70 :~euroscience Letler.v, 144 (1992) 70 "~4 ~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.0(I

NSL 08923

Expression of c-fos protein in the medulla oblongata of conscious rabbits in response to baroreceptor activation

Yu-Wen Li and R .A.L . D a m p n e y

Department of Physiology, University o# Sydney, NSW (Australia)

(Received 14 April 1992; Revised version received 1 June 1992; Accepted 2 June 1992)

K~:v words'." Ventrolateral medulla; Nucleus tractus solitarii; Area postrema; Arterial pressure; Baroreceptor reflex; Proto-oncogene; Tyrosine hydroxylase

Neuronal expression of c-fos protein (Fos) in the medulla in response to baroreceptor activation was studied in conscious rabbits. Raising arterial pressure resulted in a marked increase, compared to control animals, in Fos immunoreactivity in the nucleus tractus solitarius, area postrema and ventrolateral medulla (VLM). Fos-immunoreactive neurons in the VLM extended from the level just rostral to the obex to 3 mm more caudal. Only a small proportion of these neurons showed tyrosine hydroxylase immunoreactivity. The results indicate that baroreceptor activation induces Fos expression in circumscribed medullary regions which have previously been shown to receive excitatory baroreceptor inputs.

The expression of Fos, the DNA binding protein from the proto-oncogene c-Jos, is rapidly and transiently in- duced in central neurons by a variety of physiological stimuli [2, 7, 8, 13, 15, 17]. Fos expression has therefore been considered to be a marker of neuronal activation [2, 7, 8, 13, 17], and immunohistochemical labelling of Fos- containing neurons has been used to map pathways in the central nervous system that subserve specific func- tions [8, 15, 17].

Previous electrophysiological studies have identified neurons within the medulla that are activated by stimula- tion of baroreceptors. Such neurons have been located within the nucleus tractus solitarii (NTS), which is the primary site of termination of baroreceptor primary af- ferent fibres (for review see ref. 9), and also in the caudal part of the ventrolateral medulla (VLM) [5, 19]. The lat- ter neurons are thought to mediate baroreceptor-induced inhibition of sympathoexcitatory vasomotor neurons in the rostral VLM [5, 19]. However, these studies have not revealed the full extent of neurons within the medulla activated by baroreceptor inputs. The purpose of this study was to use the method of Fos immunohistochemis- try to gain a more complete picture of the distribution within the medulla of neurons activated by natural stim- ulation of baroreceptors in conscious animals.

Experiments were performed on 8 New Zealand White

Correspondence: Y.-W. Li, Department of Physiology, FI 3, University of Sydney, NSW, 2006, Australia. Fax: 61-2-6922058.

rabbits (2.5-3.5 kg). A marginal ear vein was cannulated for drug administration. Rabbits were then anaes- thethised with Saffan (Glaxovet, 10 mg/kg, i.v.) and a central ear artery was exposed and cannulated for moni- toring arterial pressure. This procedure was completed within 15 min and the rabbit was then allowed to recover from anaesthesia and rest in a cage for 4 h before further procedures. In 5 rabbits, phenylephrine hydrochloride (Winthrop Pharmaceuticals, 0.6-1.0 mg/ml in Ringer so- lution) was continuously infused (3-7 ml/h, i.v.), so as to raise mean arterial pressure to a steady-state level which was 23 + 5 mmHg (mean + S.D.) above the baseline level of 82 + 7 mmHg. The raised arterial pressure was maintained for either 40 min (n=2) or 60 min (n=3). Acti- vation of the baroreceptor reflex during the period of raised pressure was confirmed by the presence of bradycardia (heart rate decreased to 79 + 28 beats/rain from a baseline of 252 + 17 beats/min, n=5). In 3 control rabbits, Ringer solution was infused intravenously at the same rate as the phenylephrine infusion, for either 40 (n=l) or 60 min (n--2). In all these cases, the arterial pressure was not altered.

After the infusion of phenylephrine or Ringer solu- tion, the rabbits were deeply anaesthetised with sodium pentobarbitone (35-50 mg/kg, i.v.) and perfused trans- cardially with 500 ml 1% sodium nitrite in phosphate buffered saline (PBS, 0.01 M, pH 7.3), followed by 1.5 litre of 4% paraformaldehyde in PBS (0.1 M, pH 7.3). The brain was removed and post-fixed overnight in 30%

71

A + l . 5 m m

¢

C o0.5mm

: .O

D -1.5mm

E -2.5mm

Fig. 1. Distribution of Fos-ir cell nuclei in representative coronal sections of the medulla in a rabbit in which the arterial pressure was raised for 40 min. Each dot represents one Fos-ir cell nucleus. X, dorsal motor nucleus of vagus; XII, hypoglossal nucleus; AP, area postrema; cc, central canal; IO, inferior olivary nucleus; LRN, lateral reticular nucleus; NA, nucleus ambiguus; PrH, prepositus hypoglossal nucleus; RfN, retrofacial nucleus;

TS, tractus solitarius; Vsp, spinal nucleus of trigeminal nerve.

