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Hypotension-induced expression of the c-fos gene in the medulla oblongata of piglets
BRAIN RESEARCH ELSEVIER
Brain Research 706 (1996) 199-209
Research report
Hypotension-induced expression of the c-los gene in the medulla oblongata of piglets D.A. Ruggiero a,b, S. Tong c, M. Anwar a, N. Gootman e, P.M. Gootman c,, a Department of Neurology and Neuroscience, Division ofNeurobiology, Cornell University College of Medicine, New York, NY 10021, USA b NeurologicalResearch Institute ofLubec, Lubec, ME 04652, USA c Department of Physiology, Box 31, SUN)" - Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2908, USA
Accepted 5 September 1995
Abstract Neural networks that mediate the reflex response to baroreceptor withdrawal were explored in Sus scrofa. Induction of c-fos was used as a monitor of synaptic activity in response to hypotension sustained by systemic administration of a peripheral vasodilator, sodium nitroprusside. Patterns of c-los gene expression were compared between Saffan-anesthetized experimental animals and age-matched normotensive controls administered vehicle. Effects of other variables were controlled including 1 h preoperative accommodation to the novel environment, anesthesia, blood gases and pH. Identical post-stimulus survival periods were allowed for accumulation of transcript. The c-los protein, Fos, was identified immunocytochemically with two rabbit antisera raised against amino acids 1-131 of Fos or residues 4-17 of synthetic human transcript. Fos was identified in catecholaminergic neurons labeled with an antiserum to tyrosine hydroxylase (TH). Fos was induced in the nucleus tractus solitarii (NTS) of hypotensive piglets. Neurons encoding Fos matched projection patterns of first order visceral afferents. Induction was prominent in the dorsolateral nucleus coinciding with the baroreceptor field. Indices of increased neuronal activity were evident in other baroreceptor terminal sites, e.g., medial subnucleus, the medial commissural field, the intermediate subnucleus and a ventral A2 noradrenergic area. In reticular formation c-los protein was induced in circumscribed columns in the lateral tegmental field (LTF) extending from facial nucleus to calamus scriptorius. Catecholaminergic (TH-positive) neurons expressed Fos in the porcine C1 and A1 areas of ventrolateral medulla. Fos was also induced in a dorsal intermediate reticular zone of LTF. Minor or inconsistent differences between experimental and control were observed in nucleus raphe pallidus, rostral paramedian reticular formation, upper thoracic intermediolateral cell column, and stellate ganglia. In conclusion, baroreceptor withdrawal in young animals induced patterns of neuronal response along established cardiovascular reflex pathways. Keywords: Neonate; Blood pressure; Immediate-early response gene; Functional activity; Gene induction; Autonomic nervous system; Catecholamine
1. Introduction Swine have been shown to be an excellent model for human development [30,54] and the study of the possible role of autonomic, i.e., sympathetic, innervation in the etiology of Sudden Infant Death Syndrome [25,61]. In addition, we have shown that the piglet model is well-suited for the study o f baroreceptor reflex alterations of sympathetic nerve activity (cf. [7,26]), At one month, coherence between sympathetic outflows is present in this model [25] and responses to complex afferent inputs can be evoked, e.g., to the Valsalva maneuver [23,24]. It was important,
* Corresponding author. Fax: (1) (718) 270-3103; e-mail: [email protected]. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0006- 8993(95 )01173-0
therefore, to determine whether the immediate early gene (IEG), c-fos could be used in the piglet, Sus scrofa, to identify specific brainstem regions with increased activity during experimental inhibition of baroreceptor afferents. l E G expression is being increasingly employed as an effective and specific marker for brain regions activated following physiological stimulation [45]. The value of this technique is resolution at the level of the single neuron of circuit-specific patterns of increased functional activity coupled to gene transcription. The specificity of anatomical expression of c-los and other proto-oncogenes, e.g., the j u n family, modulated by extracellular stimuli is differential transcription by cell groups physiologically related to the stimulated site (cf. [35]). The signal transcription-coupling cascade invokes rapid and transient induction of transcriptional regulatory proteins that act as third messen-
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gets and control genetic programs downstream. Protein products encoded by c-los and c-jun form a heterodimer, F o s / J u n complex that translocates to the nucleus and interacts with the activated protein AP-1 D N A binding site - a motif required for basal and induced levels of gene expression [9,37]. Given the effectiveness of lEGs as monitors of the cellular response to altered functional activity, neurons were sought that express the c-los gene transcript after inhibition of baroreceptors in developing swine. Previous investigations have demonstrated neuronal expression of Fos in adult mammals in response to increases in arterial blood pressure or hypotension or perturbations in blood gases (e.g., [3,5,13,36,38-40,55,59]). The present report documents patterns of neuronal activation monitored by lEG expression in specific regions of the lower brainstem following sustained hypotension in the piglet. A double immunocytochemical labeling technique was used to demonstrate whether hypotension induces Fos in medullary catecholaminergic neurons implicated in other species in cardiovascular regulation [5,13,15,39,50,59]. Hypotension was produced by systemic administration of the peripheral vasodilator, Na nitroprusside. Effects on c-fos expression were compared to data from age-matched controls. The induction patterns were uncomplicated by changes in pH, pO 2 and pCO 2.
2. Materials and methods This study was approved by the Institutional Animal Care and Use Committee. All experimental procedures were in compliance with Federal and State regulations and
the Guide for the Use and Care of Laboratory Animals approved by the Council of the American Physiological Society.
2.1. Methodology for animal preparations Animals (n = 8; ages 11, 30 and 57 days, matched sexes) were subjected to identical preoperative accommodation periods of 1 h and initially anesthetized with Saffan (Pitman-Moore, 12 m g / k g , i.m.). An external jugular vein was cannulated for continuous infusion of Saffan at 6 - 1 2 m g / k g / h , i.v. during surgery. Thereafter, Saffan was maintained at 6 m g / k g / h , i.v. throughout the experiment. All animals were tracheotomized and artificially ventilated with 100% 02 and paralyzed with decamethonium Br (0.1 c c / k g , i.v.). Needle electrodes were placed subcutaneously to monitor Lead II of the electrocardiogram (ECG). A femoral artery was cannulated and the catheter advanced into the abdominal aorta for continuous monitoring of arterial blood pressure (AoP) and periodic (@30 min) determination of arterial blood gases and pH. A femoral vein was cannulated for infusion of sodium nitroprusside (Aldrich Chemical Co.) dissolved in 5% dextrose in saline in experimental hypotensive animals or 5% dextrose in saline in control animals. Both control and experimental animals were stabilized for 30 min following surgery and decrease of Saffan infusion, pH and pCO 2 were maintained within physiological range while pO 2 was > 100 mmHg. In experimental animals, hypotension was induced with infusion of Na nitroprusside. The infusion rate was adjusted to maintain an average AoP at 6 0 - 7 0 % of the baseline level (mean AoP decrease 3 0 - 4 4 mmHg) for 45
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Fig. 1. Original polygraph traces of ECG and arterial blood pressure (AoP) recorded simultaneouslyin two 30-day-oldlittermate piglets. Top set of traces: control animal (mean AoP = 102 mmHg, heart rate (HR) = 210 bpm); bottom set of traces: experimental (nitroprusside-infused) animal (NP-A). Panel A: section of record before nitroprusside infusion (NP-A: mean AoP = 103 mmHg, HR = 200 bpm); panel B: section of record obtained during infusion of nitroprusside in experimental piglet (NP-A mean AoP = 60 mmHg, HR = 240 bpm); panel C: section of record recorded during period following nitroprusside infusion (NP-A: mean AoP = 103 mmHg, HR = 200 bpm).
