Neuronal expression of nuclear transcription factor MafG in the rat medulla oblongata after baroreceptor stimulation

Life Sciences 78 (2006) 1760 – 1766 www.elsevier.com/locate/lifescie

Neuronal expression of nuclear transcription factor MafG in the rat medulla oblongata after baroreceptor stimulation Iku Kumaki a, Dawei Yang b, Noriyuki Koibuchi c,d, Kiyoshige Takayama a,* b

a Department of Laboratory Sciences, Gunma University School of Health Sciences, 3-39-15 Showa-machi, Maebashi-shi 371-8514, Japan Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba 305-8562, Japan c Department of Integrative Physiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi-shi 371-8511, Japan d Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

Received 1 March 2005; accepted 11 August 2005

Abstract The medulla oblongata is the site of central baroreceptive neurons in mammals. These neurons express specific basic-leucine zipper transcription factors (bZIP) after baroreceptor stimulation. Previously we showed that activation of baroreceptors induced expression of nuclear transcription factors c-Fos and FosB in central baroreceptive neurons. Here we studied the effects of baroreceptor stimulation on induction of MafG, a member of small Maf protein family that functions as dimeric partners for various bZIP transcription factors by forming transcriptionregulating complexes, in the rat medulla oblongata. To determine whether gene expression of MafG is induced by stimulation of arterial baroreceptors, we examined the expression of its mRNA by semi-quantitative reverse transcription-PCR method and its gene product by immunohistochemistry. We found that the number of MafG transcripts increased significantly in the medulla oblongata after baroreceptor stimulation. MafG-immunoreactive neurons were distributed in the nucleus tractus solitarii, the dorsal motor nucleus of the vagus nerve, the ambiguous nucleus and the ventrolateral medulla. The numbers of MafG-immunoreactive neurons in these nuclei were significantly greater in test rats than in saline-injected control rats. We also found approximately 20% of MafG-immunoreactive neurons coexpress FosB after baroreceptor stimulation. Our results suggest that MafG cooperates with FosB to play critical roles as an immediate early gene in the signal transduction of cardiovascular regulation mediated by baroreceptive signals in the medulla oblongata. D 2005 Elsevier Inc. All rights reserved. Keywords: Baroreceptor; Cardiovascular control sites; Dorsal motor nucleus of vagus nerve; FosB; Immediate early genes; Nucleus tractus solitarii; MafG

Introduction The outline of the baroreceptor reflex pathway in cardiovascular control sites has been established by many electrophysiological and anatomical studies (Spyer, 1990). The first-order neurons transmit signals from arterial baroreceptors to the second-order neurons in the nucleus tractus solitarii (NTS) (Miura and Reis, 1969; Ciriello et al., 1981; Ciriello, 1983; Housley et al., 1987). The second-order neurons project to the dorsal motor nucleus of the vagus nerve (DMX) (Okada and Miura, 1997), the ambiguus nucleus (AMB) (Nosaka et al., 1979, 1982; Okada and Miura, 1997) and the caudal and rostral

* Corresponding author. Tel./fax: +81 27 220 8943. E-mail address: [email protected] (K. Takayama). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.08.030

ventrolateral medulla (CVLM/RVLM) (Willette et al., 1983, 1984; Guyenet, 1990; Miura et al., 1994b). The baroreceptorcardiac vagal reflex is transmitted via the NTS-DMX/AMB pathway (Ross et al., 1985; Okada and Miura, 1997), while the baroreceptor-sympathetic nerve reflex is transmitted via the NTS-VLM pathway (Ross et al., 1985; Urbanski and Sapru, 1988a,b; Sapru, 1989; Miura et al., 1994a,b). However, the roles of intracellular signal transduction and the gene expression of neuronal cells in the baroreceptor reflex remain unclear. We have been studying the expression of genes for nuclear transcription factors in the rat central nervous system (CNS) to assess their possible role in respiration and circulation. Expression of several basic leucine zipper (bZIP) transcription factors has been known to be induced in the CNS after baroreceptor and chemoreceptor stimulation. After stimulation of arterial baroreceptors by a blood pressure increase due to