sucrose in PBS. Coronal sections (40 p m thick) through the medulla were cut on a freezing microtome. A 1 in 5 series of sections was processed for Fos immunoreactiv- ity using the avidin-biotin peroxidase method. Sections were incubated overnight at 4°C in polyclonal sheep anti-Fos antibody (Cambridge Research Biochemicals, OA-11-823) diluted 1:2000 in 1% normal horse serum in PBS. The antibody, directed against residues 2-16 of the N-terminal region of the Fos peptide, recognises Fos (62 kDa) as well as several Fos-related antigens (at 48/49 and 70 kDa). The sections were then incubated in biotinyl- ated anti-sheep antibody (Vector Labs, 1:200 dilution) and ExtrAvidin peroxidase conjugate (Sigma, 1:200 dilu-

tion), and reacted with 0.05% diaminobenzidine solution containing 1% nickel ammonium sulphate and 0.01% hy- drogen peroxide in acetate buffer (pH 6.0).

A second series of sections was processed for demon- stration of Fos and tyrosine hydroxylase (TH) im- munoreactivity by a two-color immunoperoxidase pro- cedure. Firstly, immunohistochemical staining of Fos was performed using the same procedure as above. These sections were then incubated in mouse anti-TH antibody (Incstar, 1:4000 dilution), in biotinylated anti-mouse an- tibody (Vector Labs, 1:200 dilution) and in ExtrAvidin peroxidase conjugate (1:200 dilution), and reacted with 0.05% diaminobenzidine solution containing 0.01% hy-

72

A

B

I I

LRN I !

Fig. 2. Photomicrographs of the caudal ventrolateral medulla of baro- activated (A) and control (B) rabbits showing the location of Fos-ir cell

nuclei. For abbreviations see Fig. 1. Bar=0.25 mm (A,B).

drogen peroxide in PBS. Fos-immunoreactive (-ir) cell nuclei were stained black and TR-ir neurons brown. No Fos immunoreactivity was detected in sections incubated in a solution without the c-los primary antibody. Sec- tions were examined with an Olympus BH2 microscope, and the distribution of Fos-ir cell nuclei mapped using the Magellan image-analysis program [6]. In all baro- activated and control experiments, bilateral counts were made of Fos-ir cell nuclei in the NTS and VLM at two representative levels (0.5 mm rostral and caudal to the obex), and in the area postrema at one level (0.5 mm caudal to obex). The unpaired Student's t-test was used for statistical comparisons. Values are expressed as mean _+ S.E.M.

Figure 1 is a representative example of the distribution of Fos-ir cell nuclei in the medulla of a rabbit in which the arterial pressure was raised for 60 min. There were no detectable differences in the pattern and density of la- belling whether the pressure was raised for 40 min (n=2) or 60 min (n=3). In all 5 experiments, the great majority of labelled cell nuclei were found bilaterally in 3 regions: the NTS, area postrema and VLM. A very few scattered Fos-ir cell nuclei were also found in the raphe pallidus

nucleus and dorsal reticular formation in the medulla of both baroreceptor-activated and control rabbits.

In the NTS of baroreceptor-activated rabbits, Fos-ir cell nuclei were found throughout the entire rostro-cau-

dal extent of the nucleus, but mainly caudal to the obex (Fig. 1). In particular, there was a high density of labelled cell nuclei dorsomedial to the tractus solitarius. Some Fos-ir cell nuclei were also found within the solitary tract. In the NTS the average number of Fos-ir cell nuclei per section was 88 _+ 14 (n=5), which was significantly greater than in the control experiments (27 _+ 8, n=3: P<0.02). A large number of Fos-ir cell nuclei were found in the area postrema, particularly its rostral part (Fig. 1), which contained 123 _+ 12 (n=5) cell nuclei per section, significantly more than in control animals (18 _+ 2, n--3. P<0.01). Very few labelled cell nuclei were found in the dorsal motor nucleus of the vagus, although some were located close to its border (Fig. 3A).

In the VLM the majority of Fos-ir cell nuclei were located in a circumscribed region ventrolateral to the nu- cleus ambiguus (Figs. 1, 2A and 3B). This region formed a longitudinal column, extending approximately from the level 1.0 mm rostral to the obex to 3 mm more caudal (Fig. 1). The caudal part of this column was located be- tween the lateral reticular nucleus and nucleus ambiguus (Figs. 1, 2A and 3B). The rostral part was medial to the rostral pole of the lateral reticular nucleus (Fig. I B). The average number of Fos-ir cell nuclei following barore- ceptor activation in the two representative sections through the VLM was 50 _+ 4, significantly more than in the equivalent regions in the control animals (9 _+ 2, P<0.01). In the rostral VLM, which contains vasopres- sot neurons [3], a few Fos-ir cell nuclei were found ven- trolateral to the retrofacial nucleus (Fig. I A).