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m i n (Fig. 1). T h e a p p r o x i m a t e i n f u s i o n rate o f n i t r o p r u s side r a n g e d b e t w e e n 3 - 1 0 / ~ g / k g / m i n which was equivalent to 0 . 3 - 0 . 5 m l / m i n o f the 5 % d e x t r o s e s a l i n e s o l u t i o n in c o n t r o l a n i m a l s . A o P w a s a l l o w e d to r e t u r n to b a s e l i n e l e v e l s a n d m a i n t a i n e d for 3 - 4 h. T h e c o n t r o l a n i m a l s w e r e i n f u s e d w i t h 5 % d e x t r o s e in
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0.5 rnm Fig. 2. Camera lucida drawings of transverse sections of the medulla oblongata illustrating induction of Fos-like immunoreactivity (FLD in the nucleus tractus solitarii (NTS). Data from two 30-day-aid littermate piglets (Figs. 2-5). Fos-like immunopositive nuclei were limited to discrete subnuclei of the NTS, and prominent in the dorsal-lateral (d) and intermediate (i) subnuclei, and the medial (m), commissural and ventral (v) and interstitial (is) subnuclei; immediately bordering the dorsal motor vagal nucleus (X). The levels illustrated are rostral to obex (a), obex (b) and caudal pole of area postrema (c) immediately rostral to calamus scriptorius. Each symbol represents one labeled neuron per 35 /~m section in all camera lucida illustrations. Abbreviations: ap, area postrema; ecn, external cuneate nucleus; ic, nucleus intercalatus; ltf, lateral tegmental field; mlf, medial longitudinal fasciculus; nc, nucleus cuneatus; rig, nucleus gracilis; nps, nucleus parasolitarius; sp, nucleus subpostrema; stn, spinal trigeminal nucleus; stt, spinal trigeminal tract; tr, solitary tract; vl, ventrolateral subnucleus; IV, fourth ventricle; XII, hypoglossal nucleus.
Fig. 3. Bright field photomicrographs of transverse sections through porcine medulla demonstrate induction of Fos in the NTS. Largenumbers of neurons contain FLI in hypotensive animals (b, c) as compared to a paucity of immunolabeled nuclei in control (a). Arrows point to identical loci in the dorsal-lateral (dl) region on low (b) and higher power (c) photomicrographs. Note the Fos-positive nuclei in lateral aspect of dorsal motor vagal nucleus (dmx). Abbreviations listed in Fig. 2. Bar = 234/zm (a, b); 75 /xm (c). s a l i n e a n d the i n f u s i o n rate ( a n d v o l u m e ) m a t c h e d the i n f u s i o n p a r a m e t e r s u s e d in e x p e r i m e n t a l a n i m a l s . A o P w a s s t a b l e d u r i n g the e n t i r e p r o t o c o l (Fig. 1). E a c h g r o u p of control and experimental animals was sacrificed simultaneously.
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2.2. Methodology for the immunocytochemical detection of Fos Animals received an overdose of Na pentobarbital (70 m g / k g ) and were perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The medulla oblongata and spinal cord segments T1, 2 were removed and blocked. The stellate ganglia were removed in one experimental group (n = 2). Identical procedures were followed in control (n = 4) and experimental animals (n = 4). Tissue blocks were post-fixed for 2 - 3 h in individual glass vials containing 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) and cryoprotected overnight at 4°C in a solution of 10% sucrose in 0.1 M PBS. Frozen sections were cut on a sliding microtome at 3 5 / z m in the transverse plane and every 4th section was processed immunocytochemically for c-fos protein. Tissues from control and experimental animals were processed simultaneously in the same solutions in order to control for potential variability in immunocytochemistry. All incubations were carried out in separate test wells on a Thomas rotator table. Tissues were collected in 0.1 M PBS (pH 7.4) in spot test wells and washed in Tris-buffered saline (TBS) between each step. Non-specific binding sites were blocked by pre-incubating for 30 min in goat serum, diluted 1:30 in TBS. Thereafter, sections were incubated in primary whole rabbit antiserum raised against amino acids 1-131 of Fos diluted 1:10,000 and generously donated by Dr. Tom Curran (Roche Institute of Molecular Biology, NJ). In two experimental pairs, tissues were processed with a polyclonal antiserum diluted 1:1000 and obtained commercially (Oncogene Sciences). This antiserum was raised in rabbits against a synthetic N-terminal peptide fragment of human Fos with little homology to Fos-related antigens (FRAs). Neither antiserum is thought to recognize FRAs and both produced indistinguishable induction patterns. The antisera were diluted in TBS containing 1% goat serum, to which 0.2% Triton X-100 was added to facilitate tissue penetration. Tissues were incubated for 1 - 3 days in biotinylated goat anti-rabbit IgG secondary antibody (1:200, 3 0 - 4 5 min) and avidin-biotin peroxidase complex (1:100, 60 min) (Vector Labs, A B C Elite Kit). The bound peroxidase immunoreaction product was visualized by treating tissues with a substrate solution of the chromogen, diaminobenzidine (DAB) and hydrogen peroxide in TBS. Control sections were processed omitting incubation in primary antibody. Sections were washed, mounted, dehy-
drated, and coverslipped without counterstaining. Alternate sections were counterstained in thionin to reveal nuclear boundaries. Nuclei were also visualized on unstained immunoprocessed tissues examined with darkfield optics.
2.2.1. Data analysis for c-fos studies Data were analyzed with a Leitz microscope. Neurons that expressed Fos-like immunoreactivity (FLI) were plotted with a camera lucida and photographed on Kodak TMax 100 film. Nomenclature used followed that of Hopkins et al. [30] and Miller and Ruggiero [42].
2.3. Double immunocytochemical labeling procedure In two hypotensive piglets (11 and 30 days old) alternate sections were incubated overnight at 4°C in rabbit anti-Fos diluted 1:1000 (Oncogene Sciences), rinsed, transferred to biotinylated goat-antirabbit IgG secondary antibody (1:200, Vector Labs) followed by the A B C reagent as described above. The reaction product was visualized by incubating sections in hydrogen peroxidase and DAB with 0.01% NiC12 • 6 H 2 0 for intensification. Sections were then incubated overnight in a mouse monoclonal antiserum raised against tyrosine hydroxylase (TH) (Incstar) and biotinylated horse anti-mouse IgG (1:200, Vector Labs) followed by the A B C method and D A B / H 2 0 2 to visualize the reaction product.
3. Results
3.1. Localization of Fos-like immunoreactivity (FLI) Neurons expressing FLI in the porcine medulla and spinal cord exhibited wide variations in intensity of immunoreaction product ranging from high to low. FLI was restricted to nuclei in all areas and distinct from background levels. Constitutive Fos was expressed in both experimental and control animals in the medial and inferior vestibular nuclei and external cuneate nucleus and inconsistently in the cervicothoracic spinal cord and trigeminal complex. In hypotensive piglets nuclear expression of FLI was induced in the nucleus tractus solitarii (NTS) and lateral tegmental field (LTF). Equivalent sites in control animals were characterized by sparse and sporadic labeling for Fos confirming similar observations in c o n t r o l
Fig. 4. a, b: camera lucida drawings of transverse sections through the rostral medulla demonstrate striking induction of FLI in the rostral ventrolateral reticular formation (rvl) at levels of (a) the caudal pole of the facial nucleus (VII) and (b) nucleus ambiguus compact division (nac). c, d: intermediate medullary levels demonstrating induction of FLI in the caudal extension of the rostral ventrolateral reticular formation at the level of (c) the nucleus ambiguus semicompactdivision (nasc) and (d) nucleus ambiguus, loose formation (nal). Note in d that the majority of neurons exhibiting the gene-encoded protein form a diagonal band that traverses the lateral reticular-precerebellar relay nucleus (lrn). Abbreviations: dao, dorsal accessory olive; mao, medial accessory olive; ngcv, nucleus gigantocellularis pars ventralis; p, pyramid; pion, principal inferior olivary nucleus (dl, dorsal lamina; vl, ventral lamina); ro, raphe obscurus; rpa, raphe pallidus; st, subtrigeminal nucleus; stn, spinal trigeminal nucleus; stt, spinal trigeminal tract; XII, hypoglossal nerve.
