Differential Activation of c-fos Immunoreactivity After Hypophysectomy in Developing and Adult Rats.код для вставкиСкачать
THE ANATOMICAL RECORD 290:1050–1056 (2007) Differential Activation of c-fos Immunoreactivity After Hypophysectomy in Developing and Adult Rats QIUJU YUAN,1 DAVID E. SCOTT,2 KWOK-FAI SO,1 AND WUTIAN WU1* Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 2 Department of Pathology & Anatomy, Eastern Virginia Medical School, Norfolk, Virginia 1 ABSTRACT The aim of the present study is to compare c-fos expression in identiﬁed hypothalamic vasopressin (AVP) and oxytocin (OT) neurons in developing (PN7 and PN14) and adult rats following hypophysectomy using dual-labeled immunostaining. Our results showed that hypophysectomy induced c-fos expression in supraoptic (SON) and paraventricular (PVN) nuclei in both the developing and adult rats. Few or no positive cells were observed in the same nuclei in sham-operated animals. Quantitative analysis for c-fos and either of the above named neuropeptides revealed that almost all AVP and OT neurons in the adult and PN14 groups expressed c-fos in response to hypophysectomy. In PN7, hypophysectomy also induced all AVP neurons to express c-fos in SON and PVN. However, few OT neurons in the SON and PVN produced c-fos after hypophysectomy. In addition, the time course of c-fos expression was different in the developing and adult rats after hypophysectomy. The c-fos expression in the developing rats exhibited a more prolonged induction in which staining for c-fos persisted for at least 3 days after hypophysectomy compared with that in the adult in which c-fos immunoreactivity disappeared within 24 hr post-lesion. This study demonstrates that c-fos expression after hypophysectomy is regulated differently during development. Anat Rec, 290:1050–1056, 2007. Ó 2007 Wiley-Liss, Inc. Key words: hypothalamus; hypophysectomy; neuronal death; immediate early genes Long-lasting adaptive changes in the mammalian central nervous system (CNS) may depend on the activation of a speciﬁc group of programmed response genes (Goelet et al., 1986). The protooncogene c-fos is among the genes that have been implicated in such processes (Sheng and Greenberg, 1990; Morgan and Curran, 1991b). The c-fos is induced in neurons by a variety of extracellular stimuli in the CNS. Many investigations have analyzed Fos expression in the hypothalamic magnocellular neurons of the SON and PVN. Expression of c-fos is induced by acute stimuli such as parturition (Fenelon et al., 1993; Luckman, 1995; Lin et al., 1998), noxious stimuli (Smith and Day, 1994), restraint stress (Miyata et al., 1995), LPS-injection (Matsunaga et al., 2000), Cholecystkinin injection (Verbalis et al., 1991) and Ó 2007 WILEY-LISS, INC. chronic stimuli such as water deprivation (Miyata et al., 1994) and salt loading (Miyata et al., 1994, 2001). These observations, combined with the identiﬁcation of the Grant sponsor: University of Hong Kong; Grant sponsor: Hong Kong Research Grants Council; Grant sponsor: Jeffress Foundation; Grant number: J531. *Correspondence to: Wutian Wu, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, China. Fax: 852-281-70857. E-mail: firstname.lastname@example.org Received 5 December 2006; Accepted 18 May 2007 DOI 10.1002/ar.20570 Published online 28 July 2007 in Wiley InterScience (www. interscience.wiley.com). ACTIVATION OF c-fos IN HYPOPHYSECTOMIZED RATS c-fos protein as a transcription factor (Chiu et al., 1988), have supported the role for the c-fos gene in coupling extracellular stimuli with transcriptional events leading to chronic modiﬁcations in neuronal function (Sheng and Greenberg, 1990; Morgan and Curran, 1991b). In the adult rat, injuries to the hypothalamo-neurohyphyseal system (HNS) such as hypophysectomy also induce transient c-fos expression (Villar et al., 1991). Hypophysectomy in adult rats induced a series of changes characterized by dramatic changes in peptide expression (Villar et al., 1990) and up-regulation of NOS as well as robust regeneration (Wu and Scott, 1993). It has been suggested that the transient and early activation of Fos in injured magnocellular neurons that precedes the changes in magnocellular neurons of the HNS, participates in the regulation of those changes (Villar et al., 1991). Hypophysectomy, however, elicits different reactions in magnocellular neurons of the HNS in pups, including no or unremarkable up-regulation of NOS expression as well as no signiﬁcant regeneration on the ﬂoor of the third cerebral ventricle (Yuan et al., 2006). These different responses led us to try to compare the pattern of c-fos induction following hypophysectomy in postnatal rats with that of