Brain Research, 567 (1991) 11-24 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50

11

BRES 17211

Fos-like protein is induced in neurons of the medulla oblongata after stimulation of the carotid sinus nerve in awake and anesthetized rats Jeffery T. Erickson and David E. Millhorn Department of Physiology, Universityof North Carolina, Chapel Hill, NC 27599 (U.S.A.) (Accepted 16 July 1991)

Key words: Fos; c-los; Carotid sinus nerve; Chemoreceptor reflex; Baroreceptor reflex; Hypoxia; Rat; Immunohistochemistry

The protooncogene c-los is expressed rapidly, transiently and polysynaptically within neurons in response to synaptic activation and voltage-gated calcium entry into the cell. The nuclear protein product of this gene (Fos) is detectable immunohistochemically 20-90 min after cell activation and remains within the nucleus for hours after expression. The present study was undertaken to identify cells within the rat medulla oblongata that express Fos-like protein in response to stimulation of afferent fibers of the carotid sinus nerve (CSN). Direct electrical stimulation of the CSN in anesthetized animals or hypoxic stimulation in either anesthetized or awake animals resulted in a consistent and discrete distribution of Fos-like immunoreactivity (Fos-LI). Fos-LI was observed bilaterally within nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM), within area postrema and nucleus raphe pallidus, and bilaterally along the ventral medullary surface. Unstimulated animals were devoid of Fos-LI within the medulla oblongata. Furthermore, neither the surgical preparations alone nor the effects of anesthesia could account for the extent of Fos-LI observed. We believe these cells represent second- and higher-order neurons within the baroreceptor and chemoreceptor reflex pathways. INTRODUCTION The precise location of higher order neurons within the baroreceptor and chemoreceptor reflex pathways has been a subject of interest for many years. Results from studies using the techniques of nerve degeneration 6'4s, anterograde tracing 1'5'9'19'39 and antidromic activation 7'11'16'2a'25 generally agree that afferent fibers contained within the CSN terminate primarily within and around the NTS. Terminations have also been reported in the vicinity of nucleus ambiguus s'9. It is assumed that second-order neurons are situated in close proximity to these afferent terminal fields. Mapping studies using evoked single-unit or field potentials in response to CSN stimulation 2'8'21'26-28'34'44 have confirmed the NTS as a major relay of afferent input and have localized other CSN-responsive areas of the medulla oblongata. For a comprehensive discussion of these findings see the review by Spyer 45. A major limitation of these histological and electrophysiological approaches is the inability to simultaneously and directly identify the population of secondand higher-order cells in these reflex pathways. Consequently, the overall extent and anatomical locations of synaptically activated cells in these pathways have been inferred. Ideally, the use of a specific marker expressed

by neurons that respond to CSN afferent stimulation would allow an opportunity to identify second- and higher-order cells within the baroreceptor and chemoreceptor reflex pathways. Expression of the protooncogene c-los may provide such a selective marker, c-los is expressed rapidly and transiently within neurons in response to synaptic activation and voltage-gated calcium entry into the cell 36. The protein product of this gene, Fos, which is confined predominantly to the cell nucleus, may be detected immunohistochemically 20-90 rain after cell activation and remains within the nucleus for hours after its expression 35'3a. Synaptic activation of Fos expression is specific to neurons; it does not occur in closely associated glial, ependymal or endothelial cells 3s. In addition, expression is polysynaptic, occurring at multiple levels along specific neural pathways 4'35'4°. The role of the c-los gene and its cognate protein in cellular function is not known. Activation of c-los and other immediate-early genes may play a role as 'third messengers' in signal transduction, serving to couple short-term events at the cell surface to long-term changes in gene expression 37. Despite a poor understanding of its specific function, c-los expression has been used successfully as a metabolic marker in neuronal pathway tracing4,12,22,35,40. The purpose of the present study was to identify the

Correspondence: J.Z. Erickson, Department of Physiology, University of North Carolina, Chapel Hill, NC 27599, U.S.A. Fax: (1) (919) 966-6927.

12 population of cells within the medulla oblongata of rat which respond to stimulation of afferent fibers of the CSN. Expression of Fos-like protein was used as a specific marker for activated cells. We found Fos-LI within numerous cells of the NTS, area postrema, VLM, and in cells located within nucleus raphe pallidus and along the ventral medullary surface following either electrical or physiological (hypoxic) stimulation of CSN afferents. Maps showing the locations of cells containing Fos-LI are provided.

