The development of the medullary serotonergic system in the piglet

Autonomic Neuroscience: Basic and Clinical 110 (2004) 65 – 80 www.elsevier.com/locate/autneu

The development of the medullary serotonergic system in the piglet Mary M. Niblock a,*, Hannah C. Kinney b, Catherine J. Luce b, Richard A. Belliveau b, James J. Filiano c a

Department of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03753, USA b Department of Neurology and Pathology, Children’s Hospital and Harvard Medical School, USA c Department of Pediatrics, Children’s Hospital at Dartmouth, Dartmouth Hitchcock Medical Center, USA Received 12 September 2003; accepted 28 October 2003

Abstract The anatomy of the 5-HT system in the medulla oblongata is well defined in several vertebrate species, but not in the piglet. A detailed map and developmental profile of this system is particularly important in the piglet because this species increasingly is used as a model for physiological studies of medullary homeostatic control and its disorders in human infancy, especially the sudden infant death syndrome. Tryptophan hydroxylase immunohistochemistry was used to identify 5-HT cells and map their distribution in the medullae of piglets between postnatal days 4 and 30, the putative comparable period to early human infancy. Tritiated (3H)-lysergic acid diethylamide (LSD) binding to 5HT1A-D and 5-HT2 receptors and 3H-8-hydroxy-2-[di-N-propylamine]tetralin (8-OH-DPAT) binding to 5-HT1A receptors were used to quantify and map the distribution of these serotonin receptors between 4 and 60 postnatal days. The distribution of 5-HT cells was similar to that observed in other vertebrate species, with cell bodies in and lateral to the caudal raphe´. Tritiated-LSD and 3H-8-OH-DPAT binding both showed significant age-related changes in select raphe´ and extra-raphe´ subnuclei. Taken together, these findings suggest that while the medullary 5-HT cells are topographically in place at birth in the piglet, changes in 5-HT neurotransmission take place during the first 30 days of life, as reflected by changes in patterns of receptor binding. Therefore, the first 30 days of life represent a critical period in the development of the 5-HT system and the homeostatic functions it mediates. D 2004 Elsevier B.V. All rights reserved. Keywords: Autonomic control; Chemosensitivity; Raphe´; Respiration; Sudden infant death syndrome

1. Introduction The brainstem serotonergic (5-HT) system is one of the phylogenetically oldest and ontogenetically earliest neurotransmitter systems in the brain. This important system influences early brain growth and development in many species, as well as a number of different functions mediated by the developing brain, including homeostasis. In all vertebrates in which the brainstem 5-HT system has been analyzed, including rat (Lidov and Molliver, 1982; Takeuchi et al., 1982), mouse (Ishimura et al., 1988), cat (Takeuchi et al., 1982; Jacobs et al., 1984), rabbit (Howe et al., 1983; Bjarkam et al., 1997), sheep (Tillet, 1987), wallaby (Ferguson et al., 1999), monkey (Azmitia and Gannon, 1986;

* Corresponding author. Tel.: +1-603-650-4342; fax: +1-603-6506130. E-mail address: [email protected] (M.M. Niblock). 1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2003.10.004

Hornung and Fritschy, 1988), and human (Tork and Hornung, 1990), a similar distribution of 5-HT cells has been observed. Serotonergic cells lie predominately along the midline seam (raphe´) of the brainstem and ventrolateral to it, in the reticular subnuclei. The brainstem 5-HT system is divided into two components: a rostral portion, located in the pons and midbrain, and a caudal portion, located in the caudal pons and medulla, which we refer to as the ‘‘medullary 5-HT system’’. The earlier born rostral or superior group of 5-HT cells includes 5-HT cells located in the dorsal raphe´ nucleus, median raphe´, caudal linear nucleus, and raphe´ pontis (Tork and Hornung, 1990). These rostral cells project predominantly to the telencephalon, diencephalon, hippocampus, and limbic and paralimbic regions (Tork and Hornung, 1990). Physiological and ablation studies indicate that these rostral nuclei contribute to the regulation of the waking state (Portas et al., 1996; Monti et al., 2002), cognition (Ward et al., 1999), and mood (Santarelli et al., 2001). The later born caudal or inferior group of 5-HT cells