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methoxyflurane-anesthetized rats [5]. In the NTS, enhanced nuclear expression was prominent at intermediate and caudal levels corresponding to the general visceral afferent division (Figs. 2 and 3). In hypotensive animals FLI was expressed by a diagonally elongate population of neurons in the dorsal (lateral) subnucleus of the NTS. At intermediate levels the Fos-positive nuclei were concentrated dorsomedially to the solitary tract. At caudal levels, through the calamus scriptorius, Fos-positive neurons in
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Fig. 5. Bright field photomicrographs demonstrate enhanced nuclear expression of FLI in neurons of the rostral ventrolateral medulla (rvl) in a hypotensive piglet (b, c) as compared to a control animal (a). Arrows point to the same locus for reference in b and c. Abbreviations: vms, ventral medullary surface. Bar = 234 /xm (a, b); 75 p m (c).
Fig. 6. Camera lucida drawings of transverse sections of the porcine ventrolateral medulla. Data from ll-day-old piglet (Figs. 6-8). Sections were doubled-labeled with antisera to Fos and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Catecholaminergic (TH-positive) neurons expressed Fos (asterisks) in response to baroreceptor withdrawal in two regions: the C1 adrenergic area (a) and A1 noradrenergic area (b) at rostral and caudal levels of the ventrolateral reticular formation, respectively. Abbreviations: cvlm, caudal ventrolateral medulla; lrn, lateral reticular nucleus; ltf, lateral tegmental field; rvlm, rostral ventrolateral medulla; vms, ventral medullary surface; VII, facial nucleus.
the dorsal subnucleus formed a thin, well circumscribed horizontal lamina lining the dorsal border of the NTS. A discrete population of nuclei expressed FLI in the ventral NTS. A large percentage surrounded the dorsal motor nucleus (DMX) and extended laterally and ventrally to the solitary tract. Expression of FLI was also increased in the intermediate subnucleus bordering the solitary tract, and centrally in the medial subnucleus. The subnuclei gelatinosus and centralis [53] were unlabeled. Small numbers of lightly labeled nuclei were detected in the DMX and caudal pole of the area postrema. Nuclei were sparsely labeled in the subpostremal region, primarily at rostral
D.A. Ruggiero et al. / Brain Research 706 (1996) 199-209
levels adjacent to obex. Equivalent loci were comparatively weakly immunolabeled for Fos or on most sections devoid of the gene-encoded protein in control animals. Hypotensive piglets also exhibited prominent nuclear expression of Fos in the reticular formation of the rostral ventrolateral medulla (RVLM) (Figs. 4 and 5). Neurons in these cell columns were characterized by low levels of FLI or devoid of the phosphoprotein in controls. A high concentration of nuclei that expressed FLI in the RVLM was bordered rostrally by the facial nucleus, subjacent to the nucleus ambiguus, and situated in proximity to the ventral medullary surface. At caudal levels adjacent to obex, clusters of Fos-immunoreactive nuclei were arranged in a diagonal band extending dorsally into the intermediate reticular zone and ventrally to the lateral reticular nucleus (Fig. 4d). Enhanced nuclear expression of Fos was detected in the rostral paramedian reticular formation, nucleus raphe pallidus, nucleus interstitiales hypoglossi and thoracic intermediolateral cell column in three hypotensive animals. Differences between experimental and control animals were not possible to discern in a fourth group. The stellate ganglia processed in the latter pair (30 days old) revealed clearcut neuronal expression of FLI in the hypotensive animal but not in the control.
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3.2. FLI in medullary catecholaminergic neurons Two populations of Fos-positive neurons contained TH in the LTF. Double-labeled cells were identified in: (1)
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Fig. 7. Camera lucida drawing of a transverse section of medulla through the porcine nucleus tractus solitarii (NTS). Double-immunocytochemical staining with antisera to Fos and TH revealed few dually-labeled neurons in the A2 (ventral) or C2 (dorsal) areas of NTS or the C3 area (not illustrated). Each symbol represents one labeled neuron per 35 /zm tissue section. Two double-labeled neurons are indicated by an asterisk. These data indicate that hypotension induces c-los expression predominantly within non-catecholaminergic neurons of the neonatal porcine NTS. Abbreviations listed in Fig. 2.
Fig. 8. Photomicrographs of Fos-immunoreactivity in catecholaminergic (TH-positive) neurons in the C1 adrenergic area (a) adjacent to the facial nucleus and the A1 noradrenergic area (b) adjacent to the lateral reticular nucleus. Arrows point to double-labeled cells. (c) Single labeled TH-immunoreactive perikaryon (arrow) and Fos-immunoreactive nuclei (small arrows) in A2 area of NTS. Bar = 25 ~ m (a) and 50 /xm (b, c).
The rostral pole of the C1 cell column at a level of the RVLM bordering the facial nucleus (Fig. 6a and Fig. 8a), and (2) the A1 area at levels of the caudal ventrolateral reticular formation between the obex and calamus scriptorius (Fig. 6b and Fig. 8b). In the NTS and C3 area of the rostral dorsomedial medulla neurons containing TH or FLI
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formed spatially-segregated populations (Figs. 7 and 8c). A few double-labeled neurons (1 or 2 cells per section) were identified in the ventral subnucleus of NTS bordering the DMX.
4. Discussion c-fos expression was induced in regions of the medulla oblongata known to subserve cardiovascular reflex function. Induction of c-los protein was localized to the general visceral afferent division of the NTS and targets of integrated afferent projection in the medullary reticular formation [46,51,53]. Induction of Fos in the dorsolateral NTS of hypotensive animals was consistent with evidence from other species that this subdivision is a principal recipient of baroreceptor afferents [6,12,41,44]. 4.1. Methodological considerations The effects of environmental stress, anesthesia and nitroprusside on gene expression were precluded by our control data. In partial support was the paucity of neurons that expressed FLI in control animals administered vehicle in lieu of nitroprusside. The sole variables differentiating experimental from control animals was the induction of hypotension with the vasodilator, nitroprusside. Nitroprusside exerts actions on the peripheral vasculature but does not cross the blood-brain barrier or act centrally [32,34]. Thus a direct action of this vasodilator on the c-los induction patterns was unlikely. Since the average fall in arterial blood pressure was well within the autoregulatory range [16,28], alterations in cerebral blood flow did not contribute to the increases in c-fos protein expression. Neither nitric oxide nor the chemoreceptor stimulant, cyanide formed from nitroprusside, induced Fos in response to a similar cardiovascular challenge in rabbits [39]. Our anesthetic, Saffan, promotes anesthesia yet without significant effects on hindbrain blood flow [10], depression of phrenic high frequency oscillations or compromise of cardiovascular function [18]. Another advantage of Saffan is suggested by evidence of sparse, sporadic labeling for Fos in age-matched controls administered identical doses of anesthetic over the same time course. Unlike other anesthetics, e.g., urethane or c~-chloralose [60], Saffan provoked minimal induction of c-los in regions of the area postrema or reticular formation that demonstrated striking increases in FLI following hypotension or other homeostatic challenges, e.g., toxic insult [42].
