^656, 2002 ; 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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EXPRESSION ANALYSIS OF NEUROGLOBIN mRNA IN RODENT TISSUES S. REUSS,a S. SAALER-REINHARDT,b B. WEICH,c S. WYSTUB,c M. H. REUSS,a T. BURMESTERd and T. HANKELNc a b c
Department of Anatomy, Johannes Gutenberg University, 55099 Mainz, Germany
Institute of Physiological Chemistry, Johannes Gutenberg University, 55099 Mainz, Germany
Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University, 55099 Mainz, Germany d
Institute of Zoology, Johannes Gutenberg University, 55099 Mainz, Germany
Abstract4Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the ¢rst detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting di¡erential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is su⁄cient for its putative primary function as an oxygen-supplying protein. ; 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: globin, hypoxia, neuron, oxygen-binding protein.
true oxygen carrier are conserved within Ngb, it displays only modest sequence similarity with vertebrate hemoglobin and myoglobin (less than sequence 25% amino acid identity) (Burmester et al., 2000). Ngb rather resembles some intracellular globins from invertebrates, in particular the nerve myoglobin of the annelid Aphrodite aculeata (Dewilde et al., 1996). Similarly to hemo- and myoglobin, Ngb binds oxygen reversibly via the Fe2þ ion of the heme-group (Burmester et al., 2000; Dewilde et al., 2001). The hexacoordinate structure of the Fe2þ in deoxy-Ngb (Couture et al., 2001; Dewilde et al., 2001; Trent et al., 2001) renders the oxygen-binding kinetics more complex than in other globins (Dewilde et al., 2001). Nevertheless, the oxygen a⁄nity (P50 ) of Ngb was determined to be about 1^2 Torr (Burmester et al., 2000; Dewilde et al., 2001), which is within the range of a typical mammalian myoglobin. These oxygen-binding properties suggest a role of Ngb in oxygen homeostasis of the brain and possibly other nerve tissues. Moreover, a recent study reveals that the Ngb expression rate is increased upon hypoxia, and that the presence of Ngb may promote the survival of nerve cells in vitro (Sun et al., 2001). The total amount of Ngb in the murine brain is apparently low and was estimated to be in the range of only 1 WM (Burmester et al., 2000), while myoglobin in the cells of the striated muscle cells typically occurs in concentrations in the range of some 100 WM (Schuder et al., 1979; Wittenberg and Wittenberg, 1990). This observation was continuously di⁄cult to reconcile with a func-
The supply of the vertebrate brain and other metabolically highly active tissues with su⁄cient amounts of oxygen is an essential process that requires several physiological adaptations such as high degrees of vascularization or the presence of respiratory proteins. In the vertebrate muscle, myoglobin is thought to enhance the transfer of oxygen to the mitochondria (Wittenberg, 1992; Hochachka, 1999). On the other hand, nerve-speci¢c globins have long been known from various invertebrate species, particularly molluscs, annelids and nemertini (Strittmatter and Burch, 1963; Wittenberg et al., 1965; Kraus and Colacino, 1986; Dewilde et al., 1996; Vandergon et al., 1998). Recently, we reported the expression of a distinct globin type in the brain of mouse and man that we termed neuroglobin (Ngb) (Burmester et al., 2000). So far, Ngb has been identi¢ed in various mammalian and ¢sh species, suggesting its ubiquitous presence in all vertebrate taxa (Burmester et al., 2000; Awenius et al., 2001; Zhang et al., 2002). Although all key determinants for the function as a
*Corresponding author. Tel. : +49-6131-392-3277; fax: +49-6131392-4585. E-mail address: [email protected] (T. Hankeln). Abbreviations : DRG, dorsal root ganglion; ISH, in situ hybridization; LRt, lateral reticular nucleus ; Ngb, neuroglobin; nNOS, neuronal NO-synthase ; PBS, phosphate-bu¡ered saline; PCR, polymerase chain reaction; SCG, superior cervical ganglion; SSC, standard saline citrate. 645
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tion of Ngb in oxygen storage or transport. However, regional di¡erences in Ngb expression may be instrumental in resolving this paradox. Moreover, Ngb expression has been observed by northern analysis in tissues other than the brain, such as the gastro-intestinal tract, endocrine organs and testes (Burmester et al., 2000), posing the question whether this expression was potentially due to intrinsic or innervating neurons. Thus, the understanding of the role of Ngb in the vertebrate nervous system requires a detailed knowledge of its cellular and tissue localization.
EXPERIMENTAL PROCEDURES
Hermsen et al. (1998). In brief, RNA probes were diluted in 2USSC/50% formamide and sections were incubated with the probe at 42‡C over night. Slides were washed ¢rst in 2U standard saline citrate (SSC), then in 0.1USSC at 55‡C. Preparations were then treated with a mixture of RNAse A (25 Wg/ml) and RNase T1 (25 U/ml) for 30 min at 37‡C in a wet chamber, and thereafter washed in phosphate-bu¡ered saline (PBS)/Tween 20. After blocking for 15 min in Roche bu¡er 2, label was detected by alkaline phosphatase-coupled anti-Digoxigenin antibodies (diluted 1:100 in Roche bu¡er 2; 30 min incubation at room temperature), washing in PBS/Tween 20 and Roche bu¡er 3, and subsequent incubation with nitro-blue-tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate substrate (1:50 diluted in Roche bu¡er 3; 15^30 min at room temperature in the dark). Substrate reaction was stopped by PBS washing, and sections were covered by PBS/glycerol (1:1) solution, sealed and analyzed using a Leitz Orthoplan microscope. Photomicrographs were taken on Agfa Pan ¢lm.
Animals The procedures concerning animals reported in this study complied with German legislation for the protection of animals and were approved by the county-government o⁄ce (Bezirksregierung Rheinhessen-Pfalz, Az 177-07/961-30). Eight adult mice (Balb/C) and three adult rats (Wistar) were used. The animals were maintained under constant conditions (12-h light/dark cycle, room temperature 21 O 1‡C) with food and water ad libitum. Animals were deeply anesthetized with tribromoethanol (0.3 g/kg body weight, i.p.) at the middle of the light period and perfused transcardially with 100 ml of phosphate-bu¡ered 0.9% saline (to which 15 000 IU heparin/l were added) at room temperature followed by 200^300 ml of ice-cold ¢xative (4% paraformaldehyde, 1.37% L-lysine, 0.21% sodium-periodate in phosphate bu¡er (according to McLean and Nakane, 1974) with a constant rate of 10 ml/min. The right atrium was opened to enable venous out£ow. The brain, spinal cord, sensory ganglia, pituitary gland, pineal organ, adrenal gland, testis, esophagus, duodenum and colon were immediately taken, post¢xed for 1 h and stored at 4‡C in phosphate-bu¡ered 30% sucrose. Within a week, cryosections (15 Wm) were prepared and mounted onto glass slides coated with 3-aminopropyl-triethoxysilane according to Rentrop et al. (1986). In addition, various tissues were prepared (brain, liver, kidney, testis, lung, spleen, duodenum, colon), immediately frozen on dry ice and stored at 380‡C. For these organs, cryosections (8 Wm) were air-dried and ¢xed in ice-cold paraformaldehyde (4%) for 10 min prior to the hybridization procedures. One series of sections was stained with hematoxylin-eosin. Brain regions were identi¢ed according to a stereotaxic atlas (Paxinos and Watson, 1986). In situ-hybridization (ISH) Digoxigenin-labeled sense (negative control) and antisense RNA probes were in vitro-transcribed by T7 RNA polymerase (kit by Roche Diagnostics, Mannheim, Germany), using polymerase chain reaction (PCR)-generated templates covering the 453-bp mouse Ngb coding region (accession no. AJ245945). For the purposes of template generation, a T7 RNA polymerase promoter sequence was attached to the 5P end of the sense or antisense PCR primers. ISH was performed according to
RESULTS
We probed sections from mouse and rat brain and various other tissues by ISH for expression of Ngb mRNA. The speci¢city and selectivity of the ISH method used has been demonstrated before (Hermsen et al., 1998; Bauer et al., 1998). The perinuclear signal obtained is typical for mRNA hybridization, and the absence of sense probe hybridization (see below) con¢rms probe speci¢city. The distribution of brain hybridization signals obtained by the in vitro-transcribed RNA probe was identical to the signal distribution generated by an endlabeled synthetic 40mer oligonucleotide probe (Burmester et al., 2000). A comparison of ¢xed and un¢xed brain material gave essentially identical hybridization results. However, the preservation of perfused tissue proved to be substantially better, although it required higher probe concentrations during hybridization. No signi¢cant di¡erences in distribution or strength of the signals were found between mouse and rat. The hybridization results are summarized in Table 1. Central nervous system In the CNS, the ISH signal was distinctly seen in the gray matter, while a few positive cells were also identi¢ed in the white matter. Ngb mRNA was detected in the cytoplasm of neurons in many regions of the brain. Visual inspection of the sections suggested that the vast majority of neuronal cells were labeled. Additional use of interference contrast optics revealed that in a few neurons the ISH signal was not detectable. In the cerebrum, positive neurons appeared to be
Abbreviations used in the ¢gures Cb CC DG DH Gr LP M Mo OB
cerebellum cerebral cortex dentate gyrus dorsal horn granular layer lateral paragigantocellular nucleus medullary region molecular layer olfactory bulb
P Po Rt S SOC SpC TG VH
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Purkinje cells polymorph layer reticular nucleus cortical surface superior olivary complex spinal cord trigeminal ganglion ventral horn
Neuroglobin expression Table 1. Summary of the Ngb ISH results for the various organs and tissues