Two-color immunoperoxidase staining revealed that Fos-ir cell nuclei in the NTS partly overlapped with TH- ir cells (Fig. 3A). Furthermore, a proportion of these (19 _+ 3%, n--4) were also immunoreactive for TH (Fig. 3A,C). In the VLM, Fos-ir cell nuclei also partly over- lapped with the area containing TH-ir neurons (Fig. 3B,D), but the proportion that showed TH immunoreac- tivity was much smaller (4 _+ 2%, n=4).

The results of this study show that activation of baroreceptors in the conscious rabbit, by increasing the arterial pressure, caused a marked increase in the num- ber of Fos-ir neurons in the medulla, as compared to control animals. Furthermore, such neurons had a spe- cific distribution, being largely confined to the NTS, the area postrema, and to a restricted region within the

VLM. In the rabbit baroreceptor afferents terminate densely

within the most dorsal part of the NTS [20]. Consistent with this, Fos-ir cells were concentrated in this part of the

73

('x XII ~ [CC

o . _

( D e • 0 • 0 0

o % . .%° oO

\ o

Fig. 3. Distribution of neurons stained for Fos and/or TH immunoreactivity in (A) the dorsomedial medulla, and (B) the VLM at the level approximately 1 mm caudal to the obex in baroreceptor-activated rabbits. Black dots represent Fos-ir cell nuclei, open circles represent TH-ir neurons, and circles enclosing black dots represent neurons immunoreactive for both Fos and TH. C and D are photomicrographs of Fos-ir and TH-ir neurons in the NTS and VLM, respectively, at the level approximately 1 mm caudal to the obex. Oblique arrows indicate neurons immunoreac- tive for Fos. Horizontal arrows indicate neurons immunoreactive for both Fos and TH. For abbreviations see Fig. 1. Bar=0.15 mm (A,B); 40 pm

(C,D).

NTS. It is thus possible that many of these cells are sec- ond-order neurons activated by baroreceptor stimuli. Similarly, our finding that many Fos-ir cells were located in the area postrema supports previous electrophysiol- ogical evidence that this area receives baroreceptor in- puts [16].

In the VLM, many Fos-ir neurons were found caudal to the obex, in a region that corresponds closely to the vasodepressor area as mapped by the method of gluta- mate microinjection in baroreceptor-intact rabbits [10]. Electrophysiological studies have identified neurons in this region that are activated by baroreceptor stimula- tion [5, 19]. Consistent with this, the present results indi- cate that Fos-ir cells within this region are activated by baroreceptor inputs, and thus presumably have a vaso- depressor function.

Many Fos-ir neurons in the VLM, however, were also located rostral to the vasodepressor region as defined by

Li and Blessing [10], extending to the level just rostral to the obex. Recent studies [12, 14] have shown that in baroreceptor-denervated rabbits depressor responses are also elicited by glutamate stimulation of the VLM at this level, suggesting that vasodepressor neurons in the VLM may extend further rostrally than was first indicated by studies in baroreceptor-intact rabbits. The Fos-ir neu- rons in the VLM at and just rostral to the obex may therefore represent these more rostral vasodepressor neurons.

Very few Fos-ir cells were found in the pressor region of the rostral VLM, as defined previously in the rabbit [3]. This is consistent with electrophysiological studies, which have shown that the great majority of cells in this region that respond to baroreceptor inputs are inhibited by such inputs [11, 18]. On the other hand, a study in the cat has shown a marked increase in Fos-ir neurons in the rostral VLM in response to haemorrhage [13], a stimulus

74

which excites pressor neurons. Similarly, we have also found that a decrease in blood pressure in conscious rab- bits induced Fos expression in the rostral VLM (unpub- lished observations]. Thus, taken together, the results of the present and previous studies suggest that Fos expres- sion occurs in neurons within the VLM that are excited by specific stimuli, but not in neurons that are inhibited.

The distribution of Fos-ir neurons in the VLM partly overlapped with that of TH-ir neurons of the A1 group, but also extended more dorsally. In the region of over- lap, however, only a very small proportion of Fos-ir neu- rons were double-labelled for TH. This result indicates that vasodepressor neurons in the caudal VLM are largely separate from A1 catecholamine neurons, in agreement with suggestions from earlier physiological studies in rat [1, 4].

It should be noted that very few Fos-ir cells were found in the dorsal motor nucleus of the vagus and the nucleus ambiguus, even though a marked baroreceptor- induced reflex bradycardia was observed. Similarly, McAllen et al. [13] reported that no Fos-ir neurons were detected in spinal sympathetic preganglionic nuclei fol- lowing haemorrhage. It is possible the autonomic motor neurons do not express Fos in response to activation or, alternatively, the expression is considerably delayed, as has been reported in the case of ventral horn motor neu- rons reflexly activated by nociceptor stimulation [7].

In conclusion, a sustained rise in arterial pressure in conscious rabbits induces Fos expression in neurons within circumscribed regions in the dorsomedial and ventrolateral medulla. Such neurons may be interneu- tons specifically activated by baroreceptor stimulation.

The work was supported by the National Health and Medical Research Council of Australia. We thank Dr Kevin Keay for helpful discussions on Fos immunohis- tochemistry.

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