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

pressor agent phenylephrine, c-Fos-, FosB-, c-Jun- and junDimmunoreactive neurons were detected in specific nuclei in the CNS (Chan and Sawchenko, 1994; Miura et al., 1994b; Okada and Miura, 1997; Xiong et al., 1997, 1998). On the other hand, it has been shown that Fos protein is expressed in neurons of chemoreceptive sites such as the ventral surface of the medulla oblongata in response to an increased H+ concentration in the cerebrospinal fluid due to hypercapnic stimulation (Miura et al., 1994c; Haxhiu et al., 1996; Teppema et al., 1997). This observation was also confirmed by an in vitro study in which an increase in the extracellular H+ concentration of cultured PC 12 cells led to the induction of c-fos mRNA (Shimokawa et al., 1998). Moreover, we found that hypercapnic stimulation induced gene expression of mafG (Accession No. AB026487 in Genbank) (Shimokawa et al., 2000), a member of the small Maf protein family of bZIP transcription factor, and mafG-2, a novel splice variant of mafG (Accession No. AB050011 in Genbank) (Shimokawa et al., 2001). These results suggest that both MafG and MafG-2 may be involved in signal transduction of extracellular acidification. The MafG protein reportedly forms a heterodimer with Fos at each leucine zipper structure (Kataoka et al., 1994). Thus, MafG may be involved with Fos in the signal transduction of H+-sensitivity and respiration, either by competing for binding sites or interacting directly with Fos. In the present study, we extended our previous studies to assess the effects of baroreceptor stimulation on MafG induction in the rat medulla oblongata. We identified CNS neurons that express MafG after activation of the baroreceptor by the pressor agent phenylephrine. We found the number of MafG transcripts and the number of MafG-containing neurons increased significantly in cardiovascular control sites in the NTS and the CVLM/RVLM after baroreceptor stimulation. The spatial and temporal expression patterns of MafG suggest that it may play a critical role as an immediate early gene involved in the signal transduction of baroreceptor stimulation. Materials and methods Baroreceptor stimulation Experiments were performed using 7- to 9-week-old male Wistar rats weighing 240– 280 g (n = 24, Institute of Experimental Animal Research, Saitama, Japan). We followed the Guiding Principals for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. Eight animals (4 for baroreceptor stimulation tests and 4 for controls) were used for the analysis of MafG mRNA expression (Group A) and 16 (8 for baroreceptor stimulation tests and 8 for controls) were used for immunohistochemistry (Group B). Anesthesia was induced and maintained with intraperitoneal (i.p.) injections of pentobarbital sodium (initially 50 mg/kg and after that 20 mg/kg; Abbott Laboratories, Chicago, IL, USA). The anesthesia level was monitored and maintained as described elsewhere (Xiong et al., 1997). The trachea was cannulated with vinyl tubing (2 mm in diameter) to maintain spontaneous breathing. The right femoral artery was cannulated

1761

with a polyethylene catheter to monitor arterial blood pressure and heart rate. The right femoral vein was cannulated to inject drugs. Rectal temperature was maintained at 37 -C. In test experiments each rat received three intravenous injections of l-phenylephrine hydrochloride (Sigma, St. Louis, MO, USA) 10 Ag/kg in 50 Al saline at 5 min intervals to increase arterial blood pressure. In control experiments each rat underwent cannulation of the trachea, femoral artery and femoral vein and received three injections of 50 Al saline without phenylephrine. After these procedures, the rats in Group A were processed for analysis of mafG mRNA expression, and rats in Group B were processed for immunohistochemistry to see the effects of baroreceptor stimulation on the expression of MafG. Semiquantitative reverse transcription Total cellular RNA derived from the medulla in the brain was isolated with a commercial RNeasy kit (Qiagen, Hilden, Germany). Chromosomal DNA was removed by digestion with RNase-free DNase I (Qiagen). A 1-Ag aliquot of the RNA was used for reverse transcription-polymerase chain reaction (RTPCR) amplification using OneStep RT-PCR system according to the manufacturer’s specifications (Qiagen). Semiquantitative RT-PCR was performed under the following conditions for 30 cycles: denaturation at 94 -C for 30 s, annealing at 50 -C for 30 s, and extension at 72 -C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize input cDNA. The amplification products were separated by electrophoresis on an agarose gel, excised and sequenced to verify the identity of the transcript. Forward and reverse primer pairs used for semiquantitative PCR were MafG: 5V- CTTGCAGAGTGCCTGCTCAC-3V and 5V-TCAAAGACCTGCCTGGCAA3V; and GAPDH: 5V-GTGGCAAAGTGGAGATTGTTGCC-3V and 5V-GATGATGACCCGTTTGGCTCC-3V. Contents of MafG transcripts were expressed as the density units of the amplification products stained with ethidium bromide. Results are expressed as density units in mean T S.E. The significance of difference in density of MafG transcripts was evaluated with Mann-Whitney U-test, p < 0.05 being considered to indicate a statistically significant difference. Immunohistochemistry Ninety minutes or 180 minutes after baroreceptor stimulation with phenylephrine, rats were injected with pentobarbital sodium (50 mg/kg, i.p.) and perfused transcardially with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 (PB) and with 4% paraformaldehyde in PB. The control rats were similarly perfused 90 or 180 min after injection of saline. The whole brain was removed, fixed in 4% paraformaldehyde in PB for 1.5 h and then soaked stepwise in solutions of 10%, 20% and 25% sucrose in PB. The medulla oblongata was frozen and cut into 40-Am-thick serial frontal sections. Every fourth section was collected in PB, rinsed, incubated in 0.5% bovine serum albumin in 0.1 M Tris –HCl buffer at pH 7.4