MATERIALS AND METHODS Twenty-nine adult male Sprague-Dawley rats (250-450 g) were used in this study. Fourteen animals were anesthetized and prepared surgically for electrical stimulation of the carotid sinus nerve (n = 10), sham stimulation (n -- 2) or administration of an hypoxic gas mixture (n = 2) while recording neural activity within the phrenic nerve. Of the remaining 15 animals, 12 unanesthetized rats were placed in environmental chambers and exposed to an hypoxic gas mixture (8-10% 02; n = 8) or a normoxic gas mixture (21% 02; n = 4) following the protocol described below. Three completely unstimulated animals were anesthetized and immediately processed immunohistochemically to assess baseline Fos-LI within the medulla oblongata.

Animal preparations Anesthetized animals. Immediately after administration of anesthesia (65 mg/kg chloralose, 800 mg/kg urethane, i.p.) the animal was placed in a small closed chamber that was continuously flushed with 100% O2 to minimize unintentional stimulation of the carotid body chemoreceptors. The anesthetized animal was then removed from the chamber, placed supine on a table, rapidly intubated through the trachea, and ventilated with an hyperoxic gas mixture (40% 02, balance N2). Airway pCO2 was monitored continuously with an infra-red analyser (Traverse Medical Monitor, Dynatech Electro-Optics, San Luis Obispo, CA). End-tidal CO2 was maintained between 39 and 41 mmHg during the initial surgical preparations. A femoral artery and vein were cannulated to continuously monitor arterial pressure and to administer drugs, respectively. Body temperature was measured with a rectal thermistor and maintained at 37°C --- 0.5°C by an electronic feedback circuit and d.c. heating pad. The bladder was accessed through a small abdominal incision and cannulated to avoid overdistension during the experiment. After the initial preparations, the animal was paralyzed with pancuronium bromide (2 mg/kg i.v. initially, followed by continuous infusion at 1 mg/kg/h), and a bilateral cervical vagotomy was performed. The glossopharyngeal nerve and the CSN were exposed through a ventral approach and gently dissected free from surrounding tissue. Care was taken to minimize mechanical disturbance of the nerves. One phrenic nerve root was exposed through a ventral approach. The nerve was isolated from surrounding tissue and sectioned distally. The cut central end was covered with mineral oil and placed on a platinum bipolar recording electrode. Finally, the exposed CSN was gently draped across a small platinum bipolar stimulating electrode at a point distal to its juncture with the glossopharyngeal nerve. The nerve and electrode were isolated from adjacent muscle and nerve tissue and covered with warm mineral oil. When CSN afferents were activated with hypoxia rather than electrical stimulation, the initial preparations were identical to those described above except that the CSN was not exposed.

Experimental protocols Anesthetized animals. In all experiments, phrenic nerve activity,

an index of central inspiratory drive 13, was amplified and integrated by a sample-and-hold integrator (Gould no. 13-4615-70). The threshold for phrenic nerve activity was determined by slowly decreasing end-tidal CO 2 until rhythmic discharge of the nerve disappeared. End-tidal CO 2 was then increased 3-5 mmHg above this initial level and maintained constant for the remainder of the experiment by minor adjustments of the ventilator. Integrated phrenic nerve activity, arterial pressure, and airway pCO 2 were all recorded on magnetic tape and on a strip chart recorder. Electrical stimulation. After the surgical preparations were completed and phrenic nerve activity had stabilized, the CSN was electrically stimulated for a 10-min period via a stimulator (Grass $88) connected in series with a stimulus isolation unit (Grass SIU 5) and fixed resistance. The stimulation parameters were either 25 Hz (n = 2) or 50 Hz (n --- 8) with a pulse duration of 0.05 ms; these parameters were maintained constant throughout the stimulation period. The intensity of stimulation was adjusted in each experiment to obtain a noticeable increase in integrated phrenic nerve activity. At the end of the 10-rain stimulation period, the stimulus was discontinued and the animal was allowed to recover for 2 h. During this period body temperature and end-tidal CO 2 were kept constant. Hypoxic stimulation. The protocol for these experiments (n = 2) was identical to that outlined above except that the CSN was not exposed surgically. The stimulation consisted of ventilating the anesthetized animal with an hypoxic gas mixture (12% 02, balance N2). After a 10-min stimulation period, the animal was maintained as above on the hyperoxic gas mixture for the remainder of the 2 h recovery period. Control experiments. Two sets of control experiments were performed. In the first set (n = 2), animals were prepared as for electrical CSN stimulation, but electrical pulses were not delivered through the CSN electrode. These controls were performed to determine if non-specific input due to the surgical preparations resulted in Fos-LI within the medulla oblongata. In the second set, 3 additional animals were anesthetized with sodium pentobarbital and immediately perfused and processed immunohistochemically as described below. These animals served as anesthetized controls to assess the level of constitutively expressed Fos-like protein in the absence of surgical trauma.