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includes those situated in the raphe´ obscurus nucleus, raphe´ pallidus nucleus, raphe´ magnus nucleus, lateral paragigantocellular nucleus, and gigantocellular nucleus (Tork and Hornung, 1990). These caudal 5-HT cells primarily project to other brainstem nuclei, cerebellum, and spinal cord (Tork and Hornung, 1990) and receive projections from the spinal cord and forebrain, notably the hypothalamus (Tork and Hornung, 1990). Importantly, these cells have extensive connections with sensory and autonomic motor nuclei through which they are able to sense changes in the internal and external environment, and modulate motor output to maintain homeostasis. The homeostatic functions of the nuclei that comprise the medullary (caudal) 5-HT system include state-dependent modulation of respiration (Bernard, 1998; Herman et al., 2001), upper airway reflexes (Haxhiu et al., 1998), responses to changes in oxygen and carbon dioxide levels (Coates et al., 1993; Bernard et al., 1996; Dreshaj et al., 1998; Wang et al., 1998, 2001; Curran et al., 2000, 2001; Nattie and Li, 2001; Richerson et al., 2001), responses to changes in blood pressure (Lindsey et al., 1988; Gao and Mason, 2001; Curran et al., 2002), thermoregulation (Morrison, 1999; Morrison et al., 1999), and arousal from sleep (Portas et al., 2000). This is the first of two studies from our laboratory that examine the comparative chemical anatomy of the medullary 5-HT system between piglets and human infants. The emphasis in these studies is on the medullary 5-HT system because of its relevance to human brainstem homeostatic disorders in early life, especially the sudden infant death syndrome (SIDS). SIDS is the leading cause of postneonatal infant mortality in the United States. Studies from our laboratory indicate abnormalities in 5-HT receptor binding in the medullary 5-HT system, specifically in raphe´ and extra-raphe´ regions that contain 5-HT neurons (Panigrahy et al., 2000; Kinney et al., 2001). Increasingly, the piglet is used for analyses of homeostatic physiology in regards to possible brainstem mechanisms of SIDS and other autonomic disorders of early life (Galland et al., 1993; Curran et al., 2000, 2001, 2002; Darnall et al., 2001). Yet, virtually nothing is known about neurochemical development in the piglet brainstem, particularly of the 5-HT system that is thought to be a contributing factor in SIDS. Thus, these studies were designed to provide baseline information about the medullary 5-HT system in the piglet for the pertinent physiological studies, and to compare it to the human to establish the relevance of data collected in piglets to human physiology. We postulated in the following study that the piglet has similar distributions of 5-HT cells and receptor binding patterns in the medullary raphe´ and extra-raphe´ nuclei to those in other commonly used experimental animals and humans. We also postulated that there might be early postnatal refinements in medullary 5-HT neurotransmission as this important brainstem homeostatic modulating system matures, and that these refinements would be evident in changes in 5-HT cell topography, number, or receptor

binding. Because 5-HT is secreted and degraded rapidly, 5-HT cells typically are identified based on the presence of synthetic enzymes for 5-HT, notably tryptophan hydroxylase (TPOH). We examined the topography of 5-HT neurons in the piglet medulla using immunohistochemistry with antibodies to TPOH, the rate-limiting synthetic enzyme for 5-HT. In addition, we examined the distribution and levels of 5-HT1A – D and 5-HT2, and 5-HT1A receptor binding using 3H-LSD and 3H-8-OH-DPAT, respectively. We chose these two radioligands because we have used them extensively in our human studies, and therefore their use allows us to make comparisons between the piglet and human infant.

2. Materials and methods 2.1. Animals Piglets aged postnatal day (P) 4, P12, P30, and P60, were obtained from Parson’s Farms in Massachusetts. Piglets were maintained in the animal facility at Dartmouth Hitchcock Medical Center until they were euthanized. All animal protocols used in this study are consistent with National Institutes of Health standards, and were approved by the Institutional Animal Care and Use Committee at Dartmouth Medical School, Lebanon, NH, and Children’s Hospital, Boston, MA, where some piglet tissues were processed. 2.2. Piglet tissue collection for immunohistochemistry Anesthesia was induced with halothane gas (3% in oxygen). Piglets then were deeply anesthetized with sodium pentobarbitol and perfused through the heart with normal saline at 37 jC, followed by 4% paraformaldehyde (pH 7.0– 7.5) at 4 jC. Brainstems were removed and immersed in 4% paraformaldehyde 24 –48 h at 4 jC. They then were cryoprotected by immersion in 30% sucrose at 4 jC. Serial sections (40 Am), beginning approximately 2 mm caudal to the obex and extending rostrally to the level of the inferior colliculus, were cut on a Leica CM3050 cryostat and collected in phosphate buffered saline (PBS). Sections of the medulla were used for this study. 2.3. Tryptophan hydroxylase immunohistochemistry in the piglet brainstem Tissue sections were washed three times with 0.1M Tris Buffer (TB; Sigma, pH 7.4) for 15 min at room temperature (RT). Sections were treated three times for 15 min in 50% ethanol in 0.1M TB, treated for 20 min in 3% H2O2 in 50% ethanol in 0.1 M TB, and treated for 20 min with 10% normal horse serum in TB at RT to block immunoglobulins. The monoclonal mouse anti-rabbit tryptophan hydroxylase (Sigma) was diluted 1:10,000, and sections were incubated in the primary antibody for 48 h at 4 jC. After 2 days