1762

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

A

Table 1 Parameters of circulation in the pressor (n = 8) and control (n = 8) experiments

Saline

MAP 125 (mmHg) 100 AP (mmHg)

PE 10 Ag/kg

150 SP (mmHg) DP (mmHg) MAP (mmHg) HR (bpm)

100

HR 400 (bpm) 350

B MAP 125 (mmHg) 100 AP 150 (mmHg) 100

HR 400 (bpm) 350

15 sec

*

*

40 20

20 0 -20

HR SP

DP

MAP

0

∆HR (bpm)

60

60

*

∆SP, ∆DP, ∆MAP (mmHg)

C

*

Before

After

Before

After

120 T 4 87 T 4 98 T 3 378 T 9

152 T 6* 116 T 6* 129 T 6* 363 T 9*

111 T 3 79 T 2 89 T 2 367 T 10

114 T 3 82 T 2 93 T 2 366 T 9

DP, diastolic pressure (mmHg); HR, heart rate (bpm, brats per min); MAP, mean arterial blood pressure (mmHg); PE, phenylephrine; SP, systolic pressure (mmHg). Values are means T SE; *P < 0.05.

Phenylephrine

40

Saline

-20

Fig. 1. Reflex responses evoked by intravenous saline or phenylephrine injection. (A, B) Example of the pressor experiments that induced the baroreceptor reflex. Similar pressor and reflex bradycardiac responses were elicited by three injections of phenylephrine at 5min intervals. (C) Averaged peak change in mean systolic pressure, diastolic pressure, mean arterial blood pressure and heart rate. Abbreviations: AP, arterial blood pressure (mmHg); DP, diastolic pressure (mmHg); HR, heart rate (bpm; beats per min); MAP, mean arterial blood pressure (mmHg); SP, systolic pressure (mmHg). * P < 0.05, estimated by t-test.

(TS) at 20 -C for 20 min, and then incubated with rabbit anti-MafG polyclonal antiserum raised against a peptide (EEIIQLKQRRR) at 4 -C overnight. The next day, the sections were incubated in TS containing biotinylated antirabbit IgG antiserum (Vector Laboratories, Burlingame, CA, USA) at 20 -C for 1 h. Then, the sections were incubated in avidin – biotin peroxidase complex solution (ABC solution, Vector Laboratories.) at 20 -C for 1 h, and treated with diaminobenzidine (DAB) solution containing 0.003% H2O2. Sections were rinsed and mounted on glass slides and covered with Permount (Fisher Scientific, Fair Lawn, NJ, USA). MafG-immunoreactive neurons were observed under a light-field microscope and counted. The specificity of immunostaining for MafG was verified by a preabsorption control (150 AM synthetic MafG peptide) and substitution of preimmune for immune sera. Visualization of MafG/FosB double-immunoreactive neurons was carried out as follows. Ninety minutes after intravenous injection of phenylephrine, the medulla oblongata (n = 2) was prepared for 15-Am thick serial sections. Every serial section was processed for immunohistochemical staining of FosB using anti-FosB antiserum. The sections were treated with DAB in a solution consisting of nickel