adults. MATERIALS AND METHODS Animals PN7, PN14, or adult male SD rats (200–220 g) were used in the present experiments. They were housed in a temperature-controlled room under daily light and dark cycle (12:12) with free access to laboratory chow and tap water. Hypophysectomy Animals at age of PN14 and adults were performed by removing both the anterior and posterior pituitary by the peripharyngeal approach (Thorngren et al., 1973; Wu and Scott, 1993). Brieﬂy, animals were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (8 mg/kg) and mounted upside-down in a stereotaxic apparatus. The skin on the ventral aspect of the neck was incised and the infrahyoid musculature separated on the midline and retracted to either side. The sella turcica was approached by blunt separation, and a hole was drilled at the basal occipital suture to allow visualization of the pituitary gland. The anterior and posterior pituitaries were removed by suction. The wound was packed with gelfoam, and the skin incision closed with suture. The animals in the control group received sham operation, which involved the entire surgical procedure with the exception of neural lobe ablation. In PN7 animals, hypophysectomy was performed according to Glasscock’s method (Glasscock, 1980). Brieﬂy, after pups were anesthetized with ice, the pup’s head was held between the thumb and index ﬁnger so that the skull was parallel to the tabletop. A needle 20-G tip with a length of 15 mm was probed into the region of the ear where the ear canal develops and then the needle was pushed through the skull. Once the needle penetrated the skull, the syringe was elevated approximately 45 degrees (from the horizontal plane) until the needle was parallel to the ﬂoor of the skull. The needle was then pushed toward the midline until its movement was 1051 stopped by the needle against the ear. The plunger of the syringe was withdrawn slightly (approximately 0.1 ml), removing the pituitary without disrupting the diaphragma sella. The needle was withdrawn from the pup’s head and the aspirated anterior and posterior pituitaries were examined to conﬁrm the completeness of hypophysectomy. Sham-operated pups were anesthetized and probed with the needle, but once the top had reached the pituitary region, it was withdrawn without damage to the gland. After surgery, animals were allowed to survive 3 (n 5 5), 12 (n 5 5), 24 (n 5 5) hr, and 1 (n 5 5), 3 (n 5 5), 7 (n 5 5), 14 (n 5 5) days. Perfusion and Tissue Processing After survival, animals were anesthetized with an overdose of ketamine and xylazine and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and post-ﬁxed overnight at 48C. The sella turcica was examined at autopsy to ensure that hypophysectomy was complete. Immunocytochemistry Forty-micrometer-thick frozen sections of hypothalamus were collected in wells containing phosphate-buffered saline (PBS). After rinsing, sections were treated for ﬂuorescence immunostaining. The primary antibodies used included (1) rabbit IgG polyclonal antibody against c-fos (1:1,000, Oncogene) and (2) rabbit IgG polyclonal antibodies against AVP (1:6,000, Peninsula Labs) and OT (1:3,000, Peninsula Labs). For c-fos, primary antibody (1:1,000, Oncogene) was incubated with the sections overnight followed by incubation with the Alexa Fluor 568-conjugated goat antirabbit immunoglobins (1:400) for 2 hr. For double-labeling experiments, some sections were completely washed with PBS for several times and then incubated with either the rabbit polyclonal antibody against AVP or a polyclonal antibody against OT overnight and followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit immunoglobins (1:400) for 2 hr. Procedures for immunohistochemistry carried out under room temperature. Sections were then mounted on slides and observed under a confocal laser scanning microscope. Two lines emitting at 488 and 568 nm were used for exiting Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies, respectively. To test the speciﬁcity of the immunoreactions, we have performed several tests. First, a few sections from each animal were incubated without the primary antibodies, and all sections showed negative immunoreactions. Second, to test the speciﬁcity of AVP and OT, we incubated sections which contain only AVP or OT neurons with both antibodies against AVP and OT, and the results showed that only the right type of neurons stained. For example, incubation of sections from suprachiasmatic nucleus with both antibodies against AVP and OT produced only AVP labeling, which is consistent with previous studies (Nylen et al., 2001; Van den Pol and Tsujimoto, 1985; Hou-Yu et al., 1986). Third, we incubated sections with two polyclonal antibodies against c-fos and AVP and found that there was not cross-reaction because c-fos is located in nucleus and OT/AVP is located in cytoplasm. Therefore, these antibodies were used in the present study. 