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incubation with the primary antibody, sections were washed with TB 3 times for 15 min, and then were incubated for 1 h with diluted biotinylated horse anti-mouse Immunoglobulin (IgG) solution in 0.1M TB (1:200 dilution) with 150 Al of normal horse serum (Vector) at RT. Sections were washed with 0.1M TB three times and then incubated for 60 min in VECTASTAIN ABC Reagent (Vector) at RT. Sections then were washed again with 0.1M TB three times for 15 min each time. All steps were performed with continuous shaking. Sections were incubated for 30 s in diaminobenzadine (DAB)-H2O2 solution (5 ml dH2O, 2 drops buffer, 4 drops DAB, 2 drops H2O2, 2 drops nickel; Vector). Sections were transferred from DAB solution into distilled H2O and then washed with TB. They were mounted on gelatin coated slides, allowed to air dry 2– 24 h, dehydrated through a graded series of ethanol, cleared with xylene, and coverslipped with Permount (Fisher). For control experiments, the primary antibody was omitted from immunohistochemical reactions. Sections adjacent to those immunohistochemically stained were stained with cresyl violet for anatomical comparisons and identification of medullary nuclei and landmarks. 2.4. Volumetric counting of 5-HT neurons The distribution of 5-HT cells and the number and density of raphe´ versus extra-raphe´ 5-HT neurons were determined in serially sectioned caudal brainstems from a total of eight piglets. Two medullae were analyzed at postnatal day 4, three at postnatal day 12, and three at postnatal day 30. In the piglet caudal brainstem, a few TPOH immunoreactive cells were scattered outside the raphe´ and lateral brainstem reticular subnuclei. These scattered 5-HT cells were observed just lateral to the rostral central canal of the spinal cord, and ventrolateral to and clearly separate from the paragigantocellular nucleus (PGCL), and were not included in the cell counting. Immunolabeled cell bodies were counted only if they were morphologically identifiable as neurons, axon and dendrite(s) were visible, and the neuronal cytoplasm had a dense distribution of reaction product that excluded the nucleus if visible. Tryptophan hydroxylase immunoreactive cells that we counted were classified as raphe´ (located in the raphe´ pallidus, magnus, and obscurus combined) or extraraphe´ (located in gigantocellular nucleus (GC), paragigantocellular nucleus (PGCL), and parapyramidal nucleus (PPY) combined). Cells were counted in every sixth section (every 240 Am). 2.5. Estimate of TPOH immunoreactive cell size Cell size was measured in four sections at four different standardized levels at postnatal days 4 (n = 2), 12 (n = 3), and 30 (n = 3). Because the brain grows between 4 and 30 days of age in the piglet, levels were defined based on a range of distances relative to the obex, as well as specific

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anatomical landmarks. Level A was defined as being 500– 1000 Am rostral to the obex and containing the raphe´ obscurus, levels B and C were defined as being between 1500 – 2000 and 2000 –2500 Am rostral to the obex, respectively, and containing the raphe´ magnus, and level D was defined as being 2500– 3000 Am rostral to the obex and containing the raphe´ pallidus. For each case, 10 cells in the raphe´ and 10 cells in the extra-raphe´ at each level were outlined at 200  magnification using Neurolucida software (Microbrightfield). At level D, additional measurements were made of cells in TPOH immunoreactive cell clusters at the medial and lateral ventral surface (n = 10 cells per section, 5 per cluster). The area (Am2) of each cell was calculated by the Neurolucida software. The average area of cells in each nucleus at each age was compared by single factor ANOVA. 2.6. Three-dimensional mapping and computer reconstruction of TPOH-positive cell topography Three-dimensional reconstruction was performed using Neurolucida and Neuroexplorer software (Microbrightfield). Two-dimensional plots of sections 240 Am apart were stacked serially and aligned using a reference point assigned during the mapping procedure. The sections were registered with each another to re-establish their original positions in three dimensions. We used the fourth ventricle, central canal, ventromedial sulcus, and the ventral surface of the inferior cerebellar peduncles as landmarks for registration. We also referred to a piglet brainstem atlas with 3-D reconstruction developed in our laboratory, as well as atlases for the rat (Paxinos and Watson, 1986) and cat (Snider and Niemer, 1961). 2.7. Tissue collection for 5-HT receptor binding Piglets aged P4 (n = 3), P12 (n = 4), P30 (n = 7), and P60 (n = 7), were euthanized with sodium pentobarbitol. Their brains were removed rapidly and frozen in isopentane on dry ice. Frozen brainstem sections were cut at 20 Am on a Leica CM3050 cryostat and collected on glass slides. Frozen sections were stored at 80 jC. 2.8. Serotonin receptor binding in the piglet brainstem The procedure for 3H-LSD binding to 5-HT1A – D and 5HT2 receptors is based upon methods published by us in the human brainstem, and previously has been described in detail (Zec et al., 1996; Panigrahy et al., 2000). The procedure for 3H-8-OH-DPAT binding to 5-HT1A receptors was based upon previously published methods in experimental animals (Mengod et al., 1996; Talley et al., 1997; Knapp et al., 1998). For the 3H-8-OH-DPAT incubation, unfixed, slide mounted 20 Am sections of the brainstem were preincubated in 0.17 M Tris – HCl buffer (pH 7.6), containing 4 mM CaCl2, 0.01% ascorbic acid for 30 min at