647 Table 1 (continued).
Tissue Tissue
CNS olfactory bulb glomeruli periglomerular neurons mistral cell layer neurons granule cell layer lateral olfactory nucleus cerebrum (cortical layers II^VI) motor cortex sensory cortex primary olfactory cortex subcortical regions hippocampal formation (CA1^CA4) pyramidal cells granule cells (dentate gyrus) subiculum islands of Calleja striatum thalamus subthalamic nucleus hypothalamus suprachiasmatic nucleus supraoptic nucleus paraventricular nucleus anterior hypothalamus ventromedial hypothalamus arcuate nucleus brain stem nuclei inferior colliculi superior colliculi mesencephalic trigeminal reticular nucleus lower brain stem superior olivary complex facial nucleus lateral paragigantocellular nucleus lateral reticular nucleus pontine nuclei inferior olive cerebellum cortical molecular layer cortical granule layer medullary layer deep nuclei spinal cord (cervical/thoracic segments) dorsal horn ventral horn lateral horn central autonomic region ventricle ependymal cells choroid plexus PNS sensory nervous system trigeminal ganglion** dorsal root ganglion* autonomic nervous system superior cervical ganglion* intramural ganglia esophagus duodenum colon* entero-endocrine cells** Endocrine system pituitary gland adenohypophysis neurohypophysis adrenal gland
ISH signal
Figure
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Figure
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The expression was tested in mice, exceptions are indicated by * (studied only in rat) and ** (studied in mouse and rat).
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cortex glomerular layer fascicular layer reticular layer medulla pineal** testis spermatogonia, spermatocytes Other organs liver spleen lung (bronchiolar/alveolar epithelium) kidney esophagus epithelium duodenum epithelium colon epithelium skeletal muscle
ISH signal
5a^c 5b 6a 6b, c 6b 6d
evenly distributed over cortical laminae II^VI. Distribution and amount of positive neurons were apparently similar in di¡erent cortical areas, e.g. motor and sensory cortex. A typical example demonstrating the frontal cerebral cortex is given in Fig. 1. In addition to neuronal somata, ¢bers showed the ISH signal (arrow in Fig. 1b). In the cerebellum, most of the small neurons of the granular layer exhibited Ngb mRNA (Fig. 1c, d). Interneurons of the molecular layer were also stained. Apparently, all Purkinje cells exhibited strong signals. We also observed many labeled ¢bers in the granule cell layer (arrow in Fig. 1d) and some ¢bers in the vicinity of Purkinje cells. In addition, neurons of the cerebellar nuclei located in the white matter showed the ISH signal. Strong signal was also seen in subcortical regions. In the hippocampal formation, pyramidal cells of the regions CA1^CA3 and granule cells of the dentate gyrus exhibited strong cytoplasmic signal (Fig. 2a^c), and scattered neurons of the subiculum were also labeled. In addition, neuronal somata in the islands of Calleja, striatum and thalamic nuclei showed the ISH signal. Many brainstem nuclei exhibited strong hybridization of neuronal cytoplasm. In particular, neurons were stained in both inferior and superior colliculi, the mesencephalic trigeminal nucleus and the reticular nuclei complex (Fig. 2d, e). In the lower brainstem (Fig. 3a), the auditory superior olivary complex (Fig. 3b), the nucleus of the facial nerve (Fig. 3c, d), lateral paragigantocellular nucleus, lateral reticular nucleus (LRt), pontine nuclei and the inferior olive (Fig. 3e) exhibited distinct hybridization of neuronal somata. Some of these cells were conspicuous because of their large cell bodies (see Fig. 3, facial nucleus), but the strength of the cytoplasmic signal was similar to that in other neurons. In the olfactory bulb (Fig. 3f^h), glomeruli were unstained, while periglomerular neurons as well as neurons of the mitral cell layer and of the granule cell layer exhibited the ISH signal (Fig. 3g). Stained ¢bers were detected in the periglomerular zone and in the region
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Fig. 1. Parasagittal section of a mouse brain showing the in situ-hybridization (ISH) signal (a) in neurons of area 1 of the frontal cerebral cortex (CC) in laminae I^VI, and (b) in higher magni¢cation, where arrows point to stained ¢bers. The cerebellum (Cb) is shown in (c), taken from lobule 5. In the cortex, interneurons are stained in the molecular layer (Mo) adjacent to the cortical surface (S). Apparently all Purkinje cells (P) and many cells in the granular layer (Gr) exhibit the ISH signal, while the medullary region (M) was unstained. In the higher magni¢cation (d), ¢bers (arrow) and groups of neurons (asterisk) are labeled. Scale bars = 50 Wm (a, c), 10 Wm (b, d).
between mitral and granular cells (Fig. 3h, arrow). In the spinal cord, positive neurons were observed throughout the gray matter in cervical and thoracic segments (Fig. 4a, stemming from segment Th1). In sections cut in the axial plane, the signal was seen in the laminae of the dorsal horn (Fig. 4d) and in the central autonomic region surrounding the central canal (Fig. 4b). Stained neurons were also seen in the lateral horn (presumptive preganglionic sympathetic neurons of the intermediolateral
nucleus and lateral funicle; arrow in Fig. 4d) and in the ventral horn (Fig. 4c). In the latter, some large neurons (probably alpha-motoneurons) were labeled. Peripheral nervous system The trigeminal (Gasserian) ganglion of mouse and rat and the rat dorsal root ganglia of lower cervical and upper thoracic segments were studied as examples of
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Fig. 2. Parasagittal section of a mouse brain showing the ISH signal (a) in the hippocampal formation, where the pyramidal (P) cell layer of ¢elds CA1^3, neurons of the granular layer of the dentate gyrus (DG) as well as scattered cells of the subiculum (S) were stained. The higher magni¢cations showed (b) stained neurons of the polymorph layer (Po) of the dentate gyrus and (c) the caudal end of the DG which is marked by an arrow in panels a and c. The reticular nucleus (Rt) below the cerebellum (Cb) is shown in (d) and a higher magni¢cation from the parvocellular part of the reticular nucleus in (e). Scale bars = 200 Wm (a, d), 50 Wm (b, e), 20 Wm (c).
sensory ganglia. From the autonomic nervous system, we investigated the rat sympathetic superior cervical ganglion (SCG) and intramural ganglia of several parts of the mouse and rat gastrointestinal tract. In the trigeminal ganglion, virtually every neuronal perikaryon was stained (Fig. 4e^g), while in the ¢ber regions between neuronal groups no speci¢c signal was seen. In some peripheral ¢bers, in particular in the part of the ganglion that gives rise to the ophthalmic nerve, a punctated signal was seen (Fig. 4g). However, in the dorsal root ganglion, cells exhibited only weak signals. Similar ¢ndings were obtained from the rat SCG (data not shown). The enteric nervous system was studied in cross sections of the esophagus, duodenum and colon of both species. In each intestinal region, distinct hybridization of neurons was found. This is demonstrated for the rat colon as a typical example in Fig. 5. In particular, the myenteric plexus (Auerbach’s plexus; arrows in Fig. 5a,
c, d) located between the circular and longitudinal layers of the tunica muscularis (large asterisks in Fig. 5a^d) was stained. We also observed scattered neurons in the submucosal layer belonging to the submucosal plexus (Meissner’s plexus; Fig. 5a). In addition, stained cells that are probably enteroendocrine cells were seen in the mucosal layer (arrows in Fig. 5a, b). Endocrine system From the endocrine system, we studied mouse and rat pituitary glands and pineal, mouse and rat adrenal glands and mouse testes. No signi¢cant di¡erences were observed in the distribution of the ISH signal in a distinct organ or region between the species tested. In the testis, expression of Ngb mRNA was restricted to the spermatogonia and, probably, to primary spermatocytes of the outer zones of the tubule walls (Fig. 5e). In the pituitary gland (Fig. 6a), hybridization was restricted
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Fig. 3. Parasagittal section of a mouse brain showing the ISH signal (a) in some brainstem nuclei, i.e. the superior olivary complex (SOC), facial nerve nucleus (7), lateral paragigantocellular nucleus (LP) and lateral reticular nucleus (LRt). The higher magni¢cations depict neurons labeled by ISH in the SOC (b), anterior part of the facial nucleus (c, d; arrows point to the same cell) and in the inferior olive (e). The olfactory bulb (OB) is seen in panels f^h. While glomeruli (asterisks) were unstained, the ISH signal is found in periglomerular cells (P) as well as in neurons of the mitral (M) and granular (Gr) cell layers. Scale bars = 200 Wm (a, f), 50 Wm (g), 10 Wm (b^e, h).