ammonium sulfate and 0.003% H2O2, which produced black reaction products. After visualization of the FosB-immunoreactive elements, sections on the slides were washed thoroughly in Tris – HCl (10 min per wash, 5 washes), treated with rabbit anti-MafG polyclonal antiserum in TS overnight at 4 -C, and stained as described above. Finally, the sections were dried and covered with Permount. MafG/ FosB double-immunoreactive neurons were counted under a light-field microscope. Brain histology was checked against the rat brain atlas of Swanson (1992). The significance of changes in parameters of circulation was evaluated using the paired t-test. The significance of differences in the numbers of labeled neurons was

-

A kb 0.75

-

0.5

-

1.23

-

-

+ +

+

stimulation RNA

MafG

GAPDH

B % units 400

*

300 200 100 0

-

+

+ +

stimulation RNA

Fig. 2. Effect of baroreceptor stimulation on MafG mRNA levels in the medulla and pons in the rat brain. (A) Total RNAs were isolated from brain tissues and converted to cDNA by the RT-PCR. Semiquantitative amplification was performed for 30 cycles with MafG and GAPDH. Water was used instead of a RNA sample as a control. Positions of standard molecular masses (kb) are indicated on the left. (B) Relative abundance of MafG transcripts were measured as described in Materials and methods and were normalized to GAPDH mRNA. Dark column represents mRNA levels after baroreceptor stimulation and white column represents mRNA levels without baroreceptor stimulation. The data represents mean values, and error bars indicate SEM for measurements from four independent experiments. The significance of difference was evaluated using the Mann-Whitney U-test. *P < 0.05.

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

1763

arterial blood pressure were observed in the test compared to control experiments ( P < 0.05), and significant decreases in heart rate was also observed between test and control experiments ( P < 0.05). Expression of MafG after baroreceptor stimulation We performed semiquantitative RT-PCR to confirm the regulation of MafG expression following stimulation of arterial baroreceptors by blood pressure increase after phenylephrine treatment (Fig. 2). There was a significant elevation of MafG transcripts within 30 min after baroreceptor stimulation. Fig. 2B shows histograms of these densities. The density of MafG

Number of MafG-labeled neurons

A 200

*

150 100 *

50

* *

0 NTS

DMX

AMB

CVLM/RVLM

evaluated using the Mann –Whitney U-test. The probability level taken to indicate significance was P < 0.05. Results Physiological conditions of rats Fig. 1A and B shows examples of abrupt increases in arterial blood pressure, and mean arterial blood pressure and a reflex decrease in heart rate induced by intravenous injection of phenylephrine or saline. Table 1 shows measured values of cardiovascular responses in the test experiments (n = 8) and the control experiments (n = 8) after injection of phenylephrine or saline. The intravenous injection of phenylephrine significantly elevated systolic arterial pressure, diastolic arterial pressure, mean arterial pressure, and decreased the heart rate before stimulation ( P < 0.05). No significant cardiovascular responses were observed after the injection of saline. Fig. 1C shows the average peak response to phenylephrine or saline. Between test and control experiments, significant increases in systolic pressure, diastolic pressure, and mean

C Number of MafG-labeled neurons

Fig. 3. Photomicrographs of MafG-immunoreactive neurons in the NTS, DMX, AMB and CVLM/RVLM after baroreceptor stimulation with phenylephrine. (A, B) Neurons without baroreceptor stimulation. (C, D) Neurons 90 min after baroreceptor stimulation. (E, F) Neurons 180 min after baroreceptor stimulation. MafG-immunoreactive neurons are indicated by arrows a – d. Abbreviations: AMBd, nucleus ambiguus, dorsal division; AMBv, nucleus ambiguus, ventral division. Bars: 200 Am in A – F, 20 Am in a – d.