1052 YUAN ET AL. Quantitative Analysis Sections in the medial level of the SON were observed under laser confocal microscope because of its cellular homogeneity (the SON is composed essentially of magnocellular AVP and OT neurons and almost all of them project to pituitary). In this study, a quantitative analysis of the PVN was not performed because it contains numerous parvocellular elements, which project to the brainstem or median eminence (ME) and thus would not be lesioned by hypophysectomy. The quantitative analysis was performed on at least four sections in each animal of ﬁve rats in each age group. The percentage of Fos-positive nuclei in the OT- or AVP-positive neurons in the medial level of SON was compared between different age groups and between OT- and AVP-positive neurons with one-way analysis of variance and Tukey– Kramer multiple comparisons test. RESULTS In sham control adult rats, few or no c-fos–positive cells were found in the SON or PVN (Fig. 1A,B). Expression of c-fos was induced following hypophysectomy (Fig. 1). Intensive nuclear staining for c-fos was already observed by 3 hr in both SON (Fig. 1C) and PVN (Fig. 1D) post-lesion and sustained in the group of animals up to 12 hr survival time (Fig. 1E for SON and 1F for PVN). The c-fos expression diminished at 24 hr (Fig. 1G, H), which was comparable to that of controls. These ﬁndings are consistent with a previous report of transient c-fos induction in rat magnocellular hypothalamic neurons after hypophysectomy (Villar et al., 1991). Similar to adult rats, c-fos–immunoreactive nuclei were not detected in the SON or PVN in the sham control PN14 and PN7 rats (Fig. 2A,B, 3A,B). After hypophysectomy, intensive c-fos induction was observed in the above areas (Figs. 2, 3) in both PN7 and PN14 groups. The time course of c-fos expression was signiﬁcantly prolonged compared with the adult. The intensive c-fos stained nuclei were observed by 3 hr after hypophysectomy (Figs. 2C,D, 3C,D) and maintained up to 3 days post-lesion (Figs. 2E,F, 3E,F). Occasional c-fos immunoreactivity was still detected in both the SON and PVN at 1 week after hypophysectomy (Figs. 2G,H, 3G,H). Double-labeling experiments showed that almost all magnocellular neurons in both populations of AVP and OT neurons in the SON and PVN were induced to express c-fos in PN14 and in the adult as well (Figs. 4, 5, 7). Hypophysectomy also induced almost all AVP neurons in the SON and PVN to express c-fos in the PN7 group (Fig. 6C,D), but there was little evidence of c-fos induction in OT neurons in either PVN or SON after hypophysectomy (Figs. 6A,B, 7). DISCUSSION These results demonstrate that hypophysectomy induces c-fos in both immature and adult rats in magnocellular neurons of the PVN and SON. This ﬁnding is in striking contrast to the observations in other models in the brain that some stimuli such as kainic acid (KA) treatment (Schreiber et al., 1992), cortical brain injury (Herrera et al., 1993), and hypobaric hypoxia (Luo et al., 2000), which could elicit intense c-fos expression in the adult, do not induce signiﬁcant c-fos at neonatal rats. Schreiber et al. (1992) explained their results of age-dependent induction of c-fos in hippocampal and cortical neurons after KA treatment by several mechanisms. One of the explanations is that perhaps hippocampal and cortical neurons do not participate in KA-related seizure activity during the early postnatal period, reﬂected by changes in metabolic activity following systemic KA injection. If this is the case, it would suggest that the magnocellular neurons, at least the AVP neurons, as early as PN7 participate in the response to hypophysectomy. However, so far there is no report about changes in peptide expression in magnocellular hypothalamic neurons in the PVN or SON in postnatal rats after hypophysectomy. Induction of c-fos in OT neurons after hypophysectomy did not occur until PN14, suggesting that these neurons only become engaged in the response to hypophysectomy after further maturation. Indeed, it is well documented that maturation of OT neurons is delayed compared with that of AVP neurons (Choy and Watkins, 1979; Lazcano et al., 1990). Schreiber et al. (1992) also suggested that alteration of the transcriptional apparatus that facilitates stimulustranscription coupling occurs between the second and third week of postnatal development. However, our results show that hypophysectomy induced c-fos expression at as early as PN7 and demonstrate that this is not