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room temperature. For determination of total binding, sections were incubated with 4 nM 3H-8-OH-DPAT (New England Nuclear) in 0.17 M Tris –HCl (pH 7.6) containing 4 mM CaCl2, 0.01% ascorbic acid and 10 AM pargyline for 1 h at room temperature. For determination of nonspecific binding, adjacent sections were incubated with10 AM 5-HT added to the buffer. 2.9. Quantitative analysis of medullary autoradiograms Following both the 3H-LSD and 3H-8-OH-DPAT binding incubations, the sections were placed in cassettes, and exposed to 3H-sensitive film (3H-Hyperfilm, Amersham or

Kodak BMR) for 8 weeks along with a set of 3H-standards (Amersham) for conversion of optical density of silver grains to femtomoles/milligram of tissue (fmol/mg tissue). Receptor binding density (expressed as the specific activity of tissue-bound ligand) was analyzed in 11 nuclei the medulla in each specimen. For each specimen, nuclei were analyzed at a defined level of the medulla (2 sections/ nucleus). The piglet medulla was divided into seven levels based on defined anatomic landmarks at each level, and these levels were comparable to medullary levels previously defined by us in the human brainstem (Kinney et al., 2001), except that the facial nucleus in the piglet is located in the medulla, whereas in the human it is located in the pons. The

Fig. 1. Serotonergic cell morphology in the piglet. There are four different 5-HT morphological cell types, pyramidal, multipolar, fusiform, and granular (A – D), in the piglet medulla. Each medullary 5-HT nucleus displays a heterogeneous population of subtypes, although some nuclei contain predominantly one cell type. There are no apparent age-related differences in the distribution or proportion of morphological cell types. (Scale bar = 20 Am).

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Fig. 2. Two-dimensional distribution of 5-HT cells in the piglet at representative levels of the medulla. Serotonergic cells are concentrated in the raphe´ and extra-raphe´ of the medulla, in the midline (raphe´) and lateral to it. Each circle represents a single TPOH immunoreactive cell, and the red indicates unique clusters of TPOH immunoreactive cells found in the piglet.

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following levels were used: Level 1, high cervical spinal cord; Level 2, caudal medulla, level of the nucleus gracilis and nucleus cuneatus medialis; Level 3, caudal medulla, level of the first appearance of the principal inferior olive, caudal to the obex; Level 4, level of the obex and (just rostral) area postrema; Level 5, mid-medulla, level of the hypoglossal nucleus and principal inferior olive; Level 6,

rostral medulla, level of nucleus prepositus, and the first appearance of the facial nucleus; Level 7, rostral medulla, level of mid-facial nucleus, and absence of the inferior olive; and Level 8, rostral medulla, level of the rostral pole of the facial nucleus, and absence of the inferior olive. Quantitative densitometry of autoradiograms was performed using a MCID 5+ imaging system (Imaging Research,

Fig. 3. Three-dimensional reconstructions of the piglet medulla and caudal pons. These computer reconstructions graphically display and correlate the location of TPOH immunoreactive neurons with the location of raphe´ and selected paramedian nuclei, as defined by classic cytoarchitectonic criteria. The facial nucleus and obex are included for orientation. The images in the left column (A – C) are plots of the locations of each TPOH immunoreactive cell. The images in the right column (D – F) are plots of boundary contours of nuclei using traditional nomenclature based on Nissl-stained sections. Note that there are scattered, single 5-HT cells on the dorsal, ventral and lateral surfaces, as well as throughout the tegmentum.

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Ontario, Canada), as described previously (Panigrahy et al., 2000). 2.10. Three-dimensional mapping and computer reconstruction of 5-HT receptor binding in the piglet medulla To quantify 5HT1A receptor binding, piglet medullae were incubated in 4 nM 3H-8-OH-DPAT for 60 min. These sections were then washed, rinsed, air-dried, and exposed to 3 H-sensitive Hyperfilm (Amersham) for 3 weeks. Using the MCID M5+ Image Analysis System (Imaging Research, Ontario, Canada) sections were digitized and calibrated according to previously published standard methods (Zec et al., 1996). In one representative piglet at P12, threedimensional (3-D) computer reconstructions of the medulla were made, and volumetric measurements were performed through the rostrocaudal length of selected nuclei. Thirty sections of the medulla separated by 200– 800 Am were used. Quantitative densitometric analysis of binding density and receptor distribution mapping for each individual section was performed, and the sections were then aligned according to cytoarchitectonic boundaries. The MCID + 5 system then rendered a 3-D reconstruction of the aligned