4.2. Patterns of c-fos gene expression in hypotensive swine Neurons in the NTS that expressed the immediate-early gene encoded protein matched in distribution, projections of primary cardiovascular afferents (cats: [12]; rats: [6,31]). Sites inferred as functionally-activated in hypotensive
swine: dorsal-lateral, medial and commissural subnuclei, demonstrated increases in FLI in response to carotid sinus [14] or aortic depressor [41] nerve stimulation. The medial commissural division of NTS also demonstrated enhanced FLI. Data in the cat [12] and rat [51] suggest that this subnucleus receives input preferentially from chemoreceptors although afferents from baroreceptors have also been described [6]. Two possible explanations may account for induction in cells characterized in other adult animals as the 'chemoreceptor strip' of NTS. Although our animals were not hypoxic as revealed by measurement of blood gases, compromised perfusion of chemoreceptors due to experimentally induced falls in aortic pressure might have caused localized ischemia. This interpretation is supported by our observations that systemic blood gases and pH were unchanged in the hypotensive animals and equivalent to values in control littermates. An alternative interpretation is that the neurons expressing Fos are not recipients of chemoreceptor input. Sites that harbor noradrenergic neurons in the porcine A2 area of the NTS [48] also demonstrated striking induction of Fos as compared to controls. That catecholaminergic neurons may have responded to the stimulus was initially suggested by overlap with neurons that express catecholamine-synthesizing enzymes in piglets [48]. Also in support was direct evidence of c-los gene expression in catecholaminergic neurons in the A2 and C2 areas following nitroprusside-induced hypotension in the adult rat [5] and rabbit [39]. Neurons that revealed Fos expression in the porcine NTS were recognized as predominantly noncatecholaminergic in our dual labeling experiments. A technical artifact was precluded by our observation of numerous double-labeled, F o s / T H positive neurons in the C1 and A1 areas of the ventrolateral medulla. This served as an internal control of our experimental procedures. A question to emerge from these observations is whether the discrepancy is related to a species difference or protracted development of the catecholamine phenotype in the piglet A2 area. Expression of Fos in the NTS suggests that nuclear signaling mechanisms were activated in response to baroreceptor withdrawal, an observation consistent with similar findings in the conscious rabbit [39] and anesthetized rat [5,13]. The specificity of our findings was supported by an entirely different distribution pattern of c-fos expression encompassing the area postrema, the nucleus subpostrema and medial parvicellular division of NTS in response to hypovolemia [5], activation of visceral osmoreceptors [33] or vomiting induced by the chemotherapeutic agent, cisplatin and other emetics [42]. Induction of FLI was also observed in neurons of the reticular formation. Significant increases in FLI were observed in the RVLM in hypotensive piglets. This observation is consistent with evidence of induction in equivalent regions in other mammals in response to hemorrhage or nitroprusside or hydralazine infusion [5,13,40,59]. The
D.A. Ruggiero et al. / Brain Research 706 (1996) 199-209
markedly lower densities of FLI in the C1 vasopressor area of RVLM [50] following phenylephrine-induced hypertension [39,44] support the idea that Fos may not be expressed by neurons which are reflexively inhibited. Enhanced expression in the RVLM might underlie the role these neurons play in maintaining sympathetic neuronal discharge. The distribution pattern of neurons expressing FLI in the RVLM corresponded to the origins of neurons that are pulse modulated, barosensitive and which are thought to generate basal levels of sympathetic nerve discharge in other species [29,58]. Our observations are also in accord with evidence that a large fraction of Fos-positive neurons in hypotensive animals are catecholaminergic and belong to the C1 cell group [5,39]. A population of neurons demonstrating indices of increased cellular activity in the present study contained TH and corresponded to the porcine C1 cell column previously identified as adrenergic [48]. Induction of gene expression in a region of RVLM equivalent to the C1 vasopressor area in rats [50] may have been driven by disinhibition of local intrareticular (GABAergic) afferents to the RVLM [49]. In support of this hypothesis is evidence that disinhibition with a GABA A receptor agonist of the RVLM produced increases in arterial blood pressure and c-los expression in the C1 area [43]. Neurons at levels of reticular formation caudal to the RVLM are critical in mediating baroreceptor reflex inhibition of the RVLM and in consequence decreases in sympathetic nerve discharge [27]. Arrays of neurons were identified in an equivalent (ventral) region of LTF in the hypotensive piglet, which allows speculation that disinhibition may have driven transcriptional induction of c-los. Since GABAergic neurons are also present in the C1 vasopressor area of RVLM [52], Minson et al. [43] hypothesized that a source of the local inhibition of sympathetic drive may also derive from neurons intermingled with sympathetic bulbospinal populations. In support are our earlier observations that a GABA-agonist injected in the C1 area or applied to the ventral subpial surface decreased arterial blood pressure [47]. Given the controversy as to the origin of sympathetic outflow and location of the generator of sympathetic outflow between the RVLM and dorsal LTF [1,2,4,1922,29,47,58], our results would support the theory of at least two regions involved incardiovascular regulation and generation of sympathetic outflow, i.e. both the C1 alea of the RVLM and a dorsal region of the LTF. Both regions of the piglet brainstem showed evidence of activation in response to experimentally induced hypotension. At least two theories explicitly agree that the RVLM is involved in generating sympathetic nerve discharge and cardiovascular regulation [1,2,4,29,47,49,51,58,62]. There is evidence for adrenaline containing cells in this region which project axons to spinal sympathetic preganglionic neurons in the thoracolumbar intermediolateral cell column (IML) [47,50]. One theory places 'endogenous bursters'
207
(pacemaker cells with pulse-synchronized discharge) within the RVLM [29]. The other theory claims the origin of sympathetic nerve discharge is derived from two groups of brainstem neurons, i.e. non-linear oscillators, one in the RVLM and the other in the LTF [1,2,58]. Yet another attempts to integrate and create a hybrid model for generation of the sympathetic discharge patterns [62]. This theory suggests that the 'pacemakers' fire tonically in a chaotic fashion and are linked with neural circuits shaping the patterns observed in sympathetic activity. Enhanced FLI in the stellate ganglia is consistent with anatomical and physiological evidence that the vasopressor region of the RVLM gives rise to direct sympathoexcitatory projections to the IML [47,50,58]. Hemorrhage-induced hypotension in the cat provided no evidence of induction in the IML [40]. Sparse and inconsistent induction of Fos was observed in the IML of hypotensive piglets. Li and Dampney [39] propose that significant induction of gene-encoded protein in visceral as in somatic motoneurons may require relatively longer post-stimulus survival intervals. Minson et al. [43] observed after 120 min intervals, shorter than those allowed here, that disinhibition of the rostral ventral medulla evoked 3- to 5-fold increases in the incidence of Fos-immunoreactive neurons in the IML of rabbits. The nature of the extracellular stimuli required for c-los expression in visceromotor neurons as well as other experimental variables require systematic exploration. Enhanced expression of FLI was also identified in the A1 region of the CVLM in the hypotensive piglet. Neurons containing TH were identified in the A1 area in our previous immunocytochemical study of neonatal swine [48] and demonstrated induction of Fos in response to nitroprusside-induced hypotension in conscious rabbits [39]. c-los gene induction in catecholaminergic neurons of the porcine A1 nucleus was confirmed by our work. In other species A1 noradrenergic neurons in the caudal ventrolateral medulla ascend to and mediate vagal excitation of magnocellular neurosecretory neurons of the hypothalamus [8,11]. In the rat disproportionately greater increases in Fos in A1 neurons occurred in response to hypovolemia (hemorrhagic) than isovolemic (nitroprusside-evoked) hypotension [5]. Induction of c-los expression after nitroprusside or hemorrhage, however, was reported in neurons synthesizing arginine vasopressin (AVP) in hypothalamus and A1 neurons that modulate their neurosecretory activity [39,56,57]. AVP released from terminals in the C1 area restores arterial pressure in response to hemorrhage via a V1 receptor mechanism [17]. Whether a similar mechanism operates in our experimental model requires more extensive analysis.
Acknowledgements This study was supported by NIH grants HL-20864 (P.M.G.), HD-28931 (P.M.G.), HL18974 and NS-28200
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(D.A.R.). The authors would like to thank Mr. Isaac Frasier and Mrs. Susan Ingenito for their skilled technical support.