a generalized phenomenon. This ﬁnding suggests that even if the stimulus-transcription coupling cascade is activated, induced c-fos expression is neuronal- and/or stimulus-dependent. In our present study, many c-fos–positive magnocellular neurons were found in the SON and PVN at 3 hr and 12 hr but no immunoreactivity was seen at 24 hr after hypophysectomy in the adult. This result is similar to the observation made by others that c-fos immunoreactivity was observed in magnocellular neurons for the ﬁrst 24 hr after hypophysectomy in the adult (Villar et al., 1991). In this experiment, however, it was shown that c-fos immunoreactivity was persistently expressed for at least 3 days after hypophysectomy in immature rats. Although there is a hypothesis that c-fos protein is transiently expressed and is not useful for studying chronic stimuli (Morgan et al., 1987; Morgan and Curran, 1991a), the later reports demonstrated that c-fos– positive magnocellular neurons were persistently observed in the SON and PVN of rats that were chronically stimulated by the drinking of hypertonic NaCl solution instead of water or by water deprivation (Miyata et al., 1994). These observations are inconsistent with the hypothesis, suggesting that chronic stimuli cause persistent expression of Fos expression. It has been postulated that c-fos is expressed persistently to constitute neural components and/or architectures if animals require large and gradual changes of neural components and/ or architectures in the response to stimuli, for example, in development and/or neuronal plasticity (Miyata et al., 1994). Indeed, sustained increase of cell size and the elevation of OT and AVP and dynorphin mRNA has been demonstrated during chronic stimulation of dehydration and lactation (Theodosis and Poulain, 1984; Sherman et al., 1986). This hypothesis is also supported by the observation that persistent expression of c-fos mRNA was found in the spinal cord, forebrain, cerebellum, and retina in the developing mouse (Caubet, ACTIVATION OF c-fos IN HYPOPHYSECTOMIZED RATS 1053 Fig. 1. Photomicrographs showing the inﬂuence of hypophysectomy on c-fos–immunoreactivity in the SON and PVN of adult rats. C– F: Intensive c-fos–immunoreactive stained cellular nuclei began to be observed at 3 hr in SON (C) and PVN (D) and sustain at 12 hr (E,F) after hypophysectomy. A,B,G,H: The c-fos-IR diminished at 24 hr in both SON (G) and PVN (H), which was comparable to sham control (A and B, respectively). Scale bar 5 50 mm. Fig. 2. Photomicrographs showing the inﬂuence of hypophysectomy on c-fos–immunoreactivity in the SON and PVN of PN14 rats. A– D: No c-fos–immunoreactivity could be seen in both SON (A) and PVN (B) of sham-operated rats. Intensive c-fos–immunoreactive stained cellular nuclei began to be observed at 3 hr in SON (C) and PVN (D) after hypophysectomy. E,F: Even at 3 days postlesion, there are numerous c-fos–immunoreactive stained nuclei in SON (E) and PVN (F). G,H: C-fos–immunoreactivity diminished at 1 week postlesion, a small number of c-fos–immunoreactive nuclei are scattered in SON (G) and PVN (H). Scale bar 5 50 mm. 1989). Hence, c-fos protein was persistently expressed to enhance the transcription of other genes such as AVP or OT gene. In the present study, the same stimulus of hypophysectomy induced different expression of c-fos between the immature and adult rats. We suggest that hypophysectomy induces different cellular demands that correspond to different gene expression in magnocellular neurons in immature and adult rats qualitatively and 1054 YUAN ET AL. Fig. 3. A–H: Photomicrographs showing the inﬂuence of hypophysectomy on c-fos–immunoreactivity in the SON (A,C,E,G) and PVN (B,D,F,H) of PN7 rats. A,B: No c-fos–immunoreactivity could be seen in both SON (A) and PVN (B) of sham-operated rats. C,D: intensive c-fos–immunoreactive stained nuclei began to be observed at 3 hr in SON (C) and PVN (D). E,F: Even at 3 days, there are numerous cfos–immunoreactive stained nuclei in SON (E) and PVN (F). G,H: Cfos–immunoreactivity diminished at 1 week, a small number of c-fos– immunoreactive nuclei are scattered in SON (G) and PVN (H). Scale bar 5 50 mm. Fig. 4. A–D: Laser scanning confocal microscopy showing double labeling for OT (green) and c-fos (red nuclear; A,B) or AVP (green) and c-fos (red nuclear; C,D) in SON and PVN of hypophysectomized adult rats at 3 hr after hypophysectomy. Almost all OT (A,B) or AVP (C,D) neurons express c-fos in both SON and PVN. Scale bar 5 50 mm. Fig. 5. A–D: Laser scanning confocal microscopy showing double labeling for OT (green) and c-fos (red nuclear; A,B) or AVP (green) and c-fos (red nuclear; C,D) in SON and PVN of hypophysectomized PN14 