Number of MafG-labeled neurons

B 200 150 *

100 50

*

*

*

0

NTS

200

DMX

AMB

CVLM/RVLM

*

150 *

100 50

~ ~

CVLM/RVLM

*

40 * *

20

* * *

NTS AMB DMX

0 Control

90 min

180 min

Fig. 4. Comparison of the densities of MafG-immunoreactive neurons in the NTS, DMX, AMB and CVLM/RVLM. Dark columns represent densities after baroreceptor stimulation and white columns after saline injection. (A) Neurons 90 min after baroreceptor stimulation and saline injection. (B) Neurons 180 min after baroreceptor stimulation and saline injection. (C) Numbers of MafGimmunoreactive neurons in the NTS ( ), DMX (g), AMB () and CVLM/ RVLM (o) in the experiments without baroreceptor stimulation, 90 min and 180 min after baroreceptor stimulation. Significant difference in the numbers of MafG-immunoreactive neurons 90 and 180 min after baroreceptor stimulation were calculated against values of control.

.

1764

A

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

B

mately 20% of MafG-immunoreactive neurons coexpress FosB product in the nucleus. Discussion

Fig. 5. Photomicrographs of representative FosB single-immunoreactive neuron (A) and MafG/FosB double-immunoreactive neuron (B) in the RVLM. Bar: 20 Am.

transcripts was about 3 times higher in the test group when compared to controls. Topology of MafG-immunoreactive neurons MafG-immunoreactive neurons were observed in the medulla oblongata. In the test experiment at 90 min after baroreceptor stimulation many MafG-immunoreactive neurons were found in the NTS, DMX, AMB and CVLM/RVLM (Fig. 3C,D), while in control experiments MafG-immunoreactive neurons were few in number and sporadically distributed in the medulla oblongata (Fig. 3A,B). Fewer MafG-immunoreactive neurons were found in the test experiment at 180 min after baroreceptor stimulation (Fig. 3E,F). Dark brown particles confined to the nucleus were identified as expression products of MafG (Fig. 3a– d). The numbers of MafG-immunoreactive neurons in the NTS, DMX, AMB, and CVLM/RVLM were counted, averaged and compared between test and control experiments. Fig. 4 shows frequency histograms of the MafGimmunoreactive neurons expressed in the NTS, DMX, AMB and CVLM/RVLM at 90 min (Fig. 4A) and 180 min (Fig. 4B) after baroreceptor stimulation. The number of MafGimmunoreactive neurons in all parts of the medulla was significantly higher in test experiments than in control experiments. The distribution patterns of MafG-immunoreactive neurons were dissimilar from c-Fos-immunoreactive neurons, but similar to FosB-immunoreactive neurons after baroreceptor stimulation (Okada and Miura, 1997). The number of MafG-immunoreactive neurons at 180 min after baroreceptor stimulation decreased by 45 –70% compared to the number of MafG-immunoreactive neurons at 90 min in all nuclei (Fig. 4C). MafG and FosB coexpression Because the topology of MafG-immunoreactive neurons was quite similar to that of FosB-immunoreactive neurons after baroreceptor stimulation, we examined whether both MafG and FosB were coexpressed after baroreceptor stimulation. Fig. 5 shows photomicrograph of a typical MafG/FosB doubleimmunoreactive neuron in the RVLM. Black products of FosB (Fig. 5A) were found in the RVLM neuron. After visualization of FosB, the sections were incubated with MafG antiserum. MafG/FosB double-immunoreactive neurons were found in the NTS, DMX, AMB and CVLM/RVLM (Fig. 5B). Approxi-