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DA. Ruggiero et al. / Brain Research 706 (1996) 199-209 [39] Li, Y.-W. and Dampney, R.A.L., Expression of Fos-like protein in brain following sustained hypertension and hypotension in conscious rabbits, Neuroscience, 61 (1994) 613-634. [40] McAllen, R.M., Badoer, E., Shafton, A.D., Oldfield, B.J. and McKinley, M.J., Hemorrhage induces c-los immunoreactivity in spinally projecting neurons of cat subretrofacial nucleus, Brain Res., 575 (1992) 329-332. [41] McKitrick, M.J., Krukoff, T.L. and Calaresu, F.R., Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve, Brain Res., 599 (1992) 215-222. [42] Miller, A.D. and Ruggiero, D.A., Emetic reflex arc revealed by expression of the immediate-early gene c-los in the cat, J. Neurosci., 14 (1994) 871-888. [43] Minson, J.B., Llewellyn-Smith, l.J., Arnolda, L.F., Pilowsky, P.M., Oliver, J.R. and Chalmers, J.P., Disinhibition of the rostral ventral medulla increases blood pressure and FOS expression in bulbospinal neurons, Brain Res., 646 (1994) 44-52. [44] Miura, M., Takayama, K. and Okada, J., Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation, J. Auton. Nerv. Syst., 50 (1994) 31-43. [45] Morgan, J.I. and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci., 1 (1991) 421-251. [46] Mtui, E.P., Anwar, M., Reis, D.J. and Ruggiero, D.A., Medullary visceral reflex circuits: Local afferents to nucleus tractus solitarii synthesize catecholamines and project to thoracic spinal cord, J. Comp. Neurol., 351 (1995) 5-26. [47] Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. [48] Ruggiero, D.A., Anwar, M. and Gootman, P.M., Presumptive adrenergic neurons containing phenylethanolamine N-methyltransferase immunoreactivity in the medulla oblongata of neonatal swine, Brain Res., 583 (1992) 105-119. [49] Ruggiero, D.A., Cravo, S.L., Arango, V. and Reis, D.J., Central control of the circulation by the rostral ventrolateral reticular nucleus: Anatomical substrates. In J. Ciriello, C. Polosa and C. Caverson (Eds.), Progress in Brain Research, Elsevier, Amsterdam, 1989, pp. 49-79. [50] Ruggiero, D.A., Cravo, S.L., Golanov, E., Gomez, R., Anwar, M. and Reis, D.J., Adrenergic and non-adrenergic spinal projections of
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Brain Research 706 (1996) 199-209
Research report
Hypotension-induced expression of the c-los gene in the medulla oblongata of piglets D.A. Ruggiero a,b, S. Tong c, M. Anwar a, N. Gootman e, P.M. Gootman c,, a Department of Neurology and Neuroscience, Division ofNeurobiology, Cornell University College of Medicine, New York, NY 10021, USA b NeurologicalResearch Institute ofLubec, Lubec, ME 04652, USA c Department of Physiology, Box 31, SUN)" - Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2908, USA
Accepted 5 September 1995
Abstract Neural networks that mediate the reflex response to baroreceptor withdrawal were explored in Sus scrofa. Induction of c-fos was used as a monitor of synaptic activity in response to hypotension sustained by systemic administration of a peripheral vasodilator, sodium nitroprusside. Patterns of c-los gene expression were compared between Saffan-anesthetized experimental animals and age-matched normotensive controls administered vehicle. Effects of other variables were controlled including 1 h preoperative accommodation to the novel environment, anesthesia, blood gases and pH. Identical post-stimulus survival periods were allowed for accumulation of transcript. The c-los protein, Fos, was identified immunocytochemically with two rabbit antisera raised against amino acids 1-131 of Fos or residues 4-17 of synthetic human transcript. Fos was identified in catecholaminergic neurons labeled with an antiserum to tyrosine hydroxylase (TH). Fos was induced in the nucleus tractus solitarii (NTS) of hypotensive piglets. Neurons encoding Fos matched projection patterns of first order visceral afferents. Induction was prominent in the dorsolateral nucleus coinciding with the baroreceptor field. Indices of increased neuronal activity were evident in other baroreceptor terminal sites, e.g., medial subnucleus, the medial commissural field, the intermediate subnucleus and a ventral A2 noradrenergic area. In reticular formation c-los protein was induced in circumscribed columns in the lateral tegmental field (LTF) extending from facial nucleus to calamus scriptorius. Catecholaminergic (TH-positive) neurons expressed Fos in the porcine C1 and A1 areas of ventrolateral medulla. Fos was also induced in a dorsal intermediate reticular zone of LTF. Minor or inconsistent differences between experimental and control were observed in nucleus raphe pallidus, rostral paramedian reticular formation, upper thoracic intermediolateral cell column, and stellate ganglia. In conclusion, baroreceptor withdrawal in young animals induced patterns of neuronal response along established cardiovascular reflex pathways. Keywords: Neonate; Blood pressure; Immediate-early response gene; Functional activity; Gene induction; Autonomic nervous system; Catecholamine
1. Introduction Swine have been shown to be an excellent model for human development [30,54] and the study of the possible role of autonomic, i.e., sympathetic, innervation in the etiology of Sudden Infant Death Syndrome [25,61]. In addition, we have shown that the piglet model is well-suited for the study o f baroreceptor reflex alterations of sympathetic nerve activity (cf. [7,26]), At one month, coherence between sympathetic outflows is present in this model [25] and responses to complex afferent inputs can be evoked, e.g., to the Valsalva maneuver [23,24]. It was important,
* Corresponding author. Fax: (1) (718) 270-3103; e-mail: [email protected]. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0006- 8993(95 )01173-0
therefore, to determine whether the immediate early gene (IEG), c-fos could be used in the piglet, Sus scrofa, to identify specific brainstem regions with increased activity during experimental inhibition of baroreceptor afferents. l E G expression is being increasingly employed as an effective and specific marker for brain regions activated following physiological stimulation [45]. The value of this technique is resolution at the level of the single neuron of circuit-specific patterns of increased functional activity coupled to gene transcription. The specificity of anatomical expression of c-los and other proto-oncogenes, e.g., the j u n family, modulated by extracellular stimuli is differential transcription by cell groups physiologically related to the stimulated site (cf. [35]). The signal transcription-coupling cascade invokes rapid and transient induction of transcriptional regulatory proteins that act as third messen-
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gets and control genetic programs downstream. Protein products encoded by c-los and c-jun form a heterodimer, F o s / J u n complex that translocates to the nucleus and interacts with the activated protein AP-1 D N A binding site - a motif required for basal and induced levels of gene expression [9,37]. Given the effectiveness of lEGs as monitors of the cellular response to altered functional activity, neurons were sought that express the c-los gene transcript after inhibition of baroreceptors in developing swine. Previous investigations have demonstrated neuronal expression of Fos in adult mammals in response to increases in arterial blood pressure or hypotension or perturbations in blood gases (e.g., [3,5,13,36,38-40,55,59]). The present report documents patterns of neuronal activation monitored by lEG expression in specific regions of the lower brainstem following sustained hypotension in the piglet. A double immunocytochemical labeling technique was used to demonstrate whether hypotension induces Fos in medullary catecholaminergic neurons implicated in other species in cardiovascular regulation [5,13,15,39,50,59]. Hypotension was produced by systemic administration of the peripheral vasodilator, Na nitroprusside. Effects on c-fos expression were compared to data from age-matched controls. The induction patterns were uncomplicated by changes in pH, pO 2 and pCO 2.
2. Materials and methods This study was approved by the Institutional Animal Care and Use Committee. All experimental procedures were in compliance with Federal and State regulations and
the Guide for the Use and Care of Laboratory Animals approved by the Council of the American Physiological Society.
2.1. Methodology for animal preparations Animals (n = 8; ages 11, 30 and 57 days, matched sexes) were subjected to identical preoperative accommodation periods of 1 h and initially anesthetized with Saffan (Pitman-Moore, 12 m g / k g , i.m.). An external jugular vein was cannulated for continuous infusion of Saffan at 6 - 1 2 m g / k g / h , i.v. during surgery. Thereafter, Saffan was maintained at 6 m g / k g / h , i.v. throughout the experiment. All animals were tracheotomized and artificially ventilated with 100% 02 and paralyzed with decamethonium Br (0.1 c c / k g , i.v.). Needle electrodes were placed subcutaneously to monitor Lead II of the electrocardiogram (ECG). A femoral artery was cannulated and the catheter advanced into the abdominal aorta for continuous monitoring of arterial blood pressure (AoP) and periodic (@30 min) determination of arterial blood gases and pH. A femoral vein was cannulated for infusion of sodium nitroprusside (Aldrich Chemical Co.) dissolved in 5% dextrose in saline in experimental hypotensive animals or 5% dextrose in saline in control animals. Both control and experimental animals were stabilized for 30 min following surgery and decrease of Saffan infusion, pH and pCO 2 were maintained within physiological range while pO 2 was > 100 mmHg. In experimental animals, hypotension was induced with infusion of Na nitroprusside. The infusion rate was adjusted to maintain an average AoP at 6 0 - 7 0 % of the baseline level (mean AoP decrease 3 0 - 4 4 mmHg) for 45
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Fig. 1. Original polygraph traces of ECG and arterial blood pressure (AoP) recorded simultaneouslyin two 30-day-oldlittermate piglets. Top set of traces: control animal (mean AoP = 102 mmHg, heart rate (HR) = 210 bpm); bottom set of traces: experimental (nitroprusside-infused) animal (NP-A). Panel A: section of record before nitroprusside infusion (NP-A: mean AoP = 103 mmHg, HR = 200 bpm); panel B: section of record obtained during infusion of nitroprusside in experimental piglet (NP-A mean AoP = 60 mmHg, HR = 240 bpm); panel C: section of record recorded during period following nitroprusside infusion (NP-A: mean AoP = 103 mmHg, HR = 200 bpm).