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sections, allowing visualization of isodensity contours in regions of equivalent binding, real-time rotation and display of 3-D solids, control of surface transparency, cut-awayfrom-surface images, and manipulation of light sources, colors, and shading factors. With this device, selected specific activity levels of 3H-8-OH-DPAT were rendered solid, color-coded, shaded and viewed at different angles. 2.11. Statistical analysis Because the first section for each individual brain was located at a slightly different rostrocaudal level, the obex was used as a landmark for alignment of the cell count data in the rostrocaudal dimension. For analyses, the total numbers of raphe´ and extra-raphe´ cells were calculated. Cells counts for individual piglets were averaged for three age groups: P4, P12, and P30. Raphe´ and extra-raphe´ cell counts for the three age groups were compared by ANOVA to determine if there was an age-related change in cell number. To assess the relationship between cell counts and distance from the obex (rostrocaudal level), a linear model was fit using all the available cases, taking into account the fact that counts across brainstem locations will be correlated for each

Fig. 4. The topography of 5-HT cells in the piglet medulla. 5-HT cells in the piglet medulla are distributed in 4 major nuclei: the raphe´ pallidus (A), raphe´ obscurus (C), raphe´ magnus (B), and lateral paragigantocellularis nucleus (D). Scale bar = 200 Am.

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individual. A preliminary model was fit to assess differences between age groups, taking into account the distance from the obex. Results indicated no statistically significant difference between age groups, so these data were grouped and analyzed together.

3. Results 3.1. The topography of 5-HT neurons in the piglet medulla Serotonergic cells in the medulla of the piglet were heterogeneous in size and shape, and were subclassified into four morphological cell types: pyramidal, multipolar, fusiform, and granular (round). Pyramidal cells were medium to large-sized, with a triangular cell body and distinct basal dendrites and a single apical dendrite extending from the cell body (Fig. 1A). Multipolar 5HT cells were the largest of the four subtypes, and were characterized by their multiple processes emanating from the cell body (Fig. 1B). Fusiform cells were mediumsized with spindle-shaped cell bodies and one process emanating from each pole (Fig. 1C). Granule cells were small and either round or oval with a large nucleus and one or two cytoplasmic processes (Fig. 1D). The medullary nuclei containing 5-HT neurons typically demonstrated more than one morphological subtype. For example, there were TPOH immunoreactive multipolar cells, as well as pyramidal, fusiform, and granular cells in the raphe´ obscurus. Qualitatively similar proportions of 5-HT

Fig. 6. 5-HT cells in contact with a blood vessel in the piglet medulla. Serotonergic cells and TPOH immunoreactive fibers occur in close proximity to blood vessels. Stars indicate TPOH immunoreactive cells or processes. Scale bar = 200 Am.

cell morphologies were observed in the extra-raphe´ brainstem nuclei. The TPOH immunohistochemistry frequently revealed the number and orientation of proximal dendrites of the neurons, as well as their axons. The orientation of the dendritic arbors of TPOH immunoreactive cells varied

Fig. 5. Clusters of 5-HT cells in the piglet medulla. Clusters (arrow) of TPOH-immunolabled cells are observed at the lateral ventral medulla in the piglet (A). These cluster cells are densely packed and surrounded by dense TPOH immunoreactive neuropil. The 5-HT neurons in the clusters have a granular morphology (B). Intense 3H-LSD binding colocalizes with these 5-HT cell clusters (C). Cells with a similar morphology to that in the lateral clusters are present at the ventral aspect of the raphe´ pallidus and ventrolateral medulla (D). Scale bar = 200 Am.

M.M. Niblock et al. / Autonomic Neuroscience: Basic and Clinical 110 (2004) 65–80 Table 1 Number of TPOH-immunoreactive cells in the piglet at three ages Age

Total

Raphe´

Extra-raphe´

P4 (n = 2) P12 (n = 3) P30 (n = 3)

2553 (SD F 464) 2581 (SD F 73) 2412 (SD F 391)

581 (22.7%) 682 (26.4%) 553 (22.9%)

1973 (77.3%) 1899 (73.6%) 1859 (77.1%)

with their location in the brainstem. In the raphe´ magnus and obscurus nuclei, for example, dendrites extended in multiple orientations, including parallel to the ventral surface, as well as perpendicular to it. In contrast, in extra-raphe´ nuclei, the majority of 5-HT cell dendritic trees were oriented parallel to the ventral surface. Collections of TPOH immunoreactive fibers were observed parallel and just dorsal to the ventral surface at all levels along the medulla’s rostrocaudal extent. The overall distribution of TPOH immunoreactive cells in the piglet is illustrated in two and three dimensions (Figs. 2 and 3, respectively). The immunohistochemical staining intensity between individual piglet medullae and between age groups was similar qualitatively. The distribution, size, morphology, and dendritic arbor orientation of the TPOH immunoreactive cells likewise were qualitatively similar at the three postnatal ages studied. There were no significant differences in cell size when data were grouped by age, location, or rostrocaudal level (for extra-raphe´ nuclei only). Detailed descriptions for the individual nuclei are provided below.