to the anterior lobe, where the endocrine cells of the adenohypophysis exhibited a relatively strong signal (Fig. 6b, c). In contrast, the glia-like pituicytes of the posterior lobe (neurohypophysis) were apparently
unstained (right part of Fig. 6b). In the adrenal gland, we observed the ISH signal in the cortex and medulla (Fig. 6d). Cells of the cortical zona glomerulosa, located below the capsule, exhibited a strong signal (arrow in
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Fig. 4. Axial section of the mouse spinal cord (SpC) segment Th1 demonstrating the ISH signal in neurons throughout the gray matter (a). (b) In the central autonomic region surrounding the central canal (asterisk) as well as in the ventral horn (VH; c) and the dorsal horn (DH ; d), stained neurons are found. Note that neurons in the intermediolateral nucleus of the lateral horn (arrow in panel d) also exhibit the ISH signal. (e^g) Horizontal section of the trigeminal ganglion (TG) is shown in panel e, where the ISH signal is present in neurons, as demonstrated in higher magni¢cation in panel f, and in ¢bers (g, arrows). Scale bars = 200 Wm (a, e), 50 Wm (c, d), 10 Wm (b, f, g).
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Fig. 5. Distribution of Ngb mRNA in the rat colon and mouse testis. A cross section is shown in panel a. Labeled are ganglionic cells of the myenteric plexus (arrows in panels a, c, d) located between the circular and longitudinal layers of the tunica muscularis (large asterisks in panels a^d), neurons in the submucosal layer belonging to the submucosal plexus, and putative enteroendocrine cells in the mucosal layers (arrow in panel b). The lamina muscularis mucosa is indicated by small arrows in panels a and b. (e, f) Cryosections of mouse testis tissue. Ngb transcripts were detected in the two outer layers of the tubules where spermatogonia and primary spermatocytes are located (e). No ISH signal was observed with the complementary sense RNA probe (f). Scale bars = 50 Wm (a), 10 Wm (b, c), 50 Wm (e, f).
Fig. 6d), while cells of the zona fasciculata and those of the zona reticularis were not or only rarely stained (Fig. 6f). Apparently all medullary cells exhibited Ngb mRNA (Fig. 6e). In the pineal gland, parenchymal cells (i.e. pinealocytes) appeared unstained.
nomic and sensory parts), as well as in the endocrine system. These data also explain the presence of Ngb mRNA in tissues other than the brain (Burmester et al., 2000). At the same time, the ISH results con¢rm the absence of Ngb in various organs, as suggested before by northern hybridization (Burmester et al., 2000).
DISCUSSION
Expression of Ngb in nervous and endocrine systems Ngb is a respiratory protein that is preferentially expressed in the brain of mouse and man (Burmester et al., 2000). The ISH experiments reported here con¢rm and extend this study. Ngb mRNA was observed in both the central and peripheral nervous system (in auto-
In the CNS, we found an ubiquitous distribution of Ngb mRNA-containing neuronal somata. Apparently, the vast majority of perikarya in the neuron-bearing layers of the cerebral and cerebellar cortices were labeled
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Fig. 6. Section of mouse endocrine organs showing the ISH signal. A frontal section of the pituitary gland is shown in panel a. The higher magni¢cations (b) demonstrate the absence of the signal in the posterior pituitary (right part) while the anterior part of the gland (left, and panel c) shows the signal in the cytoplasm of endocrine cells. A horizontal section of the adrenal gland is demonstrated in panel d. The signal is present in cells of the medulla (M, see also panel e) and in the cortex, in particular in the glomerular zone (arrow in panel d, dark cells on top in panel f). Asterisk in panel d depicts unspeci¢c fat cell staining in a zone between cortex and medulla. Scale bars = 200 Wm (a, d), 20 Wm (b, e, f), 10 Wm (c).
by the Ngb probe. This distribution corresponds with relatively high (cerebral cortex) and rather low (cerebellar cortex) levels of Ngb mRNA in human tissue RNA blots (Burmester et al., 2000). The rich distribution of the
ISH signal in subcortical regions, in the brain stem, olfactory bulb and spinal cord reveals that the expression of Ngb mRNA is a general feature of nerve cells. These results con¢rm and extend previous analyses (Burmester
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et al., 2000; Zhang et al., 2002) describing that neuronal somata of the hippocampal formation and of some other brain regions contain Ngb mRNA. Our data provide evidence that the other part of the CNS, the spinal cord, also expresses Ngb in its gray substance, and that the respective mRNA is also found in the peripheral nervous system. Sensory and autonomic ganglia exhibited ^ although to varying degrees ^ neuronal perikarya that were labeled by the probe. For example, our results clearly demonstrate that in the gut intrinsic neurons (submucosal and myenteric plexus, see Fig. 5a^d) rather than innervating neurons transcribe Ngb. This observation explains the positive northern blot signals obtained from these organs (Burmester et al., 2000). Di¡erent regions of the brain exhibited positive ISH signals for Ngb in nerve ¢bers, e.g. in the granule cell layer of the cerebellum, where mossy ¢bers make complex synapses (Fig. 1d). These ¢ndings suggest that Ngb mRNA is transported in nerve cell processes to provide protein synthesis in distal regions of axons and dendrites (Job and Eberwine, 2001). The remarkable punctuated hybridization signal might also be an indication for Ngb mRNA expression in varicosities and synaptic terminals as well as in dendritic spines. This localization of Ngb transcripts could have important functional consequences for synaptic plasticity, an active metabolic process which presumably needs substantial amounts of oxygen. Moreover, we obtained evidence that cells of the endocrine system (e.g. anterior pituitary, adrenal gland) also express Ngb mRNA, suggesting that signals obtained by northern blots (Burmester et al., 2000) are most likely due to intrinsic Ngb production rather than to innervation of these tissues. In particular, pituitary cells were strongly stained in mouse and rat, which coincides well with the high Ngb expression found by northern hybridization (Burmester et al., 2000). In the adrenal gland, the cortical glomerular layer (responsible for the synthesis of mineralocorticoids such as aldosterone) was heavily stained, while the fascicular zone (producing glucocorticoids such as cortisol) and the reticular zone (sex hormones) exhibited only few labeled cells. It is conceivable that the di¡erent degrees of Ngb expression are related to the activity level of the respective tissue or region. This may also apply to our ¢ndings that neuronal somata in one sensory ganglion (i.e. trigeminal ganglion) were labeled, but those in another (i.e. dorsal root ganglion, DRG) were not or only to a minor degree. The signal was found in ¢bers in both ganglia, also suggesting that Ngb is expressed in sensory neurons. In addition, neuronal somata in the sympathetic superior cervical ganglion were lightly labeled, while some stronger signal was observed in ¢bers. If these were preganglionic, they belong to sympathetic neurons located in the spinal lateral horn, which indeed exhibited labeled somata in the present study. With regard to only weakly labeled ganglion cells such as those in the DRG, we consider that their metabolism involving Ngb was rather low and that Ngb expression may be enhanced under di¡erent circumstances. An organ known to regularly vary in its activity state is
the pineal gland. Its metabolism is regulated by the endogenous circadian clock to produce melatonin during the night (cf. Reuss, 1996). Our ¢nding that the gland’s parenchymal cells, pinealocytes, are not stained may be attributed to the fact that the animals of the present study were killed during the day. The question of di¡erent expression of Ngb, either with regard to daytime or to stimulation of metabolism in the pineal and in other organs, will be addressed in subsequent studies. Ngb expression and NO-synthesizing cells It has been postulated that Ngb may function as an NO-dioxygenase involved in NO-homeostasis (e.g. Brunori, 2001), similarly to the myoglobin in the murine muscle and heart (Flo«gel et al., 2001). However, no correlation of NO synthesis and Ngb expression was observed in our analyses. For example, in the spinal cord neuronal somata immunoreactive for the NO-producing enzyme, neuronal NO-synthase (nNOS), are restricted to dorsal and lateral horns (Reuss and Reuss, 2001), while Ngb mRNA was found throughout the whole gray matter (present study). In the cerebellar cortex, Purkinje cells express Ngb mRNA (present study) while they do not contain nNOS (cf. Reuss and Riemann, 2000, for discussion). In the cerebral cortex, nNOS neurons are a distinct subpopulation of neurons, while apparently all express Ngb mRNA. It thus appears that Ngb neurons may or may not produce nNOS, and that nNOS neurons may or may not express Ngb mRNA. Thus it remains to be shown by double-immunolabeling how both neuronal subpopulations are related to each other. Considering also that NO-producing neurons exert their in£uence on neighboring cells (Flo«gel et al., 2001), further studies must address the possible function of Ngb as NO-dioxygenase. Is Ngb expressed in glia? In some analyzed regions (e.g. spinal cord funicles, hippocampal alveus, cerebellar medulla) small labeled structures were seen, suggesting that Ngb may be expressed in some presumed glia cells. Fiber-containing parts of the trigeminal ganglion and the neurohypophysis, however, appeared unstained. In the latter, pituicytes are specialized glial cells, indicating that not all glia cells may produce Ngb. The question in how far Ngb expression is a feature not only of neurons, but also of glia cells, must ultimately be addressed by experiments combining the detection of Ngb mRNA or protein and a glial cell marker such as the glial ¢brillary acidic protein. Implications for Ngb function At ¢rst glance, it is di⁄cult to assume that the low concentration of Ngb estimated in total brain tissue of mouse and man (Burmester et al., 2000) is su⁄cient to support a function in oxygen homeostasis. In contrast, nerve-speci¢c globins of several invertebrates typically occur in much higher concentrations (Kraus and Colacino, 1986; Dewilde et al., 1996; Vandergon et al.,