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201
m i n (Fig. 1). T h e a p p r o x i m a t e i n f u s i o n rate o f n i t r o p r u s side r a n g e d b e t w e e n 3 - 1 0 / ~ g / k g / m i n which was equivalent to 0 . 3 - 0 . 5 m l / m i n o f the 5 % d e x t r o s e s a l i n e s o l u t i o n in c o n t r o l a n i m a l s . A o P w a s a l l o w e d to r e t u r n to b a s e l i n e l e v e l s a n d m a i n t a i n e d for 3 - 4 h. T h e c o n t r o l a n i m a l s w e r e i n f u s e d w i t h 5 % d e x t r o s e in
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0.5 rnm Fig. 2. Camera lucida drawings of transverse sections of the medulla oblongata illustrating induction of Fos-like immunoreactivity (FLD in the nucleus tractus solitarii (NTS). Data from two 30-day-aid littermate piglets (Figs. 2-5). Fos-like immunopositive nuclei were limited to discrete subnuclei of the NTS, and prominent in the dorsal-lateral (d) and intermediate (i) subnuclei, and the medial (m), commissural and ventral (v) and interstitial (is) subnuclei; immediately bordering the dorsal motor vagal nucleus (X). The levels illustrated are rostral to obex (a), obex (b) and caudal pole of area postrema (c) immediately rostral to calamus scriptorius. Each symbol represents one labeled neuron per 35 /~m section in all camera lucida illustrations. Abbreviations: ap, area postrema; ecn, external cuneate nucleus; ic, nucleus intercalatus; ltf, lateral tegmental field; mlf, medial longitudinal fasciculus; nc, nucleus cuneatus; rig, nucleus gracilis; nps, nucleus parasolitarius; sp, nucleus subpostrema; stn, spinal trigeminal nucleus; stt, spinal trigeminal tract; tr, solitary tract; vl, ventrolateral subnucleus; IV, fourth ventricle; XII, hypoglossal nucleus.
Fig. 3. Bright field photomicrographs of transverse sections through porcine medulla demonstrate induction of Fos in the NTS. Largenumbers of neurons contain FLI in hypotensive animals (b, c) as compared to a paucity of immunolabeled nuclei in control (a). Arrows point to identical loci in the dorsal-lateral (dl) region on low (b) and higher power (c) photomicrographs. Note the Fos-positive nuclei in lateral aspect of dorsal motor vagal nucleus (dmx). Abbreviations listed in Fig. 2. Bar = 234/zm (a, b); 75 /xm (c). s a l i n e a n d the i n f u s i o n rate ( a n d v o l u m e ) m a t c h e d the i n f u s i o n p a r a m e t e r s u s e d in e x p e r i m e n t a l a n i m a l s . A o P w a s s t a b l e d u r i n g the e n t i r e p r o t o c o l (Fig. 1). E a c h g r o u p of control and experimental animals was sacrificed simultaneously.
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2.2. Methodology for the immunocytochemical detection of Fos Animals received an overdose of Na pentobarbital (70 m g / k g ) and were perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The medulla oblongata and spinal cord segments T1, 2 were removed and blocked. The stellate ganglia were removed in one experimental group (n = 2). Identical procedures were followed in control (n = 4) and experimental animals (n = 4). Tissue blocks were post-fixed for 2 - 3 h in individual glass vials containing 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) and cryoprotected overnight at 4°C in a solution of 10% sucrose in 0.1 M PBS. Frozen sections were cut on a sliding microtome at 3 5 / z m in the transverse plane and every 4th section was processed immunocytochemically for c-fos protein. Tissues from control and experimental animals were processed simultaneously in the same solutions in order to control for potential variability in immunocytochemistry. All incubations were carried out in separate test wells on a Thomas rotator table. Tissues were collected in 0.1 M PBS (pH 7.4) in spot test wells and washed in Tris-buffered saline (TBS) between each step. Non-specific binding sites were blocked by pre-incubating for 30 min in goat serum, diluted 1:30 in TBS. Thereafter, sections were incubated in primary whole rabbit antiserum raised against amino acids 1-131 of Fos diluted 1:10,000 and generously donated by Dr. Tom Curran (Roche Institute of Molecular Biology, NJ). In two experimental pairs, tissues were processed with a polyclonal antiserum diluted 1:1000 and obtained commercially (Oncogene Sciences). This antiserum was raised in rabbits against a synthetic N-terminal peptide fragment of human Fos with little homology to Fos-related antigens (FRAs). Neither antiserum is thought to recognize FRAs and both produced indistinguishable induction patterns. The antisera were diluted in TBS containing 1% goat serum, to which 0.2% Triton X-100 was added to facilitate tissue penetration. Tissues were incubated for 1 - 3 days in biotinylated goat anti-rabbit IgG secondary antibody (1:200, 3 0 - 4 5 min) and avidin-biotin peroxidase complex (1:100, 60 min) (Vector Labs, A B C Elite Kit). The bound peroxidase immunoreaction product was visualized by treating tissues with a substrate solution of the chromogen, diaminobenzidine (DAB) and hydrogen peroxide in TBS. Control sections were processed omitting incubation in primary antibody. Sections were washed, mounted, dehy-
drated, and coverslipped without counterstaining. Alternate sections were counterstained in thionin to reveal nuclear boundaries. Nuclei were also visualized on unstained immunoprocessed tissues examined with darkfield optics.
2.2.1. Data analysis for c-fos studies Data were analyzed with a Leitz microscope. Neurons that expressed Fos-like immunoreactivity (FLI) were plotted with a camera lucida and photographed on Kodak TMax 100 film. Nomenclature used followed that of Hopkins et al. [30] and Miller and Ruggiero [42].
2.3. Double immunocytochemical labeling procedure In two hypotensive piglets (11 and 30 days old) alternate sections were incubated overnight at 4°C in rabbit anti-Fos diluted 1:1000 (Oncogene Sciences), rinsed, transferred to biotinylated goat-antirabbit IgG secondary antibody (1:200, Vector Labs) followed by the A B C reagent as described above. The reaction product was visualized by incubating sections in hydrogen peroxidase and DAB with 0.01% NiC12 • 6 H 2 0 for intensification. Sections were then incubated overnight in a mouse monoclonal antiserum raised against tyrosine hydroxylase (TH) (Incstar) and biotinylated horse anti-mouse IgG (1:200, Vector Labs) followed by the A B C method and D A B / H 2 0 2 to visualize the reaction product.