Unanesthetized animals Hypoxic stimulation. Unanesthetized rats (n = 8) were placed individually within small closed environmental chambers which were flushed with an hypoxic gas mixture for 3 h. Oxygen and carbon dioxide levels within the chambers were monitored with an oxygen analyser and infra-red CO z analyser, respectively. The desired level of hypoxia was obtained by mixing room air with nitrogen at rates sufficient to produce an oxygen level of 8-10% while minimizing CO2 buildup within the chamber. The gases were humidified by bubbling the gas mixture through water before it entered the chamber. After the 3 h hypoxic exposure, the animals were immediately anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg, i.p.), perfused, and the medulla was removed and processed immunohistochemieally as described below. Control experiments. To test the effects of the chamber protocol (handling of the animal, confinement within the chamber, etc.) on the expression of Fos-like protein in unanesthetized animals, rats (n = 4) were placed individually in the environmental chambers and subjected to a 3 h exposure to room air (normoxia). Tissue from these animals was processed in parallel with tissue from rats that received hypoxia.

Tissue preparation and immunohistochemistry At the end of each experiment the animal was perfused transcardially with calcium-free Tyrode's solution followed by picric acid (0.3%)-containing paraformaldehyde (4%) in 0.16 M phosphate buffer51. The brain was removed and postfixed at 4°C in the same fixative for 60-90 min, then stored in 0.1 M phosphate buffer containing 10% sucrose at 4°C for 18-24 h.

13 RESULTS

Transverse sections (30 /~m) were cut in a eryostat (Mierom, Heidelberg, Germany) from the rostralmost extent of the pyramidal decussation to the pontomedullary border. Sections were floated in 0.1 M PBS (pH 7.5), then processed for Fos-like protein using an avidin-biotin technique 2°. The antibody to Fos-like protein was raised in sheep using an immunogen derived from an amino acid sequence common to both mouse and human (Cambridge Research Biochemical; 0A-11-823, batch no. 04286A). Whenever possible, tissue from control and experimental animals was processed together. Sections were thoroughly rinsed in 0.1 M PBS and ethanol pretreated to minimize endogenous peroxidase activity3° (10 rain in 50% ETOH, 15 rain in 70% ETOH, 10 rain in 50% ETOH). The sections were then rinsed in PBS (2 x 10 rain), pretreated for 1 h in 0.1 M PBS containing 2% normal rabbit serum (NRS) and 0.3% Triton X-100, then incubated for 18-24 h at 4°C with the Fos antiserum (1:8000 in 0.1 M PBS, 2% NRS, 0.3% Triton X-100). The sections were then rinsed (0.1 M PBS, 2% NRS, 2 x 10 min) and incubated with biotinylated anti-sheep IgG secondary antibody followed by ABC reagent (Vector Labs, Burlingame, CA). Reaction product was made visible by incubating sections with hydrogen peroxide and diaminobenzidine using a nickel intensification procedure 3°. Finally, sections were rinsed in PBS, floated onto gelatin-coated slides and air dried overnight. The following day, the sections were cleared in xylene and coverslipped with DPX mounting medium for viewing. In some cases, the sections were counterstained with Neutral red. Immunohistochemical control experiments consisted of preadsorption of the antisera with an excess of antigen (Fos peptide, Cambridge Research Biochemical; OP-11-3210) or omission of the primary antiserum. Staining was absent in all immunohistochemical control experiments. Kodak Tmax 400 and Kodacolor Gold 100 print film was used for photography of immunostalned material. In addition, camera lucida drawings (x 125) were made of representative material to record the precise anatomical locations of Fos-LI within the medulla oblongata.

A

ARTERIAL PRESSURE (mmHg)

Carotid sinus nerve afferent fibers were activated with either electrical or physiological stimulation in anesthetized animals (Fig. 1). The effectiveness of the stimulus was assessed by monitoring phrenic nerve activity. During electrical stimulation, the increase in phrenic nerve discharge was sometimes, but not always, accompanied by a change in arterial blood pressure. In the example shown in Fig. 1A, arterial pressure did not change during electrical stimulation, although phrenic nerve discharge increased substantially as the intensity of the stimulus was increased from 1 V to 2 V. End-tidal CO2 remained constant during the stimulation and throughout the remainder of the experiment. Physiological stimulation with hypoxia resulted in an increase in phrenic nerve discharge throughout the stimulation period (Fig. 1B). Blood pressure and end-tidal CO 2 both decreased during hypoxia, and were elevated above control levels during the initial 10-20 min of the recovery period. Locations of FOS-LI in stimulated animals Carotid sinus nerve afferent stimulation resulted in a consistent and discrete distribution of Fos-LI, regardless of the method of stimulation in either anesthetized or