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3.2. Raphe´ pallidus The raphe´ pallidus in the piglet is located in the rostral medullary midline, at the ventral surface and extending dorsally to the border of the raphe´ magnus. The majority of TPOH immunolabeled cells in the raphe´ pallidus were densely packed granular cells, with a population of relatively more densely packed, less intensely stained cells at its ventral aspect (Fig. 4). The cell somata were very small (145 F 80 Am2) at the ventral aspect relative to other medullary serontonergic cells and average-sized (511 F 112 Am2) in the dorsal aspect. Very few, typically two or three, short dendrites emanated from cells in the dorsal aspect. There was no apparent orientation bias of these dendrites. A relatively more dense reaction product was observed consistently in the neuropil surrounding the ventral aspect of the raphe´ pallidus. Whether this was an artifact resulting from the close proximity of the ventral neurons, or is a manifestation of abundant TPOH immunoreactive fibers crowded together was difficult to distinguish in our preparations. 3.3. Raphe´ magnus In the piglet, the raphe´ magnus is located in the rostral half of the medullary midline, and extends ventrodorsally about 6 mm, to the level of the dispersion of the medial lemniscus. The majority of TPOH immunoreactive cells had dense dendritic trees, and were multipolar, with more

Fig. 7. The distribution of 5-HT cells along the rostrocaudal axis in the piglet medulla. The numbers of 5-HT cells in the raphe´ and extra-raphe´ increase rostrally ( p < 0.0001 and p < 0.001, respectively).

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than four primary dendrites extending from the soma (data not shown). The morphology of 5-HT neurons, however, was heterogeneous, with a predominance of multipolar and pyramidal cells, combined with a relatively lower concentration of fusiform and granular cells (Fig. 4B). The average size of cells was 540 F 107 Am2. The orientation of dendritic trees also was heterogeneous, with dendritic arbors extending in the ventrodorsal direction (vertical in the coronal plane), as well as the mediolateral direction (horizontal in the coronal plane). 3.4. Raphe´ obscurus The raphe´ obscurus in the piglet is located primarily in the caudal medulla. The topography of 5-HT neurons in this nucleus is unique compared with that in the raphe´ pallidus and raphe´ magnus in that the cells formed two distinctive paramedian columns (Fig. 4C). TPOH immunoreactive cells tended to be slightly smaller than those observed in the raphe´ magnus, with an average soma size

of 505 F 62 Am2, but they exhibited a similar morphological heterogeneity. In contrast to the raphe´ magnus, however, the dendritic arbors of 5-HT neurons in the raphe´ obscurus were oriented primarily in the ventrodorsal direction (vertical in the coronal plane, parallel to the midline). 3.5. Extra-raphe´ nuclei Tryptophan hydroxylase immunoreactive cells were observed in several extra-raphe´ nuclei, including the lateral reticular nucleus (LRN), GC, PGCL, retrotrapezoid nucleus (RTN), and PPY (Fig. 2B – E). Serotonergic cells in the PGCL were heterogeneous in both shape and size (Fig. 4D), while 5-HT cells in the PPY were smaller and always granular in morphology. In cresyl violet stained sections at corresponding levels, PPY neurons were near the midline rostrally, and lateral in more caudal sections, extending laterally like a wing, rostrocaudally, from the raphe´ pallidus. At the lateral aspect of the PPY, TPOH immunoreactive

Fig. 8. Tritiated-LSD binding at representative levels of the piglet medulla at P12. Images from caudal (A) to rostral (H) illustrate the distribution of 5-HT1A – D and 5-HT2 receptor binding to 3H-LSD. The intense binding in the raphe´ pallidus (ventral midline, E – H) also was seen in 3H-8-OH-DPAT binding.

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neurons are found, in some sections, in a dense cluster very close ( < 100 Am) to the ventral surface amidst closely apposed, dorsomedially extending TPOH immunoreactive fibers (Fig. 5A). Bilaterally near the ventral raphe´ pallidus, lightly stained TPOH immunoreactive neurons were found in compact collections similar to those observed lateral to the PPY (Fig. 5). These cells typically were smaller (average area = 199 F 96 Am2) and substantially more densely packed than TPOH immunoreactive cells elsewhere in the medulla. The lightly stained neurons adjacent to the raphe´ pallidus appeared to be continuous with the ventral cell group of the raphe´ pallidus. The locations of these cells were consistent with the topography of strikingly intense 3H-LSD binding (Fig. 5D). The dendritic arbors of immunostained neurons in the lateral paragigantocellular nucleus typically were oriented in the mediolateral direction. 5-HT neurons near the ventral surface (within 100 Am) in the parapyramidal region, however, typically had an asymmetrical dendritic arbor, with many dendrites extending toward the pial surface. In the mid-medulla, where the inferior olive was at its largest size, extra-raphe´ TPOH immunoreactive neurons and an abundant collection of TPOH immunoreactive fibers were closely adherent to the amiculum of the inferior olive, with a ‘‘cap’’ of 5-HT neurons overlying the amiculum. Rostrally, where the inferior olive was absent, the extra-raphe´ neurons were more dispersed.