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1998). However, here we show that Ngb mRNA is in fact expressed in neuronal somata the volume of which makes up only a small part of total brain volume (see Fig. 1b). Although at present we have no means available to determine the intracellular amount of Ngb protein in single cells, these data suggest that local concentrations of Ngb within nerve cells can be considerably higher than the 1 WM previously estimated for total brain (Burmester et al., 2000) and may in fact reach intracellular values similar to those which have been observed for myoglobin in muscles (Schuder et al., 1979; Wittenberg and Wittenberg, 1990). Accordingly, Ngb is expressed, at apparently high levels, in secretory cells of the endocrine system (Fig. 6). It can be assumed that these metabolically highly active cells require large amounts of oxygen, and the presence of the oxygen-binding Ngb may sustain this need. Cladistic analyses indicate that Ngb belongs to an ancient branch of intracellular globins (Burmester et al., 2000; Awenius et al., 2001), which includes at least the nerve myoglobin of the annelid Aphrodite aculeata (Dewilde et al., 1996). The early emergence of Ngb-like globins in the evolution of the Metazoa suggests a conserved function of Ngb in the animal nerval system. However, while the vertebrate Ngb expression is pronounced in neurons, most invertebrate nerve globins that are expressed at high levels are located in glia cells (Wittenberg et al., 1965; Vandergon et al., 1998;
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Dewilde et al., 1996). In molluscs, species living under especially hypoxic conditions display high levels of nerval globin expression in glia cells, while related species from oxygen-rich habitats seem to feature a low-level neuronal expression (Weber and Vinogradov, 2001). Pronounced changes in the strength and cellular localization of nerve globin expression may therefore have accompanied the adaptation for enduring severe hypoxia in invertebrates. Future studies on species adapted to low-oxygen environments will show whether this is also true for vertebrates. Given the recent evidence for nerve cell survival-promoting features of Ngb upon hypoxia (Sun et al., 2001), our expression analyses con¢rm previous notions that Ngb may indeed act as an intracellular oxygen storage or transport protein of nerve cells (Burmester et al., 2000). Other proposed functions of Ngb (see Dewilde et al., 2001) such as oxygen sensing, NO-dioxygenase (see above) and protection from reactive oxygen species can thus be regarded less likely.
Acknowledgements)This work is supported by the Deutsche Forschungsgemeinschaft (Grants Ha2103/3 and Bu956/5). The authors thank Katja Loppe-Ho¡mann and Ursula DisqueKaiser for expert technical assistance. T.H. wishes to thank Erwin R. Schmidt for excellent working facilities and support.
REFERENCES
Awenius, C., Hankeln, T., Burmester, T., 2001. Neuroglobins from the zebra¢sh Danio rerio and the pu¡er¢sh Tetraodon nigroviridis. Biochem. Biophys. Res. Commun. 287, 418^421. Bauer, U., Schneider-Hirsch, S., Reinhardt, S., Bonavente, R., Maelicke, A., 1998. The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes. FEBS Lett. 208, 208^214. Brunori, M., 2001. Nitric oxide, cytochrome-c oxidase and myoglobin. Trends Biochem. Sci. 26, 21^23. Burmester, T., Weich, B., Reinhardt, S., Hankeln, T., 2000. A vertebrate globin expressed in the brain. Nature 407, 520^523. Couture, M., Burmester, T., Hankeln, T., Rousseau, D.L., 2001. The heme environment of mouse neuroglobin : Evidence for the presence of two conformation of the heme pocket. J. Biol. Chem. 276, 36377^36382. Dewilde, S., Blaxter, M., Van Hauwaert, M.L., Van£eteren, J., Esmans, E.L., Marden, M., Gri¡on, N., Moens, L., 1996. Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata. J. Biol. Chem. 271, 19865^19870. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M.C., Caubergs, R., Moens, L., 2001. Biochemical characterization and ligand-binding properties of neuroglobin, a novel member of the globin family. J. Biol. Chem. 276, 38949^38955. Flo«gel, U., Merx, M.W., Go«decke, A., Decking, U.K., Schrader, J., 2001. Myoglobin : A scavenger of bioactive NO. Proc. Natl. Acad. Sci. USA 98, 735^740. Hermsen, B., Stetzer, E., Thees, R., Heiermann, R., Schrattenholz, A., Ebbinghaus, U., Kretschmer, A., Methfessel, C., Reinhardt, S., Maelicke, A., 1998. Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression. J. Biol. Chem. 273, 18394^18404. Hochachka, P.W., 1999. The metabolic implications of intracellular circulation. Proc. Natl. Acad. Sci. USA 96, 12233^12239. Job, C., Eberwine, J., 2001. Localization and translation of mRNA in dendrites and axons. Nat. Rev. Neurosci. 2, 889^898. Kraus, D.W., Colacino, J.M., 1986. Extended oxygen delivery from the nerve hemoglobin of Tellina alternata (Bivalvia). Science 232, 90^92. McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde ¢xative. A new ¢xation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077^1083. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA. Rentrop, M., Knapp, B., Winter, H., Schweizer, J., 1986. Aminoalkylsilane-treated glass slides as support for in situ hybridisation of keratin cDNA to frozen tissue sections under varying ¢xation and penetration conditions. Histochem. J. 18, 271^276. Reuss, M.H., Reuss, S., 2001. Nitric oxide synthase neurons in the rodent spinal cord: distribution, relation to substance P ¢bers, and e¡ects of dorsal rhizotomy. J. Chem. Neuroanat. 21, 181^196. Reuss, S., 1996. Components and connections of the circadian timing system in mammals. Cell Tissue Res. 285, 353^378. Reuss, S., Riemann, R., 2000. Distribution and projections of nitric oxide synthase neurons in the rodent superior olivary complex. Microsc. Res. Tech. 51, 318^329. Schuder, S., Wittenberg, J.B., Haseltine, B., Wittenberg, B.A., 1979. Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography. Anal. Biochem. 92, 473^481. Strittmatter, P., Burch, H.B., 1963. The heme protein in ganglia of Spisula solidissima. Biochim. Biophys. Acta 78, 562^563. Sun, Y., Jin, K., Mao, X.O., Zhu, Y., Greenberg, D.A., 2001. Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. Proc. Natl. Acad. Sci. USA 98, 15306^15311. Trent, J.T., III, Watts, R.A., Hargrove, M.S., 2001. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J. Biol. Chem. 276, 30106^30110.
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Vandergon, T.L., Riggs, C.K., Gorr, T.A., Colacino, J.M.RiggsA.F., 1998. The mini-hemoglobins in neural and body wall tissue of the nemertean worm, Cerebratulus lacteus. J. Biol. Chem. 273, 16998^17011. Weber, R.E., Vinogradov, S.N., 2001. Nonvertebrate hemoglobins: Functions and molecular adaptations. Physiol. Rev. 81, 569^628. Wittenberg, B.A., Briehl, R.W., Wittenberg, J.B., 1965. Haemoglobins of invertebrate tissues - nerve haemoglobins of Aphrodite, Aplysia, and Halosydna. Biochem. J. 96, 363^371. Wittenberg, J.B., Wittenberg, B.A., 1990. Mechanisms of cytoplasmic hemoglobin and myoglobin function. Annu. Rev. Biophys. Biophys. Chem. 19, 217^241. Wittenberg, J.B., 1992. Functions of cytoplasmatic hemoglobins and myohemerythrin. Adv. Comp. Environ. Physiol. 13, 60^85. Zhang, C., Wang, C., Deng, M., Li, L., Wang, H., Fan, M., Xu, W., Meng, F., Qian, L., He, F., 2002. Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin. Biochem. Biophys. Res. Commun. 290, 1411^1419. (Accepted 9 August 2002)
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Neuroscience Vol. 115, No. 3, pp. 645^656, 2002 ; 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
EXPRESSION ANALYSIS OF NEUROGLOBIN mRNA IN RODENT TISSUES S. REUSS,a S. SAALER-REINHARDT,b B. WEICH,c S. WYSTUB,c M. H. REUSS,a T. BURMESTERd and T. HANKELNc a b c
Department of Anatomy, Johannes Gutenberg University, 55099 Mainz, Germany
Institute of Physiological Chemistry, Johannes Gutenberg University, 55099 Mainz, Germany
Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University, 55099 Mainz, Germany d
Institute of Zoology, Johannes Gutenberg University, 55099 Mainz, Germany
Abstract4Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the ¢rst detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting di¡erential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is su⁄cient for its putative primary function as an oxygen-supplying protein. ; 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: globin, hypoxia, neuron, oxygen-binding protein.