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Fig. 1. Phrenic nerve response to stimulation of carotid sinus nerve afferent fibers. A: electrical stimulation (50 Hz, 0.05 ms pulse duration, 10 rain continuous stimulation). First arrow: beginning of electrical stimulation. Second arrow: increased stimulus intensity eficited an increase in phrenic nerve activity without a change in blood pressure. Third arrow: end of stimulation. B: hypoxic stimulation (12% 02 balance N2, 10 min continuous exposure). First arrow: hypoxic stimulation increased phrenic nerve activity and decreased blood pressure. Second arrow: beginning of recovery period breathing 40% O 2 balance N 2.

14 unanestetized animals. For the purposes of description, labeled cells were conveniently divided into 4 areas: (1) dorsal aspects of medulla: (2) ventrolateral aspects of medulla (VLM); (3) nucleus raphe pallidus; and (4) a small bilateral and superficial group of cells located just lateral to the pyramids, which we will refer to as the parapyramidal cell group.

Electrical stimulation of CSN afferents Dorsal medulla. Most Fos-LI was located bilaterally in and around the caudal and intermediate portions of NTS.

Labeled cells were found within the commissural subnucleus of NTS extending from the rostral extent of the pyramidal decussation to the level of area postrema (Fig. 2A, sections 1-3). The number of labeled cells within the commissural subnucleus was highest caudal to area postrema, where a large number of immunopositive cell nuclei were observed above the central canal and extended laterally toward the solitary tracts (Fig. 3A,B). At the level of area postrema, the greatest number of immunopositive cells within the commissural subnucleus were confined to its lateral aspects (Fig. 2A, section 3).

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Fig. 2. Camera lucida drawings (×125) of single transverse sections of the rat medulla oblongata illustrating the distribution of Fos-LI at selected caudal (1) to rostral (7) levels. Each black dot represents one labeled cell nucleus. A: carotid sinus nerve stimulation. Stimulation parameters: 50 I-Iz, 0.05 ms pulse duration, 1-4 V, 10 min total stimulation. B: surgery control. Animal was prepared exactly as for CSN stimulation, but no electrical pulses were delivered through the CSN electrode. AP, area postrema; Cu, cuneate nucleus; Gr, gracile nucleus;

15 A high density of immunoreactive cells was also found in the dorsomedial, medial, and ventromedial aspects of NTS, especially at the level of area postrema. Fewer immunoreactive cells were located ventral and ventrolateral to the solitary tract, in some cases extending into the subjacent dorsal reticular formation. In particular, in caudal sections, a number of immunopositive cells were observed extending in an arc from the ventrolateral aspect of NTS toward the VLM (Fig. 2A, sections 1-3). Labeled cells were also observed within the solitary tract itself while the lateral and dorsolateral aspects of NTS were largely devoid of staining. The number of cells containing Fos-LI diminished in sections rostral to area postrema. Very few labeled cells were seen rostral to the rostralmost extent of the hypoglossal nucleus. A significant number of immunopositive cells were observed within area postrema (Fig. 2A, section 3). In addition, labeled cells were seen along the dorsal aspect

of, and in some cases extending slightly into, the dorsal motor nucleus of the vagus. Rostral to the hypoglossal nucleus, sparse but consistent labeling occurred bilaterally in both the medial vestibular nucleus (Fig. 2A, sections 5-7) and the cochlear nucleus (data not shown). Occasionally, a few labeled cells were observed at the dorsal pole of the spinal trigeminal nucleus. Labeling was absent in the hypoglossal, gracile and cuneate nuclei. Except for those cells arcing toward the VLM from NTS described above, the dorsal reticular formation was also devoid of Fos-LI. Ventrolateral medulla. A distinct bilateral cluster of immunoreactive cells was observed in the VLM, extending from the rostralmost extent of the pyramidal decussation to the caudalmost pole of the facial nucleus. In caudal sections this group of cells appeared relatively compact, lying just dorsal to, and occasionally extending into, the lateral reticular nucleus (Fig. 2A, sections 1-4;

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ION, inferior olivary nucleus; LRt, lateral reticular nucleus; mlf, medial longitudinal fasciculus; MVe, medial vestibular nucleus; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; pyr, pyramidal tract; pyrx, pyramidal decussation; RM, nucleus raphe magnus; RO, nucleus raphe obscurus; RP, nucleus raphe pallidus; sol, solitary tract; spV, spinal trigeminal tract; VII, facial nucleus; X, dorsal motor nucleus of vagus; XII, hypoglossal nucleus.