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Fig. 7). Because the discrete ventral clusters of TPOH immunoreactive cells were limited to a small number of sections within individual cases and were not observed in every case, these cells were not included in the extra-raphe´ cell counts and were not analyzed statistically. 3.8. Serotonergic receptor binding in the piglet medulla Receptor binding patterns were analyzed in piglets between 4 and 60 days of age using the radioligands 3H-LSD

3.6. 5-HT cells in contact with blood vessels Penetrating blood vessels in contact with TPOH immunoreactive cells were observed in the rostral, ventral raphe´ pallidus, occasionally in the raphe´ obscurus, and among the dense clusters of TPOH immunoreactive neurons at the lateral ventral surface, at the level of the facial nucleus. These TPOH immunoreactive cells had processes that extended to the walls of the vessels and appeared to contact them (Figs. 4 and 6). 3.7. Numbers of 5-HT neurons in the piglet medulla The number of TPOH immunoreactive cells was essentially the same at 4, 12, and 30 days of age (Table 1). In piglets of all ages studied, the majority of 5-HT cells were located bilaterally in the reticular subnuclei. On average, 24% of the 5-HT cells were located in the raphe´ regions, with the remaining 76% located in the extra-raphe´ regions (LRN, PPY, PGCL, GC, RTN) combined. At all ages, there was a statistically significant increase in 5-HT cell number in the extra-raphe´ ( p < 0.001), that was most evident from the caudal medulla to the rostral pole of the facial nucleus, at which point 5-HT cell number began to decrease (Fig. 7). The highest concentration of 5-HT cells was observed at levels containing the facial nucleus, approximately 1.5 –3.5 mm rostral to the obex. In the raphe´ nuclei, 5-HT cell number increased from the caudal to rostral levels, from 1.4 mm caudal to 4.32 mm rostral to the obex ( p < 0.0001;

Fig. 9. Three-dimensional reconstruction of 3H-8-OH-DPAT binding to 5HT1A receptors in the piglet medulla. High levels of 5-HT1A receptor binding can be seen in the major nuclei of the caudal and rostral 5-HT system divisions in the axial (A), ventral (B), and lateral (C) views of the reconstructed brainstem. Locations of the raphe´ obscurus and raphe´ pallidus are indicated for orientation.

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and 3H-8-OH-DPAT, that bind to the 5-HT1A-D and 5-HT2 receptors, and to 5-HT1A receptors, respectively. The regional distribution of these binding patterns is illustrated at eight representative levels of the P12 piglet medulla (Fig. 8). Very high 3H-LSD binding (>45 fmol/mg tissue) was present in the raphe´ pallidus, vagal nuclei (NTS and dorsal motor nucleus of the vagus), and spinal trigeminal nucleus (caudal pole). In contrast, very high 3H-8-OH-DPAT binding (>45 fmol/mg tissue) was present only in the raphe´ pallidus (Figs. 9 and 10). The proportional differences between 3H-LSD and 3 H-8-OH-DPAT binding among medullary nuclei suggested that non-5-HT1A receptors, which bind to 3H-LSD, are predominant in the NTS, dorsal motor nucleus of the vagus, and spinal trigeminal nucleus, and subdivisions of the reticular formation (Tables 2 and 3). Essentially, 5-HT1A receptor binding was restricted to the raphe´ nuclei and parapyramidal nucleus, in contrast to low (10 – 19 fmol/mg tissue) or negligible ( < 10 fmol/mg tissue) binding in cranial nerve nuclei, subnuclei of the reticular formation, and the inferior olive (Table 3). The retrotrapezoid nucleus at the

Table 2 Tritiated-LSD binding in piglets at four ages Nucleus

P4

P12

P30

P60

SG HG ION NTS DMX PGCL GC Fac RTN ROb RPa

37.8 F 2.1 46.1 F 14.3 14.6 F 6.1 49.2 F 17.3 60.9 F 20.6 43.1 F 3.1 35.6 F 4.8 43.2 F 1.9 21.2 F 4.3 22.3 F 9.7 88.8 F 14.1