true oxygen carrier are conserved within Ngb, it displays only modest sequence similarity with vertebrate hemoglobin and myoglobin (less than sequence 25% amino acid identity) (Burmester et al., 2000). Ngb rather resembles some intracellular globins from invertebrates, in particular the nerve myoglobin of the annelid Aphrodite aculeata (Dewilde et al., 1996). Similarly to hemo- and myoglobin, Ngb binds oxygen reversibly via the Fe2þ ion of the heme-group (Burmester et al., 2000; Dewilde et al., 2001). The hexacoordinate structure of the Fe2þ in deoxy-Ngb (Couture et al., 2001; Dewilde et al., 2001; Trent et al., 2001) renders the oxygen-binding kinetics more complex than in other globins (Dewilde et al., 2001). Nevertheless, the oxygen a⁄nity (P50 ) of Ngb was determined to be about 1^2 Torr (Burmester et al., 2000; Dewilde et al., 2001), which is within the range of a typical mammalian myoglobin. These oxygen-binding properties suggest a role of Ngb in oxygen homeostasis of the brain and possibly other nerve tissues. Moreover, a recent study reveals that the Ngb expression rate is increased upon hypoxia, and that the presence of Ngb may promote the survival of nerve cells in vitro (Sun et al., 2001). The total amount of Ngb in the murine brain is apparently low and was estimated to be in the range of only 1 WM (Burmester et al., 2000), while myoglobin in the cells of the striated muscle cells typically occurs in concentrations in the range of some 100 WM (Schuder et al., 1979; Wittenberg and Wittenberg, 1990). This observation was continuously di⁄cult to reconcile with a func-
The supply of the vertebrate brain and other metabolically highly active tissues with su⁄cient amounts of oxygen is an essential process that requires several physiological adaptations such as high degrees of vascularization or the presence of respiratory proteins. In the vertebrate muscle, myoglobin is thought to enhance the transfer of oxygen to the mitochondria (Wittenberg, 1992; Hochachka, 1999). On the other hand, nerve-speci¢c globins have long been known from various invertebrate species, particularly molluscs, annelids and nemertini (Strittmatter and Burch, 1963; Wittenberg et al., 1965; Kraus and Colacino, 1986; Dewilde et al., 1996; Vandergon et al., 1998). Recently, we reported the expression of a distinct globin type in the brain of mouse and man that we termed neuroglobin (Ngb) (Burmester et al., 2000). So far, Ngb has been identi¢ed in various mammalian and ¢sh species, suggesting its ubiquitous presence in all vertebrate taxa (Burmester et al., 2000; Awenius et al., 2001; Zhang et al., 2002). Although all key determinants for the function as a
*Corresponding author. Tel. : +49-6131-392-3277; fax: +49-6131392-4585. E-mail address: [email protected] (T. Hankeln). Abbreviations : DRG, dorsal root ganglion; ISH, in situ hybridization; LRt, lateral reticular nucleus ; Ngb, neuroglobin; nNOS, neuronal NO-synthase ; PBS, phosphate-bu¡ered saline; PCR, polymerase chain reaction; SCG, superior cervical ganglion; SSC, standard saline citrate. 645
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tion of Ngb in oxygen storage or transport. However, regional di¡erences in Ngb expression may be instrumental in resolving this paradox. Moreover, Ngb expression has been observed by northern analysis in tissues other than the brain, such as the gastro-intestinal tract, endocrine organs and testes (Burmester et al., 2000), posing the question whether this expression was potentially due to intrinsic or innervating neurons. Thus, the understanding of the role of Ngb in the vertebrate nervous system requires a detailed knowledge of its cellular and tissue localization.
EXPERIMENTAL PROCEDURES
Hermsen et al. (1998). In brief, RNA probes were diluted in 2USSC/50% formamide and sections were incubated with the probe at 42‡C over night. Slides were washed ¢rst in 2U standard saline citrate (SSC), then in 0.1USSC at 55‡C. Preparations were then treated with a mixture of RNAse A (25 Wg/ml) and RNase T1 (25 U/ml) for 30 min at 37‡C in a wet chamber, and thereafter washed in phosphate-bu¡ered saline (PBS)/Tween 20. After blocking for 15 min in Roche bu¡er 2, label was detected by alkaline phosphatase-coupled anti-Digoxigenin antibodies (diluted 1:100 in Roche bu¡er 2; 30 min incubation at room temperature), washing in PBS/Tween 20 and Roche bu¡er 3, and subsequent incubation with nitro-blue-tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate substrate (1:50 diluted in Roche bu¡er 3; 15^30 min at room temperature in the dark). Substrate reaction was stopped by PBS washing, and sections were covered by PBS/glycerol (1:1) solution, sealed and analyzed using a Leitz Orthoplan microscope. Photomicrographs were taken on Agfa Pan ¢lm.
Animals The procedures concerning animals reported in this study complied with German legislation for the protection of animals and were approved by the county-government o⁄ce (Bezirksregierung Rheinhessen-Pfalz, Az 177-07/961-30). Eight adult mice (Balb/C) and three adult rats (Wistar) were used. The animals were maintained under constant conditions (12-h light/dark cycle, room temperature 21 O 1‡C) with food and water ad libitum. Animals were deeply anesthetized with tribromoethanol (0.3 g/kg body weight, i.p.) at the middle of the light period and perfused transcardially with 100 ml of phosphate-bu¡ered 0.9% saline (to which 15 000 IU heparin/l were added) at room temperature followed by 200^300 ml of ice-cold ¢xative (4% paraformaldehyde, 1.37% L-lysine, 0.21% sodium-periodate in phosphate bu¡er (according to McLean and Nakane, 1974) with a constant rate of 10 ml/min. The right atrium was opened to enable venous out£ow. The brain, spinal cord, sensory ganglia, pituitary gland, pineal organ, adrenal gland, testis, esophagus, duodenum and colon were immediately taken, post¢xed for 1 h and stored at 4‡C in phosphate-bu¡ered 30% sucrose. Within a week, cryosections (15 Wm) were prepared and mounted onto glass slides coated with 3-aminopropyl-triethoxysilane according to Rentrop et al. (1986). In addition, various tissues were prepared (brain, liver, kidney, testis, lung, spleen, duodenum, colon), immediately frozen on dry ice and stored at 380‡C. For these organs, cryosections (8 Wm) were air-dried and ¢xed in ice-cold paraformaldehyde (4%) for 10 min prior to the hybridization procedures. One series of sections was stained with hematoxylin-eosin. Brain regions were identi¢ed according to a stereotaxic atlas (Paxinos and Watson, 1986). In situ-hybridization (ISH) Digoxigenin-labeled sense (negative control) and antisense RNA probes were in vitro-transcribed by T7 RNA polymerase (kit by Roche Diagnostics, Mannheim, Germany), using polymerase chain reaction (PCR)-generated templates covering the 453-bp mouse Ngb coding region (accession no. AJ245945). For the purposes of template generation, a T7 RNA polymerase promoter sequence was attached to the 5P end of the sense or antisense PCR primers. ISH was performed according to
RESULTS
We probed sections from mouse and rat brain and various other tissues by ISH for expression of Ngb mRNA. The speci¢city and selectivity of the ISH method used has been demonstrated before (Hermsen et al., 1998; Bauer et al., 1998). The perinuclear signal obtained is typical for mRNA hybridization, and the absence of sense probe hybridization (see below) con¢rms probe speci¢city. The distribution of brain hybridization signals obtained by the in vitro-transcribed RNA probe was identical to the signal distribution generated by an endlabeled synthetic 40mer oligonucleotide probe (Burmester et al., 2000). A comparison of ¢xed and un¢xed brain material gave essentially identical hybridization results. However, the preservation of perfused tissue proved to be substantially better, although it required higher probe concentrations during hybridization. No signi¢cant di¡erences in distribution or strength of the signals were found between mouse and rat. The hybridization results are summarized in Table 1. Central nervous system In the CNS, the ISH signal was distinctly seen in the gray matter, while a few positive cells were also identi¢ed in the white matter. Ngb mRNA was detected in the cytoplasm of neurons in many regions of the brain. Visual inspection of the sections suggested that the vast majority of neuronal cells were labeled. Additional use of interference contrast optics revealed that in a few neurons the ISH signal was not detectable. In the cerebrum, positive neurons appeared to be
Abbreviations used in the ¢gures Cb CC DG DH Gr LP M Mo OB
cerebellum cerebral cortex dentate gyrus dorsal horn granular layer lateral paragigantocellular nucleus medullary region molecular layer olfactory bulb
P Po Rt S SOC SpC TG VH
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Purkinje cells polymorph layer reticular nucleus cortical surface superior olivary complex spinal cord trigeminal ganglion ventral horn
Neuroglobin expression Table 1. Summary of the Ngb ISH results for the various organs and tissues