16

Fig. 3. Comparison of photomicrographs of Fos-LI within tissue sections of the rat medulla oblongata from an anesthetized, electrically stimulated (50 Hz) animal (A,B) and an anesthetized, surgery control animal (C,D). Low power (A,C) and higher power (B,D) photomicrographs were taken from comparable sections within the medulla at the level of the commissural nucleus. Labeled cell nuclei appear as dark rounded structures within the cell body (arrows). Note the absence of labeled nuclei within the hypoglossal, gracile, and cuneate nuclei. Tissue was counterstained with Neutral red. Bar: 100/*m (A,C), 40/~m (B,D). cc, central canal; Comm, commissural nucleus; Gr, gracile nucleus; sol, solitary tract; XII, hypoglossal nucleus.

Fig. 4A,B). At this level, a number of labeled ceils were observed in the reticular formation arcing from the ventrolateral aspects of NTS toward the VLM. Labeled ceils appeared to be sandwiched between the lateral reticular nucleus ventrally and nucleus ambiguus/retroambigualis dorsally. Fos-LI within this area corresponds with the location of the CI/A1 adrenergic and noradrenergic cell groups 17.

In more rostral sections, the location of immunopositive cells in the VLM appeared to shift ventrally within

the lateral paragigantocellular and rostroventrolateral reticular nuclei (Fig. 2A, section 5). Most labeled cells were located ventral to the compact portion of nucleus ambiguus. Very few labeled ceils were located within the compact portion of nucleus ambiguus itself. Labeled cells were absent from the facial nucleus, and areas of the reticular formation other than those described above were virtually devoid of staining. Nucleus raphe pallidus. Immunoreactive cells were observed in nucleus raphe pallidus, beginning near the ros-

Fig. 4. Photomicrographs of Fos-LI within tissue sections of the rat medulla oblongata of anesthetized, electrically stimulated (50 Hz) animals (A,B,F,I) and unanesthetized animals exposed to hypoxia (C,D,E,G,H,J; 8-10% 02, 3 h). The photomicrographs document Fos-LI within the ventrolateral medulla (A-D), area postrema (E), the parapyramidal cell group (F,G), along the ventral surface of the medulla, lateral to the parapyramidal cell group (H), and within nucleus raphe pallidus (I,J). Note that Fos-LI was confined to the cell nucleus (B, arrows) which typically varied in the intensity of staining (E, arrows). A,B: low-power and higher-power photomicrographs of the ventrolateral medulla from an electrically stimulated animal. Note the similar location of labeled nuclei in a comparable section from an animal exposed to hypoxia (C,D). Tissue was counterstalned with Neutral red (except for C,D). Bar: 100/zm (A,C), 40/~m (D,E,I), or 20/zm (B,F,G,H,J). AP, area postrema; Gr, gracile nucleus; LRt, lateral reticular nucleus; pyr, pyramidal tract.

17

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18 tralmost extent of the inferior olives and extending rostraUy to the pontomeduUary b o r d e r (Fig. 2A, sections 5 - 7 ; Fig. 4I). No labeling was o b s e r v e d in the m o r e caudal portions of this nucleus. L a b e l e d cells were not observed in either nucleus raphe obscurus or nucleus raphe magnus.

A CHAMBER HYPOXIA

Parapyramidal cell group. Fos-LI was observed bilaterally in a small, distinct cell group located at the ventral surface and just lateral to the pyramids (Fig. 2 A , sections 5 - 7 ; Fig. 4F). The caudal to rostral extent of labeling within this superficial cell group c o r r e s p o n d e d approximately with that seen in nucleus raphe pallidus, begin-

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Fig. 5. Camera lucida drawings (x125) of single transverse sections of the rat medulla oblongata illustrating the distribution of Fos-LI at selected caudal (1) to rostral (7) levels. Each black dot represents one labeled cell nucleus. A: chamber hypoxia. Animal was placed within an environmental chamber and exposed to 8-10% 0 2 (hypoxia) for 3 h. B: chamber control. Animal was placed within an environmental chamber and exposed to 21% 02 (normoxia) for 3 h. AP, area postrema; Comm, commissural nucleus; Cu, cuneate nucleus; Gr, gracile

19 ning near the rostral extent of the inferior olives and extending to the pontomedullary border. Labeled cells were often seen superficially along the ventral surface of the pyramids and appeared to bridge these two cell groups (Fig. 2A, sections 5 and 7). Control experiments. Anesthetized and surgically prepared control animals showed a greatly reduced pattern of Fos-LI (compare Fig. 2A with 2B; Fig. 3A,B with 3C,D). In addition to sparse labeling within NTS, consistent but sparse labeling was observed bilaterally within the medial vestibular nucleus (Fig. 2B, sections 5-7) and the cochlear nucleus (data not shown). In the absence of CSN afferent stimulation, therefore, neither surgical trauma nor mechanical disturbance of the nerve was sufficient to induce extensive Fos-LI within the medulla.