To understand the function of the baroreceptor reflex, it is important to trace the neuronal pathway of the reflex. The c-fos gene, one of the proto-oncogenes, and its product Fos protein (c-Fos), has been used to identify activated neurons within the central nervous system (Dragunow and Faull, 1989; Sagar et al., 1988; Dragunow and Robertson, 1987). Several investigators surveyed the neuronal expression of c-Fos protein after baroreceptor stimulation. Electrical stimulation of the axotomized carotid sinus nerve and aortic depressor nerve of rats (Rutherfurd et al., 1992; McKitrick et al., 1992) and baroreceptor stimulation by phenylephrine injections into rabbit (Li and Dampney, 1992) resulted in neuronal expression of Fos within the NTS, AP, CVLM and RVLM of the medulla. On the contrary, complete denervation of both carotid sinus nerve and aortic depressor nerve reduced the number of neurons exhibiting phenylephrine-induced Fos expression by 90% (Chan et al., 2000). In our recent study, after repeated stimulation of baroreceptors by phenylephrine, we found the correlation coefficient of the dose – response relationship between the dose of phenylephrine and Fos expression was high, and significant only in the medial part of the NTS in the medulla and periaqueductal gray (PAG) in the midbrain. The correlation coefficient was comparatively high but insignificant in the commissure and lateral parts of the NTS, caudal and rostral VLM, periambiguus nucleus, dorsal and ventral medullary reticula nuclei, lateral parabrachial nucleus, paraventricular nucleus thalamus, and dorsomedial nucleus hypothalamus (Miura et al., 1994b). These observations are unquestionably important, but other signaling molecules in neuronal cells involved in the baroreceptor reflex remain to be elucidated. Okada and Miura (1997) reported that expression of FosB in neuronal cells of the medulla was more sensitive to baroreceptor stimulation than expressions of any other immediate early genes such as c-Fos, c-Jun, and JunD. They also compared the distribution of FosB-immunoreactive neurons in the medulla after baroreceptor stimulation with that of neurons labeled by the retrograde transport of chorea toxin (CT) conjugated horseradish peroxidase (HRP) placed on the sino-atrial node. The distribution patterns of the FosBimmunoreactive neurons in the DMX and AMB were similar to that of the CT-HRP-labeled neurons of the medulla. From these observations, it appears that the cardioinhibitory baroreceptor reflex is mediated via the DMX and AMB. In our previous study (Xiong et al., 1997, 1998), we examined the difference in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), and we determined the topology and numbers of barosensitive neurons. We found that FosB is expressed in the AMB, DMX and CVLM/RVLM after baroreceptor stimulation and that in the AMB and DMX, induction of c-Fos was very low. In the

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

present study, MafG was expressed in the AMB and DMX including the NTS and CVLM/RVLM after baroreceptor stimulation. The distribution of MafG was similar to that of FosB-immunoreactive neurons. We then examined by the double-staining method to determine whether MafG and FosB were expressed in the same cells after baroreceptor stimulation. Our experiments confirmed coexpression of MafG and FosB in neurons of the medulla oblongata and pons after baroreceptor stimulation. This result suggests that a combination of MafG and FosB might be more dominant than MafG and c-Fos in the AMB and DMX for signal transduction after baroreceptor stimulation. MafG has been shown to forms heterodimers with c-Fos (Kataoka et al., 1994). Recently, we found that MafG is partially co-localized with FosB in the nucleus of cells and MafG can form heterodimers with FosB (Shimokawa et al., 2005). These results suggest that MafG may play critical roles as an immediate early gene with the cooperation of FosB in signal transduction of cardiovascular regulation mediated by baroreceptive signals in the medulla oblongata. In general, expression of the protein product of immediate early genes, especially c-Fos, peaks 1.5 – 2.0 h after stimulation and thereafter approaches control levels in a few hours. In 15% cases of hypotensive hemorrhage, the numbers of Fosimmunoreactive neurons in the medulla peak 2.0 h after the hemorrhage, and returned to baseline within 4 h (Chan and Sawchenko, 1994). In rats, light pulses can stimulate the expression of immediate early genes in the suprachiasmatic nucleus (Schwartz et al., 2000). The number of light-induced cFos-immunoreactive neurons in the suprachiasmatic nucleus reach maximum numbers in 2.0 h after light onset, and then decrease by 31% 4 h after stimulation by light. We detected MafG expression in neurons in the brainstem 90 min after baroreceptor stimulation and then showed that the number of MafG-immunoreactive neurons diminished by 50% 180 min after the stimulation. This result indicates that MafG has a similar temporal expression pattern to those of immediate early gene such as c-Fos as described above. Recently, To¨ro¨csik et al. (2002) found that MafK, a member of the Maf transcription factor family plays a role in neurite outgrowth as a new neural growth factor-responsive immediate early gene. These collective results strongly suggest that MafG may serve signal transduction and gene expression as an immediate early gene in response to baroreceptor stimulation in the brain. In conclusion, the present findings support the notion that baroreceptor stimulation by phenylephrine can induce MafG expression in cardiovascular control sites involved in the central processing of baroreceptor input. The spatial and temporal expression patterns suggest that MafG may play critical functions in signal transduction as an immediate early gene in response to baroreceptor stimulation. Future functional analysis of MafG may provide new insights into cardiovascular regulation by the central nervous system. Acknowledgements We are very grateful to Dr. Noriaki Shimokawa, Department of Integrative Physiology, Gunma University Graduate School