647 Table 1 (continued).
Tissue Tissue
CNS olfactory bulb glomeruli periglomerular neurons mistral cell layer neurons granule cell layer lateral olfactory nucleus cerebrum (cortical layers II^VI) motor cortex sensory cortex primary olfactory cortex subcortical regions hippocampal formation (CA1^CA4) pyramidal cells granule cells (dentate gyrus) subiculum islands of Calleja striatum thalamus subthalamic nucleus hypothalamus suprachiasmatic nucleus supraoptic nucleus paraventricular nucleus anterior hypothalamus ventromedial hypothalamus arcuate nucleus brain stem nuclei inferior colliculi superior colliculi mesencephalic trigeminal reticular nucleus lower brain stem superior olivary complex facial nucleus lateral paragigantocellular nucleus lateral reticular nucleus pontine nuclei inferior olive cerebellum cortical molecular layer cortical granule layer medullary layer deep nuclei spinal cord (cervical/thoracic segments) dorsal horn ventral horn lateral horn central autonomic region ventricle ependymal cells choroid plexus PNS sensory nervous system trigeminal ganglion** dorsal root ganglion* autonomic nervous system superior cervical ganglion* intramural ganglia esophagus duodenum colon* entero-endocrine cells** Endocrine system pituitary gland adenohypophysis neurohypophysis adrenal gland
ISH signal
Figure
3f, g, h ^ + + + + + + + +
+ + + + + + +
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2a 2a 2b, c 2a
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2d, e 3a, b, e 3a, c, d 3a 3a
1c, d + + ^ + + + + + ^ +
+ (+)
4a 4d 4c 4d 4b
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+ ^
Figure
+ ^ ^ + ^
6f
6e 5e, f
+ ^ ^ ^ ^ ^ ^ ^ ^
The expression was tested in mice, exceptions are indicated by * (studied only in rat) and ** (studied in mouse and rat).
+ + + + + + + + + +
cortex glomerular layer fascicular layer reticular layer medulla pineal** testis spermatogonia, spermatocytes Other organs liver spleen lung (bronchiolar/alveolar epithelium) kidney esophagus epithelium duodenum epithelium colon epithelium skeletal muscle
ISH signal
5a^c 5b 6a 6b, c 6b 6d
evenly distributed over cortical laminae II^VI. Distribution and amount of positive neurons were apparently similar in di¡erent cortical areas, e.g. motor and sensory cortex. A typical example demonstrating the frontal cerebral cortex is given in Fig. 1. In addition to neuronal somata, ¢bers showed the ISH signal (arrow in Fig. 1b). In the cerebellum, most of the small neurons of the granular layer exhibited Ngb mRNA (Fig. 1c, d). Interneurons of the molecular layer were also stained. Apparently, all Purkinje cells exhibited strong signals. We also observed many labeled ¢bers in the granule cell layer (arrow in Fig. 1d) and some ¢bers in the vicinity of Purkinje cells. In addition, neurons of the cerebellar nuclei located in the white matter showed the ISH signal. Strong signal was also seen in subcortical regions. In the hippocampal formation, pyramidal cells of the regions CA1^CA3 and granule cells of the dentate gyrus exhibited strong cytoplasmic signal (Fig. 2a^c), and scattered neurons of the subiculum were also labeled. In addition, neuronal somata in the islands of Calleja, striatum and thalamic nuclei showed the ISH signal. Many brainstem nuclei exhibited strong hybridization of neuronal cytoplasm. In particular, neurons were stained in both inferior and superior colliculi, the mesencephalic trigeminal nucleus and the reticular nuclei complex (Fig. 2d, e). In the lower brainstem (Fig. 3a), the auditory superior olivary complex (Fig. 3b), the nucleus of the facial nerve (Fig. 3c, d), lateral paragigantocellular nucleus, lateral reticular nucleus (LRt), pontine nuclei and the inferior olive (Fig. 3e) exhibited distinct hybridization of neuronal somata. Some of these cells were conspicuous because of their large cell bodies (see Fig. 3, facial nucleus), but the strength of the cytoplasmic signal was similar to that in other neurons. In the olfactory bulb (Fig. 3f^h), glomeruli were unstained, while periglomerular neurons as well as neurons of the mitral cell layer and of the granule cell layer exhibited the ISH signal (Fig. 3g). Stained ¢bers were detected in the periglomerular zone and in the region
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Fig. 1. Parasagittal section of a mouse brain showing the in situ-hybridization (ISH) signal (a) in neurons of area 1 of the frontal cerebral cortex (CC) in laminae I^VI, and (b) in higher magni¢cation, where arrows point to stained ¢bers. The cerebellum (Cb) is shown in (c), taken from lobule 5. In the cortex, interneurons are stained in the molecular layer (Mo) adjacent to the cortical surface (S). Apparently all Purkinje cells (P) and many cells in the granular layer (Gr) exhibit the ISH signal, while the medullary region (M) was unstained. In the higher magni¢cation (d), ¢bers (arrow) and groups of neurons (asterisk) are labeled. Scale bars = 50 Wm (a, c), 10 Wm (b, d).
between mitral and granular cells (Fig. 3h, arrow). In the spinal cord, positive neurons were observed throughout the gray matter in cervical and thoracic segments (Fig. 4a, stemming from segment Th1). In sections cut in the axial plane, the signal was seen in the laminae of the dorsal horn (Fig. 4d) and in the central autonomic region surrounding the central canal (Fig. 4b). Stained neurons were also seen in the lateral horn (presumptive preganglionic sympathetic neurons of the intermediolateral
nucleus and lateral funicle; arrow in Fig. 4d) and in the ventral horn (Fig. 4c). In the latter, some large neurons (probably alpha-motoneurons) were labeled. Peripheral nervous system The trigeminal (Gasserian) ganglion of mouse and rat and the rat dorsal root ganglia of lower cervical and upper thoracic segments were studied as examples of
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Fig. 2. Parasagittal section of a mouse brain showing the ISH signal (a) in the hippocampal formation, where the pyramidal (P) cell layer of ¢elds CA1^3, neurons of the granular layer of the dentate gyrus (DG) as well as scattered cells of the subiculum (S) were stained. The higher magni¢cations showed (b) stained neurons of the polymorph layer (Po) of the dentate gyrus and (c) the caudal end of the DG which is marked by an arrow in panels a and c. The reticular nucleus (Rt) below the cerebellum (Cb) is shown in (d) and a higher magni¢cation from the parvocellular part of the reticular nucleus in (e). Scale bars = 200 Wm (a, d), 50 Wm (b, e), 20 Wm (c).
sensory ganglia. From the autonomic nervous system, we investigated the rat sympathetic superior cervical ganglion (SCG) and intramural ganglia of several parts of the mouse and rat gastrointestinal tract. In the trigeminal ganglion, virtually every neuronal perikaryon was stained (Fig. 4e^g), while in the ¢ber regions between neuronal groups no speci¢c signal was seen. In some peripheral ¢bers, in particular in the part of the ganglion that gives rise to the ophthalmic nerve, a punctated signal was seen (Fig. 4g). However, in the dorsal root ganglion, cells exhibited only weak signals. Similar ¢ndings were obtained from the rat SCG (data not shown). The enteric nervous system was studied in cross sections of the esophagus, duodenum and colon of both species. In each intestinal region, distinct hybridization of neurons was found. This is demonstrated for the rat colon as a typical example in Fig. 5. In particular, the myenteric plexus (Auerbach’s plexus; arrows in Fig. 5a,
c, d) located between the circular and longitudinal layers of the tunica muscularis (large asterisks in Fig. 5a^d) was stained. We also observed scattered neurons in the submucosal layer belonging to the submucosal plexus (Meissner’s plexus; Fig. 5a). In addition, stained cells that are probably enteroendocrine cells were seen in the mucosal layer (arrows in Fig. 5a, b). Endocrine system From the endocrine system, we studied mouse and rat pituitary glands and pineal, mouse and rat adrenal glands and mouse testes. No signi¢cant di¡erences were observed in the distribution of the ISH signal in a distinct organ or region between the species tested. In the testis, expression of Ngb mRNA was restricted to the spermatogonia and, probably, to primary spermatocytes of the outer zones of the tubule walls (Fig. 5e). In the pituitary gland (Fig. 6a), hybridization was restricted
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Fig. 3. Parasagittal section of a mouse brain showing the ISH signal (a) in some brainstem nuclei, i.e. the superior olivary complex (SOC), facial nerve nucleus (7), lateral paragigantocellular nucleus (LP) and lateral reticular nucleus (LRt). The higher magni¢cations depict neurons labeled by ISH in the SOC (b), anterior part of the facial nucleus (c, d; arrows point to the same cell) and in the inferior olive (e). The olfactory bulb (OB) is seen in panels f^h. While glomeruli (asterisks) were unstained, the ISH signal is found in periglomerular cells (P) as well as in neurons of the mitral (M) and granular (Gr) cell layers. Scale bars = 200 Wm (a, f), 50 Wm (g), 10 Wm (b^e, h).