Hypoxic stimulation of CSN afferents In both anesthetized (data not shown) and unanesthe-

tized animals, the location of labeled cells observed after hypoxia (Fig. 5A) was very similar to that found in animals in which CSN afferents were stimulated electrically (Fig. 2A). Fos-LI was consistently observed within NTS (Fig. 5A, sections 1-4; Fig. 6A,B), area postrema (Fig. 4E; Fig. 5A, section 3) and the VLM (Fig. 4C,D; Fig. 5A, sections 1-5). In unanesthetized animals, labeled cells within nucleus raphe pallidus and the parapyramidal cell group began to appear at more caudal levels than in anesthetized animals (Fig. 4G,J, Fig. 5A, sections 4-7). In addition, labeled ceils were often observed superficially along the ventral surface of the medulla lateral to the parapyramidal cell group (Fig. 4H; Fig. 5A, sections 5-7). In anesthetized animals, Fos-LI was observed in the medial vestibular and cochlear nuclei, as in animals in which the CSN was stimulated electrically. In contrast, in unanesthetized animals, immunopositive cells were consistently observed within the cochlear nucleus but

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nucleus; ION, inferior olivary nucleus; LRt, lateral reticular nucleus; mlf, medial longitudinal fasciculus; MVe, medial vestibular nucleus; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; pyr, pyramidal tract; pyrx, pyramidal decussation; RM, nucleus raphe magnus; RO, nucleus raphe obscurus; RP, nucleus raphe paUidus; sol, solitary tract; spV, spinal trigeminal tract; VII, facial nucleus; X, dorsal motor nucleus of vagus; XII, hypoglossal nucleus.

20

Fig. 6. Comparison of photomicrographsof Fos-LI within tissue sections of the rat medulla oblongata from an unanesthetized animal exposed to hypoxia (A,B; 8-10% Oz, 3 h) and an unanesthetized control animal exposed to normoxia (C,D; 21% Oz, 3 h). Lower power (A,C) and higher power (B,D) photomicrographswere taken from comparable sections within the medulla at the level of the commissural nucleus. This material was not counterstained. Bar: 100/zm (A,C) or 40/~m (B,D). cc, central canal; Comm, commissural nucleus; Gr, gracile nucleus; sol, solitary tract; XII, hypoglossal nucleus.

were not observed within the medial vestibular nucleus. Control experiments. The extent of Fos-LI in animals exposed to normoxia in environmental chambers was greatly reduced compared to animals breathing an hypoxic gas mixture (compare Fig. 5A,B; Fig. 6A,B with 6C,D). In these animals, Fos-LI was consistently observed within the cochlear nucleus but was completely absent within the medial vestibular nucleus. Thus, stimulation resulting from handling the animal, confinement within the environmental chamber, or some other nonspecific aspect of the experimental protocol was not sufficient to induce extensive Fos-LI within the medulla. Anesthetized animals which received no further treatment were virtually devoid of Fos-LI (data not shown). In these animals, only in the cochlear nucleus was Fos-LI consistently observed. These results indicated a very low level of constitutive expression of Fos-like protein within the rat medulla.

DISCUSSION In the present study, we have utilized the expression of Fos-like protein to identify groups of cells within the rat medulla oblongata which respond to stimulation of CSN afferent fibers. We have demonstrated that both direct electrical stimulation of the CSN as well as physiological stimulation of the carotid bodies with hypoxia result in the expression of Fos-LI in similar locations within the medulla oblongata. We have mapped the locations of these cells and believe they represent secondand higher-order neurons within the baroreceptor and chemoreceptor reflex pathways. This approach has allowed the simultaneous and direct identification of polysynaptically activated cells along these reflex pathways. An extensive number of immunopositive cells within the medulla oblongata was observed only in animals receiving some form of CSN afferent input. Except for the vestibular nucleus, electrical stimulation of the CSN in