1765

of Medicine, for helping in the preparation of the manuscript. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to N.K. and K.T. References Chan, R.K.W., Sawchenko, P.E., 1994. Spatially and temporally differentiated pattern of c-fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. Journal of Comparative Neurology 348 (3), 433 – 460. Chan, R.K.W., Jarvina, E.V., Sawchenko, P.E., 2000. Effects of selective sinoaortic denervations on phenylephrine-induced activational responses in the nucleus of the solitary tract. Neuroscience 101 (1), 165 – 178. Ciriello, J., 1983. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neuroscience Letters 36 (1), 37 – 42. Ciriello, J., Hrycyshyn, A.W., Calaresu, F.R., 1981. Glossopharyngeal and vagal afferent projections to the brain stem of the cat: a horseradish peroxidase study. Journal of the Autonomic Nervous System 4 (1), 63 – 79. Dragunow, M., Faull, R.J., 1989. The use of c-fos as metabolic marker in neuronal pathway tracing. Journal of Neuroscience Methods 29 (3), 261 – 265. Dragunow, M., Robertson, H.A., 1987. Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyrus. Nature 329 (6138), 441 – 442. Guyenet, P.G., 1990. Role of ventral medulla oblongata in blood pressure regulation. In: Loewy, A.D., Spyer, K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford University Press, Oxford, pp. 145 – 167. Haxhiu, M.A., Yung, K., Erokwu, B., Cherniack, N.S., 1996. CO2-induced cfos expression in the CNS catecholaminergic neurons. Respiratory Physiology 105 (1 – 2), 35 – 45. Housley, G.D., Martin-Body, R.L., Dawson, N.J., Sinclair, J.D., 1987. Brainstem projections of the glossopharyngeal nerve and its carotid sinus branch in the rat. Neuroscience 22 (1), 237 – 250. Kataoka, K., Noda, M., Nishizawa, N., 1994. Maf nuclear oncoprotein recognizes sequences related to an AP-1 site and forms heterodimers with both Fos and Jun. Molecular and Cellular Biology 14 (1), 700 – 712. Li, Y.W., Dampney, R.A.L., 1992. Expression of c-fos protein in the medulla oblongata of conscious rabbits in response to baroreceptor activation. Neuroscience Letters 144 (1 – 2), 70 – 74. McKitrick, D.J., Krukoff, T.L., Calaresu, F.R., 1992. Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve. Brain Research 599 (2), 215 – 222. Miura, M., Reis, D.J., 1969. Termination and secondary projections of carotid sinus nerve in the cat brain stem. American Journal of Physiology 217 (1), 142 – 153. Miura, M., Takayama, K., Okada, J., 1994a. Distribution of glutamate-and GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats: a double-labeling study. Brain Research 638 (1 – 2), 139 – 150. Miura, M., Takayama, K., Okada, J., 1994b. Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation. Journal of the Autonomic Nervous System 50 (1), 31 – 43. Miura, M., Okada, J., Takayama, K., Suzuki, T., 1994c. Neuronal expression of Fos and Jun protein in the rat medulla and spinal cord after anoxic and hypercapnic stimulations. Neuroscience Letters 178 (2), 227 – 230. Nosaka, S., Yamamoto, T., Yasunaga, K., 1979. Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem. Journal of Comparative Neurology 186 (1), 79 – 92. Nosaka, S., Yasunaga, K., Tamaki, S., 1982. Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges. American Journal of Physiology 243 (1), R92 – R98. Okada, J., Miura, M., 1997. Barosensitive cardioinhibitory neurons in the medulla: comparison of FosB/ChAT-positive neurons with CT-HRP-labeled neurons. Autonomic Nervous System 64 (2 – 3), 85 – 90.