to the anterior lobe, where the endocrine cells of the adenohypophysis exhibited a relatively strong signal (Fig. 6b, c). In contrast, the glia-like pituicytes of the posterior lobe (neurohypophysis) were apparently
unstained (right part of Fig. 6b). In the adrenal gland, we observed the ISH signal in the cortex and medulla (Fig. 6d). Cells of the cortical zona glomerulosa, located below the capsule, exhibited a strong signal (arrow in
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Neuroglobin expression
Fig. 4. Axial section of the mouse spinal cord (SpC) segment Th1 demonstrating the ISH signal in neurons throughout the gray matter (a). (b) In the central autonomic region surrounding the central canal (asterisk) as well as in the ventral horn (VH; c) and the dorsal horn (DH ; d), stained neurons are found. Note that neurons in the intermediolateral nucleus of the lateral horn (arrow in panel d) also exhibit the ISH signal. (e^g) Horizontal section of the trigeminal ganglion (TG) is shown in panel e, where the ISH signal is present in neurons, as demonstrated in higher magni¢cation in panel f, and in ¢bers (g, arrows). Scale bars = 200 Wm (a, e), 50 Wm (c, d), 10 Wm (b, f, g).
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Fig. 5. Distribution of Ngb mRNA in the rat colon and mouse testis. A cross section is shown in panel a. Labeled are ganglionic cells of the myenteric plexus (arrows in panels a, c, d) located between the circular and longitudinal layers of the tunica muscularis (large asterisks in panels a^d), neurons in the submucosal layer belonging to the submucosal plexus, and putative enteroendocrine cells in the mucosal layers (arrow in panel b). The lamina muscularis mucosa is indicated by small arrows in panels a and b. (e, f) Cryosections of mouse testis tissue. Ngb transcripts were detected in the two outer layers of the tubules where spermatogonia and primary spermatocytes are located (e). No ISH signal was observed with the complementary sense RNA probe (f). Scale bars = 50 Wm (a), 10 Wm (b, c), 50 Wm (e, f).
Fig. 6d), while cells of the zona fasciculata and those of the zona reticularis were not or only rarely stained (Fig. 6f). Apparently all medullary cells exhibited Ngb mRNA (Fig. 6e). In the pineal gland, parenchymal cells (i.e. pinealocytes) appeared unstained.
nomic and sensory parts), as well as in the endocrine system. These data also explain the presence of Ngb mRNA in tissues other than the brain (Burmester et al., 2000). At the same time, the ISH results con¢rm the absence of Ngb in various organs, as suggested before by northern hybridization (Burmester et al., 2000).
DISCUSSION
Expression of Ngb in nervous and endocrine systems Ngb is a respiratory protein that is preferentially expressed in the brain of mouse and man (Burmester et al., 2000). The ISH experiments reported here con¢rm and extend this study. Ngb mRNA was observed in both the central and peripheral nervous system (in auto-
In the CNS, we found an ubiquitous distribution of Ngb mRNA-containing neuronal somata. Apparently, the vast majority of perikarya in the neuron-bearing layers of the cerebral and cerebellar cortices were labeled
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Fig. 6. Section of mouse endocrine organs showing the ISH signal. A frontal section of the pituitary gland is shown in panel a. The higher magni¢cations (b) demonstrate the absence of the signal in the posterior pituitary (right part) while the anterior part of the gland (left, and panel c) shows the signal in the cytoplasm of endocrine cells. A horizontal section of the adrenal gland is demonstrated in panel d. The signal is present in cells of the medulla (M, see also panel e) and in the cortex, in particular in the glomerular zone (arrow in panel d, dark cells on top in panel f). Asterisk in panel d depicts unspeci¢c fat cell staining in a zone between cortex and medulla. Scale bars = 200 Wm (a, d), 20 Wm (b, e, f), 10 Wm (c).
by the Ngb probe. This distribution corresponds with relatively high (cerebral cortex) and rather low (cerebellar cortex) levels of Ngb mRNA in human tissue RNA blots (Burmester et al., 2000). The rich distribution of the
ISH signal in subcortical regions, in the brain stem, olfactory bulb and spinal cord reveals that the expression of Ngb mRNA is a general feature of nerve cells. These results con¢rm and extend previous analyses (Burmester
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et al., 2000; Zhang et al., 2002) describing that neuronal somata of the hippocampal formation and of some other brain regions contain Ngb mRNA. Our data provide evidence that the other part of the CNS, the spinal cord, also expresses Ngb in its gray substance, and that the respective mRNA is also found in the peripheral nervous system. Sensory and autonomic ganglia exhibited ^ although to varying degrees ^ neuronal perikarya that were labeled by the probe. For example, our results clearly demonstrate that in the gut intrinsic neurons (submucosal and myenteric plexus, see Fig. 5a^d) rather than innervating neurons transcribe Ngb. This observation explains the positive northern blot signals obtained from these organs (Burmester et al., 2000). Di¡erent regions of the brain exhibited positive ISH signals for Ngb in nerve ¢bers, e.g. in the granule cell layer of the cerebellum, where mossy ¢bers make complex synapses (Fig. 1d). These ¢ndings suggest that Ngb mRNA is transported in nerve cell processes to provide protein synthesis in distal regions of axons and dendrites (Job and Eberwine, 2001). The remarkable punctuated hybridization signal might also be an indication for Ngb mRNA expression in varicosities and synaptic terminals as well as in dendritic spines. This localization of Ngb transcripts could have important functional consequences for synaptic plasticity, an active metabolic process which presumably needs substantial amounts of oxygen. Moreover, we obtained evidence that cells of the endocrine system (e.g. anterior pituitary, adrenal gland) also express Ngb mRNA, suggesting that signals obtained by northern blots (Burmester et al., 2000) are most likely due to intrinsic Ngb production rather than to innervation of these tissues. In particular, pituitary cells were strongly stained in mouse and rat, which coincides well with the high Ngb expression found by northern hybridization (Burmester et al., 2000). In the adrenal gland, the cortical glomerular layer (responsible for the synthesis of mineralocorticoids such as aldosterone) was heavily stained, while the fascicular zone (producing glucocorticoids such as cortisol) and the reticular zone (sex hormones) exhibited only few labeled cells. It is conceivable that the di¡erent degrees of Ngb expression are related to the activity level of the respective tissue or region. This may also apply to our ¢ndings that neuronal somata in one sensory ganglion (i.e. trigeminal ganglion) were labeled, but those in another (i.e. dorsal root ganglion, DRG) were not or only to a minor degree. The signal was found in ¢bers in both ganglia, also suggesting that Ngb is expressed in sensory neurons. In addition, neuronal somata in the sympathetic superior cervical ganglion were lightly labeled, while some stronger signal was observed in ¢bers. If these were preganglionic, they belong to sympathetic neurons located in the spinal lateral horn, which indeed exhibited labeled somata in the present study. With regard to only weakly labeled ganglion cells such as those in the DRG, we consider that their metabolism involving Ngb was rather low and that Ngb expression may be enhanced under di¡erent circumstances. An organ known to regularly vary in its activity state is