3. Results
3.1. Localization of Fos-like immunoreactivity (FLI) Neurons expressing FLI in the porcine medulla and spinal cord exhibited wide variations in intensity of immunoreaction product ranging from high to low. FLI was restricted to nuclei in all areas and distinct from background levels. Constitutive Fos was expressed in both experimental and control animals in the medial and inferior vestibular nuclei and external cuneate nucleus and inconsistently in the cervicothoracic spinal cord and trigeminal complex. In hypotensive piglets nuclear expression of FLI was induced in the nucleus tractus solitarii (NTS) and lateral tegmental field (LTF). Equivalent sites in control animals were characterized by sparse and sporadic labeling for Fos confirming similar observations in c o n t r o l
Fig. 4. a, b: camera lucida drawings of transverse sections through the rostral medulla demonstrate striking induction of FLI in the rostral ventrolateral reticular formation (rvl) at levels of (a) the caudal pole of the facial nucleus (VII) and (b) nucleus ambiguus compact division (nac). c, d: intermediate medullary levels demonstrating induction of FLI in the caudal extension of the rostral ventrolateral reticular formation at the level of (c) the nucleus ambiguus semicompactdivision (nasc) and (d) nucleus ambiguus, loose formation (nal). Note in d that the majority of neurons exhibiting the gene-encoded protein form a diagonal band that traverses the lateral reticular-precerebellar relay nucleus (lrn). Abbreviations: dao, dorsal accessory olive; mao, medial accessory olive; ngcv, nucleus gigantocellularis pars ventralis; p, pyramid; pion, principal inferior olivary nucleus (dl, dorsal lamina; vl, ventral lamina); ro, raphe obscurus; rpa, raphe pallidus; st, subtrigeminal nucleus; stn, spinal trigeminal nucleus; stt, spinal trigeminal tract; XII, hypoglossal nerve.
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methoxyflurane-anesthetized rats [5]. In the NTS, enhanced nuclear expression was prominent at intermediate and caudal levels corresponding to the general visceral afferent division (Figs. 2 and 3). In hypotensive animals FLI was expressed by a diagonally elongate population of neurons in the dorsal (lateral) subnucleus of the NTS. At intermediate levels the Fos-positive nuclei were concentrated dorsomedially to the solitary tract. At caudal levels, through the calamus scriptorius, Fos-positive neurons in
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Fig. 6. Camera lucida drawings of transverse sections of the porcine ventrolateral medulla. Data from ll-day-old piglet (Figs. 6-8). Sections were doubled-labeled with antisera to Fos and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Catecholaminergic (TH-positive) neurons expressed Fos (asterisks) in response to baroreceptor withdrawal in two regions: the C1 adrenergic area (a) and A1 noradrenergic area (b) at rostral and caudal levels of the ventrolateral reticular formation, respectively. Abbreviations: cvlm, caudal ventrolateral medulla; lrn, lateral reticular nucleus; ltf, lateral tegmental field; rvlm, rostral ventrolateral medulla; vms, ventral medullary surface; VII, facial nucleus.
the dorsal subnucleus formed a thin, well circumscribed horizontal lamina lining the dorsal border of the NTS. A discrete population of nuclei expressed FLI in the ventral NTS. A large percentage surrounded the dorsal motor nucleus (DMX) and extended laterally and ventrally to the solitary tract. Expression of FLI was also increased in the intermediate subnucleus bordering the solitary tract, and centrally in the medial subnucleus. The subnuclei gelatinosus and centralis [53] were unlabeled. Small numbers of lightly labeled nuclei were detected in the DMX and caudal pole of the area postrema. Nuclei were sparsely labeled in the subpostremal region, primarily at rostral
D.A. Ruggiero et al. / Brain Research 706 (1996) 199-209
levels adjacent to obex. Equivalent loci were comparatively weakly immunolabeled for Fos or on most sections devoid of the gene-encoded protein in control animals. Hypotensive piglets also exhibited prominent nuclear expression of Fos in the reticular formation of the rostral ventrolateral medulla (RVLM) (Figs. 4 and 5). Neurons in these cell columns were characterized by low levels of FLI or devoid of the phosphoprotein in controls. A high concentration of nuclei that expressed FLI in the RVLM was bordered rostrally by the facial nucleus, subjacent to the nucleus ambiguus, and situated in proximity to the ventral medullary surface. At caudal levels adjacent to obex, clusters of Fos-immunoreactive nuclei were arranged in a diagonal band extending dorsally into the intermediate reticular zone and ventrally to the lateral reticular nucleus (Fig. 4d). Enhanced nuclear expression of Fos was detected in the rostral paramedian reticular formation, nucleus raphe pallidus, nucleus interstitiales hypoglossi and thoracic intermediolateral cell column in three hypotensive animals. Differences between experimental and control animals were not possible to discern in a fourth group. The stellate ganglia processed in the latter pair (30 days old) revealed clearcut neuronal expression of FLI in the hypotensive animal but not in the control.
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Fig. 7. Camera lucida drawing of a transverse section of medulla through the porcine nucleus tractus solitarii (NTS). Double-immunocytochemical staining with antisera to Fos and TH revealed few dually-labeled neurons in the A2 (ventral) or C2 (dorsal) areas of NTS or the C3 area (not illustrated). Each symbol represents one labeled neuron per 35 /zm tissue section. Two double-labeled neurons are indicated by an asterisk. These data indicate that hypotension induces c-los expression predominantly within non-catecholaminergic neurons of the neonatal porcine NTS. Abbreviations listed in Fig. 2.
Fig. 8. Photomicrographs of Fos-immunoreactivity in catecholaminergic (TH-positive) neurons in the C1 adrenergic area (a) adjacent to the facial nucleus and the A1 noradrenergic area (b) adjacent to the lateral reticular nucleus. Arrows point to double-labeled cells. (c) Single labeled TH-immunoreactive perikaryon (arrow) and Fos-immunoreactive nuclei (small arrows) in A2 area of NTS. Bar = 25 ~ m (a) and 50 /xm (b, c).
The rostral pole of the C1 cell column at a level of the RVLM bordering the facial nucleus (Fig. 6a and Fig. 8a), and (2) the A1 area at levels of the caudal ventrolateral reticular formation between the obex and calamus scriptorius (Fig. 6b and Fig. 8b). In the NTS and C3 area of the rostral dorsomedial medulla neurons containing TH or FLI
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D.A. Ruggieroet al. /Brain Research 706 (1996)199-209
formed spatially-segregated populations (Figs. 7 and 8c). A few double-labeled neurons (1 or 2 cells per section) were identified in the ventral subnucleus of NTS bordering the DMX.
4. Discussion c-fos expression was induced in regions of the medulla oblongata known to subserve cardiovascular reflex function. Induction of c-los protein was localized to the general visceral afferent division of the NTS and targets of integrated afferent projection in the medullary reticular formation [46,51,53]. Induction of Fos in the dorsolateral NTS of hypotensive animals was consistent with evidence from other species that this subdivision is a principal recipient of baroreceptor afferents [6,12,41,44]. 4.1. Methodological considerations The effects of environmental stress, anesthesia and nitroprusside on gene expression were precluded by our control data. In partial support was the paucity of neurons that expressed FLI in control animals administered vehicle in lieu of nitroprusside. The sole variables differentiating experimental from control animals was the induction of hypotension with the vasodilator, nitroprusside. Nitroprusside exerts actions on the peripheral vasculature but does not cross the blood-brain barrier or act centrally [32,34]. Thus a direct action of this vasodilator on the c-los induction patterns was unlikely. Since the average fall in arterial blood pressure was well within the autoregulatory range [16,28], alterations in cerebral blood flow did not contribute to the increases in c-fos protein expression. Neither nitric oxide nor the chemoreceptor stimulant, cyanide formed from nitroprusside, induced Fos in response to a similar cardiovascular challenge in rabbits [39]. Our anesthetic, Saffan, promotes anesthesia yet without significant effects on hindbrain blood flow [10], depression of phrenic high frequency oscillations or compromise of cardiovascular function [18]. Another advantage of Saffan is suggested by evidence of sparse, sporadic labeling for Fos in age-matched controls administered identical doses of anesthetic over the same time course. Unlike other anesthetics, e.g., urethane or c~-chloralose [60], Saffan provoked minimal induction of c-los in regions of the area postrema or reticular formation that demonstrated striking increases in FLI following hypotension or other homeostatic challenges, e.g., toxic insult [42].