rats at 3 hr after hypophysectomy. Almost all OT (A,B) or AVP (C,D) neurons express c-fos in both SON and PVN. Scale bar 5 50 mm. quantitatively. Indeed, our previous study showed that hypophysectomy elicited different reactions in magnocellular neurons of the HNS in pups as compared with adults. No or unremarkable up-regulation of NOS expression nor signiﬁcant regeneration on the ﬂoor of the third cerebral ventricle was observed in pups after hypophysectomy. In contrast, a marked up-regulation of NOS expression and a robust regeneration occurred following hypophysectomy in the adults (Yuan et al., 2006). Thus, in our study, transient c-fos expression corresponds to large changes in NOS expression and neuronal plasticity in the adult. However, persistent c-fos expression corresponds to no remarkable changes in NOS expression and neuronal plasticity in developing rats. We cannot exclude the possibility that the up-regulation of NOS and the structural plasticity are attributed to the activation of c-fos after hypophysectomy in the adult. However, the persistent expression c-fos is ACTIVATION OF c-fos IN HYPOPHYSECTOMIZED RATS 1055 Fig. 7. Quantitative analysis of the percentage of c-fos–immunoreactive nuclei in AVP- or OT-immunoreactive neurons in SON in 3 hr after hypophysectomy in PN7, PN14, and adult rats. Data are mean 6 SEM of four rats. *P < 0.01 vs. PN7. **P < 0.01 between OT-immunoreactive and AVP-immunoreactive neurons. Fig. 6. A–D: Laser scanning confocal microscopy showing double labeling for OT (green) and c-fos (red nuclear; A,B) or AVP (green) and c-fos (red nuclear; C,D) in SON and PVN of hypophysectomized PN7 rats at 3 hr after hypophysectomy. Almost all AVP neurons express cfos in both SON (C) and PVN (D) while few OT neurons in SON (A) and PVN (B) express c-fos. Scale bar 5 50 mm. induced to enhance other gene expression rather than NOS expression in the developing rats. This study also extends the ﬁnding that not only chronic stimuli but also axonal injury induces persistent c-fos expression in neurons. A previous study of ours demonstrated that there was substantial magnocellular neuronal degeneration in both immature and adult rats after hypophysectomy as seen in other systems of the CNS (Yuan et al., 2006). The c-fos activation has been implicated in the events associated with programmed cell death both during development (Gonzalez-Martin et al., 1991, 1992) and after several experimental brain lesion, including KA-induced seizure (Popovici et al., 1990; Smeyne et al., 1993; Kasof et al., 1995) and okadaic acid-reduced apoptosis (Afshari et al., 1994). Hafezi et al. (1997) further demonstrated the relationship between programmed cell death and cfos by providing direct evidence that the absence of c-fos (transgenic mice) prevents light-induced cell death of photoreceptors. Our quantiﬁed results do not support this model for the following reasons. In PN7 immature rats, OT neurons underwent a substantial neuronal loss after hypophysectomy (Yuan et al., 2006) without induction of c-fos. In addition, almost all AVP magnocellular neurons express c-fos, but signiﬁcant numbers of AVPIR magnocellular neurons survived hypophysectomy in PN7. In PN14 and adults, c-fos was present in almost all AVP and OT neurons that survived after hypophysectomy. These results suggest that not all c-fos–expressing neurons underwent degeneration, although it has not yet been demonstrated whether c-fos expression takes place in regenerating neurons or in those that are known to degenerate. Therefore, we suggested that c-fos induction by hypophysectomy is involved in other physiological processes (reviewed in Hughes and Dragunow, 1995) rather than neuronal death. It is well documented that removal of anterior pituitary can induce a decreased and suppressed negative feedback of glucocorticoids, resulting in the increased expression of AVP within neurons producing corticotropin-releasing hormone in PVN (Kiss et al., 1984; Wolfson et al., 1985). It is thus likely that, after removal of anterior pituitary, AVP neurons producing corticotrophinreleasing hormone in parvocellular neurons of PVN, whose axons are not lesioned by hypophysectomy, are activated to enhance AVP expression. ACKNOWLEDGMENTS D.E.S. was supported in part by a grant from the Jeffress Foundation. LITERATURE CITED Afshari CA, Bivins HM, Barrett JC. 1994. Utilization of a fos-lacZ plasmid to investigate the activation of c-fos during cellular senescence and okadaic acid-induced apoptosis. J Gerontol 49:B263– B269. Caubet JF. 1989. c-fos proto-oncogene expression in the nervous system during mouse development. Mol Cell Biol 9:2269–2272. 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