the pineal gland. Its metabolism is regulated by the endogenous circadian clock to produce melatonin during the night (cf. Reuss, 1996). Our ¢nding that the gland’s parenchymal cells, pinealocytes, are not stained may be attributed to the fact that the animals of the present study were killed during the day. The question of di¡erent expression of Ngb, either with regard to daytime or to stimulation of metabolism in the pineal and in other organs, will be addressed in subsequent studies. Ngb expression and NO-synthesizing cells It has been postulated that Ngb may function as an NO-dioxygenase involved in NO-homeostasis (e.g. Brunori, 2001), similarly to the myoglobin in the murine muscle and heart (Flo«gel et al., 2001). However, no correlation of NO synthesis and Ngb expression was observed in our analyses. For example, in the spinal cord neuronal somata immunoreactive for the NO-producing enzyme, neuronal NO-synthase (nNOS), are restricted to dorsal and lateral horns (Reuss and Reuss, 2001), while Ngb mRNA was found throughout the whole gray matter (present study). In the cerebellar cortex, Purkinje cells express Ngb mRNA (present study) while they do not contain nNOS (cf. Reuss and Riemann, 2000, for discussion). In the cerebral cortex, nNOS neurons are a distinct subpopulation of neurons, while apparently all express Ngb mRNA. It thus appears that Ngb neurons may or may not produce nNOS, and that nNOS neurons may or may not express Ngb mRNA. Thus it remains to be shown by double-immunolabeling how both neuronal subpopulations are related to each other. Considering also that NO-producing neurons exert their in£uence on neighboring cells (Flo«gel et al., 2001), further studies must address the possible function of Ngb as NO-dioxygenase. Is Ngb expressed in glia? In some analyzed regions (e.g. spinal cord funicles, hippocampal alveus, cerebellar medulla) small labeled structures were seen, suggesting that Ngb may be expressed in some presumed glia cells. Fiber-containing parts of the trigeminal ganglion and the neurohypophysis, however, appeared unstained. In the latter, pituicytes are specialized glial cells, indicating that not all glia cells may produce Ngb. The question in how far Ngb expression is a feature not only of neurons, but also of glia cells, must ultimately be addressed by experiments combining the detection of Ngb mRNA or protein and a glial cell marker such as the glial ¢brillary acidic protein. Implications for Ngb function At ¢rst glance, it is di⁄cult to assume that the low concentration of Ngb estimated in total brain tissue of mouse and man (Burmester et al., 2000) is su⁄cient to support a function in oxygen homeostasis. In contrast, nerve-speci¢c globins of several invertebrates typically occur in much higher concentrations (Kraus and Colacino, 1986; Dewilde et al., 1996; Vandergon et al.,
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1998). However, here we show that Ngb mRNA is in fact expressed in neuronal somata the volume of which makes up only a small part of total brain volume (see Fig. 1b). Although at present we have no means available to determine the intracellular amount of Ngb protein in single cells, these data suggest that local concentrations of Ngb within nerve cells can be considerably higher than the 1 WM previously estimated for total brain (Burmester et al., 2000) and may in fact reach intracellular values similar to those which have been observed for myoglobin in muscles (Schuder et al., 1979; Wittenberg and Wittenberg, 1990). Accordingly, Ngb is expressed, at apparently high levels, in secretory cells of the endocrine system (Fig. 6). It can be assumed that these metabolically highly active cells require large amounts of oxygen, and the presence of the oxygen-binding Ngb may sustain this need. Cladistic analyses indicate that Ngb belongs to an ancient branch of intracellular globins (Burmester et al., 2000; Awenius et al., 2001), which includes at least the nerve myoglobin of the annelid Aphrodite aculeata (Dewilde et al., 1996). The early emergence of Ngb-like globins in the evolution of the Metazoa suggests a conserved function of Ngb in the animal nerval system. However, while the vertebrate Ngb expression is pronounced in neurons, most invertebrate nerve globins that are expressed at high levels are located in glia cells (Wittenberg et al., 1965; Vandergon et al., 1998;
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Dewilde et al., 1996). In molluscs, species living under especially hypoxic conditions display high levels of nerval globin expression in glia cells, while related species from oxygen-rich habitats seem to feature a low-level neuronal expression (Weber and Vinogradov, 2001). Pronounced changes in the strength and cellular localization of nerve globin expression may therefore have accompanied the adaptation for enduring severe hypoxia in invertebrates. Future studies on species adapted to low-oxygen environments will show whether this is also true for vertebrates. Given the recent evidence for nerve cell survival-promoting features of Ngb upon hypoxia (Sun et al., 2001), our expression analyses con¢rm previous notions that Ngb may indeed act as an intracellular oxygen storage or transport protein of nerve cells (Burmester et al., 2000). Other proposed functions of Ngb (see Dewilde et al., 2001) such as oxygen sensing, NO-dioxygenase (see above) and protection from reactive oxygen species can thus be regarded less likely.
Acknowledgements)This work is supported by the Deutsche Forschungsgemeinschaft (Grants Ha2103/3 and Bu956/5). The authors thank Katja Loppe-Ho¡mann and Ursula DisqueKaiser for expert technical assistance. T.H. wishes to thank Erwin R. Schmidt for excellent working facilities and support.
REFERENCES
Awenius, C., Hankeln, T., Burmester, T., 2001. Neuroglobins from the zebra¢sh Danio rerio and the pu¡er¢sh Tetraodon nigroviridis. Biochem. Biophys. Res. Commun. 287, 418^421. Bauer, U., Schneider-Hirsch, S., Reinhardt, S., Bonavente, R., Maelicke, A., 1998. The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes. FEBS Lett. 208, 208^214. Brunori, M., 2001. Nitric oxide, cytochrome-c oxidase and myoglobin. Trends Biochem. Sci. 26, 21^23. Burmester, T., Weich, B., Reinhardt, S., Hankeln, T., 2000. A vertebrate globin expressed in the brain. Nature 407, 520^523. Couture, M., Burmester, T., Hankeln, T., Rousseau, D.L., 2001. The heme environment of mouse neuroglobin : Evidence for the presence of two conformation of the heme pocket. J. Biol. Chem. 276, 36377^36382. Dewilde, S., Blaxter, M., Van Hauwaert, M.L., Van£eteren, J., Esmans, E.L., Marden, M., Gri¡on, N., Moens, L., 1996. Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata. J. Biol. Chem. 271, 19865^19870. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M.C., Caubergs, R., Moens, L., 2001. Biochemical characterization and ligand-binding properties of neuroglobin, a novel member of the globin family. J. Biol. Chem. 276, 38949^38955. Flo«gel, U., Merx, M.W., Go«decke, A., Decking, U.K., Schrader, J., 2001. Myoglobin : A scavenger of bioactive NO. Proc. Natl. Acad. Sci. USA 98, 735^740. Hermsen, B., Stetzer, E., Thees, R., Heiermann, R., Schrattenholz, A., Ebbinghaus, U., Kretschmer, A., Methfessel, C., Reinhardt, S., Maelicke, A., 1998. Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression. J. Biol. Chem. 273, 18394^18404. Hochachka, P.W., 1999. The metabolic implications of intracellular circulation. Proc. Natl. Acad. Sci. USA 96, 12233^12239. Job, C., Eberwine, J., 2001. Localization and translation of mRNA in dendrites and axons. Nat. Rev. Neurosci. 2, 889^898. Kraus, D.W., Colacino, J.M., 1986. Extended oxygen delivery from the nerve hemoglobin of Tellina alternata (Bivalvia). Science 232, 90^92. McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde ¢xative. A new ¢xation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077^1083. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA. Rentrop, M., Knapp, B., Winter, H., Schweizer, J., 1986. Aminoalkylsilane-treated glass slides as support for in situ hybridisation of keratin cDNA to frozen tissue sections under varying ¢xation and penetration conditions. Histochem. J. 18, 271^276. Reuss, M.H., Reuss, S., 2001. Nitric oxide synthase neurons in the rodent spinal cord: distribution, relation to substance P ¢bers, and e¡ects of dorsal rhizotomy. J. Chem. Neuroanat. 21, 181^196. Reuss, S., 1996. Components and connections of the circadian timing system in mammals. Cell Tissue Res. 285, 353^378. Reuss, S., Riemann, R., 2000. Distribution and projections of nitric oxide synthase neurons in the rodent superior olivary complex. Microsc. Res. Tech. 51, 318^329. Schuder, S., Wittenberg, J.B., Haseltine, B., Wittenberg, B.A., 1979. Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography. Anal. Biochem. 92, 473^481. Strittmatter, P., Burch, H.B., 1963. The heme protein in ganglia of Spisula solidissima. Biochim. Biophys. Acta 78, 562^563. Sun, Y., Jin, K., Mao, X.O., Zhu, Y., Greenberg, D.A., 2001. Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. Proc. Natl. Acad. Sci. USA 98, 15306^15311. Trent, J.T., III, Watts, R.A., Hargrove, M.S., 2001. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J. Biol. Chem. 276, 30106^30110.
NSC 5873 4-11-02
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S. Reuss et al.
Vandergon, T.L., Riggs, C.K., Gorr, T.A., Colacino, J.M.RiggsA.F., 1998. The mini-hemoglobins in neural and body wall tissue of the nemertean worm, Cerebratulus lacteus. J. Biol. Chem. 273, 16998^17011. Weber, R.E., Vinogradov, S.N., 2001. Nonvertebrate hemoglobins: Functions and molecular adaptations. Physiol. Rev. 81, 569^628. Wittenberg, B.A., Briehl, R.W., Wittenberg, J.B., 1965. Haemoglobins of invertebrate tissues - nerve haemoglobins of Aphrodite, Aplysia, and Halosydna. Biochem. J. 96, 363^371. Wittenberg, J.B., Wittenberg, B.A., 1990. Mechanisms of cytoplasmic hemoglobin and myoglobin function. Annu. Rev. Biophys. Biophys. Chem. 19, 217^241. Wittenberg, J.B., 1992. Functions of cytoplasmatic hemoglobins and myohemerythrin. Adv. Comp. Environ. Physiol. 13, 60^85. Zhang, C., Wang, C., Deng, M., Li, L., Wang, H., Fan, M., Xu, W., Meng, F., Qian, L., He, F., 2002. Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin. Biochem. Biophys. Res. Commun. 290, 1411^1419. (Accepted 9 August 2002)
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