1766

I. Kumaki et al. / Life Sciences 78 (2006) 1760 – 1766

Ross, C.A., Ruggiero, D.A., Reis, D.J., 1985. Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. Journal of Comparative Neurology 242 (4), 511 – 534. Rutherfurd, S.D., Widdop, R.E., Sannajust, F., Louis, A.L., Gundlach, W.J., 1992. Expression of c-fos and NGFI-A messenger RNA in the medulla oblongata of the anesthetized rat following stimulation of vagal and cardiovascular afferents. Molecular Brain Research 13 (4), 301 – 312. Sagar, S.M., Sharp, F.R., Curran, T., 1988. Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 240 (4857), 1328 – 1331. Sapru, H.N., 1989. Cholinergic mechanism subserving cardiovascular function in the medulla and spinal cord. In: Ciriello, J., Caverson, M.M., Polosa, C. (Eds.), The Central Neural Organization of Cardiovascular Control, Progress in Brain Research vol. 81. Elsevier, Amsterdam, pp. 171 – 179. Schwartz, W.J., Carpino, A., De la Iglesia, H.O., Baler, R., Klein, D.C., Nakabeppu, Y., Aronin, N., 2000. Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus. Neuroscience 98 (3), 535 – 547. Shimokawa, N., Sugama, S., Miura, M., 1998. Extracellular H+ stimulates the expression of c-fos/c-jun mRNA through Ca2+/calmodulin in PC12 cells. Cellular Signalling 10 (7), 499 – 503. Shimokawa, N., Okada, J., Miura, M., 2000. Cloning of MafG homologue from the rat brain by differential display and its expression after hypercapnic stimulation. Molecular and Cellular Biochemistry 203 (1 – 2), 135 – 141. Shimokawa, N., Kumaki, I., Takayama, K., 2001. MafG-2 is a novel Maf protein that is expressed by stimulation of extracellular H+. Cellular Signalling 13 (11), 835 – 839. Shimokawa, N., Kumaki, I., Qiu, C.H., Ohmiya, Y., Takayama, K., Koibuchi, N., 2005. Extracellular acidification enhances DNA binding activity of MafG-FosB heterodimer. Journal of Cellular Physiology 205 (1), 77 – 85.

Spyer, K.M., 1990. The central nervous organization of reflex circulatory control. In: Loewy, A.D., Spyer, K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford University Press, Oxford, pp. 168 – 188. Swanson, L.W., 1992. Brain Maps: Structure of the Rat Brain. Elsevier, Amsterdam. Teppema, L.J., Veening, J.G., Kranenburg, A., Dahan, A., Berkenbosch, A., Olievier, C., 1997. Expression of c-fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia. Journal of Comparative Neurology 388 (2), 169 – 190. To¨ro¨csik, B., Angelastro, J.M., Greene, L.A., 2002. The basic region and leucine zipper transcription factor MafK is a new nerve growth factorresponsive immediate early gene that regulates neurite outgrowth. Journal of Neuroscience 22 (20), 8971 – 8980. Urbanski, R.W., Sapru, H.N., 1988a. Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area. Journal of the Autonomic Nervous System 23, 161 – 174. Urbanski, R.W., Sapru, H.N., 1988b. Putative neurotransmitters involved in medullary cardiovascular regulation. Journal of the Autonomic Nervous System 25, 181 – 193. Willette, R.N., Barcas, P.P., Krieger, A.J., Sapru, H.N., 1983. Vasopressor and depressor areas in the rat medulla: identification by microinjection of lglutamate. Neuropharmacology 22 (9), 1071 – 1079. Willette, R.N., Barcas, P.P., Krieger, A.J., Sapru, H.N., 1984. Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure. Brain Research 321 (1), 169 – 174. Xiong, Y., Takayama, K., Miura, M., 1997. Differences in the density of barosensitive neurons in the medulla of spontaneously hypertensive and Wistar-Kyoto rats. Clinical Expand Pharmacology and physiology 24 (6), 398 – 402. Xiong, Y., Okada, J., Tomizawa, S., Takayama, K., Miura, M., 1998. Difference in topology and numbers of barosensitive catecholaminergic and cholinergic neurons in the medulla between SHR and WKY rats. Journal of the Autonomic Nervous System 70 (3), 200 – 208.