4.2. Patterns of c-fos gene expression in hypotensive swine Neurons in the NTS that expressed the immediate-early gene encoded protein matched in distribution, projections of primary cardiovascular afferents (cats: [12]; rats: [6,31]). Sites inferred as functionally-activated in hypotensive
swine: dorsal-lateral, medial and commissural subnuclei, demonstrated increases in FLI in response to carotid sinus [14] or aortic depressor [41] nerve stimulation. The medial commissural division of NTS also demonstrated enhanced FLI. Data in the cat [12] and rat [51] suggest that this subnucleus receives input preferentially from chemoreceptors although afferents from baroreceptors have also been described [6]. Two possible explanations may account for induction in cells characterized in other adult animals as the 'chemoreceptor strip' of NTS. Although our animals were not hypoxic as revealed by measurement of blood gases, compromised perfusion of chemoreceptors due to experimentally induced falls in aortic pressure might have caused localized ischemia. This interpretation is supported by our observations that systemic blood gases and pH were unchanged in the hypotensive animals and equivalent to values in control littermates. An alternative interpretation is that the neurons expressing Fos are not recipients of chemoreceptor input. Sites that harbor noradrenergic neurons in the porcine A2 area of the NTS [48] also demonstrated striking induction of Fos as compared to controls. That catecholaminergic neurons may have responded to the stimulus was initially suggested by overlap with neurons that express catecholamine-synthesizing enzymes in piglets [48]. Also in support was direct evidence of c-los gene expression in catecholaminergic neurons in the A2 and C2 areas following nitroprusside-induced hypotension in the adult rat [5] and rabbit [39]. Neurons that revealed Fos expression in the porcine NTS were recognized as predominantly noncatecholaminergic in our dual labeling experiments. A technical artifact was precluded by our observation of numerous double-labeled, F o s / T H positive neurons in the C1 and A1 areas of the ventrolateral medulla. This served as an internal control of our experimental procedures. A question to emerge from these observations is whether the discrepancy is related to a species difference or protracted development of the catecholamine phenotype in the piglet A2 area. Expression of Fos in the NTS suggests that nuclear signaling mechanisms were activated in response to baroreceptor withdrawal, an observation consistent with similar findings in the conscious rabbit [39] and anesthetized rat [5,13]. The specificity of our findings was supported by an entirely different distribution pattern of c-fos expression encompassing the area postrema, the nucleus subpostrema and medial parvicellular division of NTS in response to hypovolemia [5], activation of visceral osmoreceptors [33] or vomiting induced by the chemotherapeutic agent, cisplatin and other emetics [42]. Induction of FLI was also observed in neurons of the reticular formation. Significant increases in FLI were observed in the RVLM in hypotensive piglets. This observation is consistent with evidence of induction in equivalent regions in other mammals in response to hemorrhage or nitroprusside or hydralazine infusion [5,13,40,59]. The
D.A. Ruggiero et al. / Brain Research 706 (1996) 199-209
markedly lower densities of FLI in the C1 vasopressor area of RVLM [50] following phenylephrine-induced hypertension [39,44] support the idea that Fos may not be expressed by neurons which are reflexively inhibited. Enhanced expression in the RVLM might underlie the role these neurons play in maintaining sympathetic neuronal discharge. The distribution pattern of neurons expressing FLI in the RVLM corresponded to the origins of neurons that are pulse modulated, barosensitive and which are thought to generate basal levels of sympathetic nerve discharge in other species [29,58]. Our observations are also in accord with evidence that a large fraction of Fos-positive neurons in hypotensive animals are catecholaminergic and belong to the C1 cell group [5,39]. A population of neurons demonstrating indices of increased cellular activity in the present study contained TH and corresponded to the porcine C1 cell column previously identified as adrenergic [48]. Induction of gene expression in a region of RVLM equivalent to the C1 vasopressor area in rats [50] may have been driven by disinhibition of local intrareticular (GABAergic) afferents to the RVLM [49]. In support of this hypothesis is evidence that disinhibition with a GABA A receptor agonist of the RVLM produced increases in arterial blood pressure and c-los expression in the C1 area [43]. Neurons at levels of reticular formation caudal to the RVLM are critical in mediating baroreceptor reflex inhibition of the RVLM and in consequence decreases in sympathetic nerve discharge [27]. Arrays of neurons were identified in an equivalent (ventral) region of LTF in the hypotensive piglet, which allows speculation that disinhibition may have driven transcriptional induction of c-los. Since GABAergic neurons are also present in the C1 vasopressor area of RVLM [52], Minson et al. [43] hypothesized that a source of the local inhibition of sympathetic drive may also derive from neurons intermingled with sympathetic bulbospinal populations. In support are our earlier observations that a GABA-agonist injected in the C1 area or applied to the ventral subpial surface decreased arterial blood pressure [47]. Given the controversy as to the origin of sympathetic outflow and location of the generator of sympathetic outflow between the RVLM and dorsal LTF [1,2,4,1922,29,47,58], our results would support the theory of at least two regions involved incardiovascular regulation and generation of sympathetic outflow, i.e. both the C1 alea of the RVLM and a dorsal region of the LTF. Both regions of the piglet brainstem showed evidence of activation in response to experimentally induced hypotension. At least two theories explicitly agree that the RVLM is involved in generating sympathetic nerve discharge and cardiovascular regulation [1,2,4,29,47,49,51,58,62]. There is evidence for adrenaline containing cells in this region which project axons to spinal sympathetic preganglionic neurons in the thoracolumbar intermediolateral cell column (IML) [47,50]. One theory places 'endogenous bursters'
207
(pacemaker cells with pulse-synchronized discharge) within the RVLM [29]. The other theory claims the origin of sympathetic nerve discharge is derived from two groups of brainstem neurons, i.e. non-linear oscillators, one in the RVLM and the other in the LTF [1,2,58]. Yet another attempts to integrate and create a hybrid model for generation of the sympathetic discharge patterns [62]. This theory suggests that the 'pacemakers' fire tonically in a chaotic fashion and are linked with neural circuits shaping the patterns observed in sympathetic activity. Enhanced FLI in the stellate ganglia is consistent with anatomical and physiological evidence that the vasopressor region of the RVLM gives rise to direct sympathoexcitatory projections to the IML [47,50,58]. Hemorrhage-induced hypotension in the cat provided no evidence of induction in the IML [40]. Sparse and inconsistent induction of Fos was observed in the IML of hypotensive piglets. Li and Dampney [39] propose that significant induction of gene-encoded protein in visceral as in somatic motoneurons may require relatively longer post-stimulus survival intervals. Minson et al. [43] observed after 120 min intervals, shorter than those allowed here, that disinhibition of the rostral ventral medulla evoked 3- to 5-fold increases in the incidence of Fos-immunoreactive neurons in the IML of rabbits. The nature of the extracellular stimuli required for c-los expression in visceromotor neurons as well as other experimental variables require systematic exploration. Enhanced expression of FLI was also identified in the A1 region of the CVLM in the hypotensive piglet. Neurons containing TH were identified in the A1 area in our previous immunocytochemical study of neonatal swine [48] and demonstrated induction of Fos in response to nitroprusside-induced hypotension in conscious rabbits [39]. c-los gene induction in catecholaminergic neurons of the porcine A1 nucleus was confirmed by our work. In other species A1 noradrenergic neurons in the caudal ventrolateral medulla ascend to and mediate vagal excitation of magnocellular neurosecretory neurons of the hypothalamus [8,11]. In the rat disproportionately greater increases in Fos in A1 neurons occurred in response to hypovolemia (hemorrhagic) than isovolemic (nitroprusside-evoked) hypotension [5]. Induction of c-los expression after nitroprusside or hemorrhage, however, was reported in neurons synthesizing arginine vasopressin (AVP) in hypothalamus and A1 neurons that modulate their neurosecretory activity [39,56,57]. AVP released from terminals in the C1 area restores arterial pressure in response to hemorrhage via a V1 receptor mechanism [17]. Whether a similar mechanism operates in our experimental model requires more extensive analysis.
Acknowledgements This study was supported by NIH grants HL-20864 (P.M.G.), HD-28931 (P.M.G.), HL18974 and NS-28200
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(D.A.R.). The authors would like to thank Mr. Isaac Frasier and Mrs. Susan Ingenito for their skilled technical support.
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