21 anesthetized animals or hypoxic stimulation in anesthetized or unanesthetized animals induced similar patterns of Fos-LI within the rat medulla. Labeled cells within the medial vestibular nucleus were observed only in animals exposed to prolonged anesthesia and appeared to be associated with the induction and/or maintenance of the anesthetized state. Surgical preparations alone did not reproduce the pattern of Fos-LI observed in stimulated animals. In all surgically prepared animals we minimized the chance of Fos-LI due to unintentional stimulation of the carotid bodies by maintaining hyperoxic conditions during the surgical preparations. Despite adequate anesthesia, however, we observed a small number of immunopositivc cells within the NTS and VLM of surgically prepared control animals. These cells represented only a small proportion of the immunopositive cells observed within the NTS and VLM of surgically prepared and stimulated animals. This labeling was likely related to the surgical procedures rather than constitutive expression of Foslike protein, since animals receiving neither surgical preparations nor CSN afferent stimulation were virtually devoid of labeled cells. Nociceptive input is well known to evoke changes in respiration and blood pressure 42'49's°. In an extensive study of Fos expression in response to peripheral noxious stimulation, Bullitt4 invariably observed Fos labeling within the NTS and VLM of lightly anesthetized rats and considered this labeling to result from a secondary response to the noxious stimulus. The residual Fos-LI we observed may therefore have resulted from nociceptive input due to the surgical preparation of these control animals. Unstimulated control animals (sacrificed immediately) and animals exposed to normoxia within environmental chambers were either devoid of or showed a drastically reduced level of Fos-LI, except within the cochlear nucleus. Labeling within this nucleus appeared to occur to the same extent in all stimulated and control animals. Thus, we considered it unrelated to CSN input, but it served as an important positive internal control for the primary antibody labeling. Taken together, the results of all stimulation and control experiments are evidence that the Fos-LI we observed within the rat medulla was induced by stimulation of CSN afferent fibers. We have not yet addressed the issue of whether the observed pattern of Fos-LI in stimulated animals may be obtained solely through activation of CSN afferent fibers. Selective electrical stimulation of the CSN in animals breathing an hyperoxic gas mixture clearly produced a pattern of labeling similar to animals receiving a more physiological stimulation of CSN afferent fibers via hypoxia. The electrical stimulation frequencies used (25 or 50 Hz) are within the range of discharge rates recorded from single

CSN afferent fibers in cat, dog and rabbit a'lS'24,4a. We cannot, however, rule out the possibility that other types of stimulation which result in an increase in respiratory activity might also produce a similar overall pattern of Fos-LI. Our results are comparable with previous mapping studies which consistently demonstrate the presence of CSN-responsive cells within NTS 2's'10'11'21'25'26 and the ventrolateral aspects of the medulla, particularly in the vicinity of nucleus ambiguus 2's'21'25'28. Both of these areas are traditionally associated with regulation of respiratory and cardiovascular function. Our data also support the observations of Miura and Reis a4 who reported a long latency input from the CSN to nucleus raphe pallidus. Localization of CSN-responsive cells within the region we refer to as the parapyramidal cell group have not been reported previously. Contrary to earlier electrophysiological studies, we did not observe CSN-responsive cells within nucleus raphe obscurus 21'34, the hypoglossal nucleus 31, the 'parahypoglossal area' just ventrolateral to the hypoglossal nucleus 25 or the paramedian reticular formation 7'21'34. Our results cannot exclude these areas as possible sites of CSN input, since the absence of Fos-labeled cells does not necessarily indicate an absence of neuronal activation. The reason for these differences is unclear. It is important to emphasize that the present study was performed in rat whereas the studies cited above used cat as an experimental model. There is thus a possibility of species differences in the locations of CSN-responsive cells within the medulla. Moreover, there is not complete agreement concerning the locations of CSN-rcsponsive cells in cat. For example, several electrophysiological studies25,46 have argued convincingly against a CSN input to the paramedian reticular formation and, apart from the hypoglossal nucleus, none of the areas listed above have been consistently reported as responsive to CSN input. In addition, Fos expression appears to be difficult to induce in large motoneurons. For example, both Hunt ct al. 22 and Bullitt 4 could detect Fos labeling in cells of the dorsal horn of the spinal cord, but not in motor neurons of the ventral horn in response to noxious stimulation of the hindlimb despite repeated withdrawal reflexes. The minimum requirements for Fos expression within neurons is not known. Some cells within specific neural pathways do not appear to express Fos protein in response to neuronal activation 4'22. This may be due to a true absence of Fos expression within these cells. Alternatively, Fos protein may be expressed, but at a level below the detection limits of current immunohistochemical techniques. In addition, the stimulus requirements for Fos expression in different neuronal cell types may

22 not necessarily be the same. Results from recent studies, however, indicate a high degree of specificity of Fos labeling within the brain in response to discrete central and peripheral stimulation 4'12'22'35'4°. These authors suggest that activation of the c-los gene and expression of Fos protein may serve as a high resolution, though possibly incomplete, metabolic marker of specific polysynaptic neural pathways. Since the type of stimulation (electrical vs hypoxic) and the duration of the stimulation (10 min vs 3 h) were so different between experimental groups in this study, we deemed it inappropriate with the present protocols to strictly quantitate Fos-LI for statistical comparisons. Nevertheless, we did observe some differences in the rostrocaudal extent of labeled cells within specific cell groups. Although the overall pattern of Fos-LI was similar in all stimulated animals, immunopositive cells along the ventral surface of the medulla, especially within nucleus raphe pallidus and the parapyramidal cell group, were more numerous and began to appear at more caudal levels in unanesthetized animals. This difference could be due in some way to the anesthetized state. Anesthetics are known to inhibit Fos expression 12. Alternatively, the more prolonged hypoxic stimulation (3 h) may have been more effective in activating these cells than the relatively short (10 min) electrical stimulation. We made no attempt to normalize the electrical and hypoxic stimulations, and we cannot discount the possibility that this rostrocaudal difference was directly related to the difference in the type and duration of stimulation. It is important to realize that induced expression of Fos-like protein does not distinguish second-order from higher-order neurons. Both retrograde tracing and antidromic activation studies indicate that CSN-afferent fibers terminate monosynaptically within NTS. Termination sites appear to be predominantly ipsilateral; contralateral projections have been shown consistently only within the commissural subnucleus. We observed extensive bilateral Fos labeling within NTS, from the pyramidal decussation to the rostralmost level of the hypoglossal nucleus, in response to unilateral stimulation of the CSN. The symmetry of this labeling was striking and, rostral to the commissural subnucleus, implies at least a disynaptic connection from the stimulated side. There have been controversial reports of a monosynaptic projection of CSN-afferent fibers to the vicinity of nucleus ambiguus 8'9. Our data support the possibility of either a mono- or polysynaptic pathway to this region of the medulla. The present protocol did not allow us to distinguish between chemoreceptor and baroreceptor activation of second- and higher-order neurons. Afferent CSN fibers of similar size originate from both baroreceptors in the

carotid sinus and chemoreceptors in the carotid body 29. It was thus impossible to activate selectively either fiber type with electrical stimulation. Hypoxic stimulation of anesthetized animals clearly increased phrenic nerve discharge (Fig. 1B), indicating activation of the carotid bodies. The subsequent transient augmentation of blood pressure during the early recovery period, however, raises the possibility of baroreceptor activation as well. We did not monitor blood pressure or phrenic nerve activity in unanesthetized animals. The pattern of Fos-LI we have described, therefore, could have resulted from activation of both types of sensory fibers. Convergence of inputs from baroreceptor and chemoreceptor fibers onto single cells within NTS has been described 27. It is possible that a subset of the labeled cells we observed received afferent input from both sources. We have demonstrated Fos expression in cells within nucleus raphe pallidus and the parapyramidal cell group only in animals receiving stimulation of CSN afferent fibers. To our knowledge, this is the first documentation of a CSN input to the parapyramidal cell group. Other evidence, however, supports the suggestion that these cell groups are integrated into the baroreceptor and chemoreceptor reflex pathways. In rat, both nucleus raphe pallidus and the parapyramidal cell group contain a high concentration of serotonergic cells which project to NTS 47, the ventrolateral medulla TM and the intermediolateral cell column of the spinal cord 41'47. Both cell groups, therefore, appear to communicate with areas of the medulla involved in respiratory and cardiovascular control. Studies from our laboratory have shown that in cat, stimulation of CSN afferent fibers or point electrical stimulation of the caudal raphe nuclei activates an endogenous central serotonergic mechanism located in the brainstem which mediates a long-lasting facilitation of respiration 32'33. A similar mechanism also appears to exist in rat 14. The present results indicate that cells in both nucleus raphe pallidus and the parapyramidal cell group receive information transmitted through the CSN and therefore could conceivably mediate this long-lasting mechanism. The neuronal tracing technique we employed in these experiments allowed, in a single animal, detection of a population of CSN-responsive cells. The ability to study this population of functionally defined cells directly is an important step in their further characterization. Evidence linking cells within the baroreceptor and chemoreceptor reflex pathways with specific neurotransmitters or neuromodulators is indirect, and the direction of information flow between cells within these pathways is not certain. Combining CSN-specific Fos expression with immunohistochemical or in situ hybridization double labeling techniques, or conventional anterograde and retrograde

23

tract tracing techniques could provide answers to these questions. Both types of information would be invaluable for a better understanding of these important autonomic reflex pathways. Acknowledgements. The authors express their appreciation to

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