47.9 F 8.9 37.8 F 4.6 16.3 F 4.7 50.6 F 6.1 69.2 F 6.8 33.4 F 4.6 28.1 F 3.6 29.9 F 5 17.5 F 5.5 19.1 F 2.2 74.6 F 8.5

55.7 F 7.5 39.5 F 2.2 20.7 F 2.6 49.9 F 3.6 64.9 F 6.2 27.7 F 3.4 35.6 F 5.2 38.9 F 4.3 23.5 F 3.2 30.5 F 4.4 71.7 F 7.5

43.7 F 3.1 28.1 F 3.2 14.9 F 1.0 34.8 F 3.2 52.7 F 3.6 15.3 F 1.8 20.1 F 1.5 23.7 F 1.9 11.3 F 1.4 21.3 F 1.9 53.6 F 5.2

Data reported as fmol/mg tissue F standard error of the mean. Italics indicate nuclei with a statistically significant change in binding with age, p < 0.05.

rostral ventral surface of the medulla and inferior olive had low or negligible binding to both 3H-LSD and 3H-8-OHDPAT (Tables 2 and 3). Three-dimensional reconstructions of 3H-LSD and 3H-8-OH-DPAT binding in a piglet at P12

Fig. 10. Graphs of 3H-LSD and 3H-8-OH-DPAT binding in selected nuclei of the piglet medulla.

M.M. Niblock et al. / Autonomic Neuroscience: Basic and Clinical 110 (2004) 65–80 Table 3 Tritiated-8-OH-DPAT binding in piglets at four ages Nucleus

P4

P12

P30

P60

SG HG ION NTS DMX PGCL GC Fac RTN ROb RPa

33.7 F 2.3 7.3 F 1.3 NSD 13.6 F 2.4 17.3 F 2.8 21.7 F 4.0 15.8 F 3.0 10.0 F 1.8 7.8 F 1.2 12.1 F 1.9 38.7 F 6.4

36.5 F 3.4 5.7 F 2.6 NSD 9.2 F 2.4 17.8 F 1.9 19.4 F 5.0 11.9 F 4.7 6.4 F 1.4 6.3 F 3.6 12.8 F 2.1 22.5 F 6.0

32.4 F 1.8 6.4 F 4.1 NSD 16.3 F 3.6 20.2 F 4.0 13.1 F 2.1 12.7 F 2.0 6.5 F 2.3 4.4 F 2.2 13.6 F 1.9 42.2 F 9.7

25.6 F 3.1 6.0 F 2.6 6.0 F 3.8 11.1 F 2.4 17.8 F 3.6 12.7 F 1.5 9.6 F 2.0 7.4 F 1.7 5.8 F 1.6 11.5 F 1.4 35.2 F 7.7

Data reported as fmol/mg tissue F standard error of the mean. Italics indicate nuclei with a statistically significant change in binding with age, p < 0.05; NSD = insufficient data.

revealed that the very intense binding in the raphe´ pallidus was confined to the cytoarchitectonic boundaries of this nucleus at the level of the facial nucleus in the rostral medulla (Fig. 9). Three ‘‘columns’’ of 5-HT receptor binding to 3H-8-OH-DPAT, one midline the other two lateral, were visualized in the ventral view of the 3-D reconstruction (Fig. 9). The anatomic distribution of these three columns of receptor binding corresponded with the three columns of TPOH positive cells visualized in the ventral view of 3-D reconstructions of cell topography (Fig. 3). These topographic columns reflected the ventral cell components of the raphe´ pallidus (midline) and PGCL. At all of the ages examined in the piglet, there was a striking differential pattern of binding in the caudal raphe´. The heaviest concentration (>70 fmol/ mg tissue) for both radioligands was observed in the raphe´ pallidus, compared to low binding ( < 20 fmol/mg) in the raphe´ obscurus (Fig. 8). Serotonergic receptor binding, demonstrated with both 3H-LSD and 3H-8-OH-DPAT, was high at the ventral surface in the parapyramidal region (Figs. 5 and 9) and co-localized with the discrete 5-HT cell clusters demonstrated by immunostaining. It is important to note that there was a striking degree of variability in receptor binding levels in individual nuclei among the pigs at each age sampled, despite identical processing of the specimens and postmortem intervals less than 1 h in all instances (Tables 2 and 3). In the raphe´ obscurus at P30 (n = 7), for example, the 3 H-LSD binding levels varied from 15.6 fmol/mg tissue to 49.1 fmol/mg tissue, which represents a three-fold difference (mean = 30.5 + 11.6 [Standard Deviation] fmol/mg tissue). The developmental profile of 5-HT receptor binding with the two selected radioligands was assessed in the pig at ages P4, P12, P30, and P60 in 11 medullary nuclei (Fig. 10). The patterns of binding of these two radioligands relative to each other varied among the nuclei with age (Fig. 10). There was a significant difference with age, albeit according to different patterns, in 3H-LSD binding in 5 of the 11 nuclei sampled. The nuclei with age-related differences in binding patterns were the PGCL ( p = 0.000), GC ( p = 0.018), facial nucleus ( p = 0.007), RTN ( p = 0.024), and raphe´ pallidus

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( p = 0.007, Fig. 10). In the PGCL and raphe´ pallidus, binding decreased progressively across the ages analyzed (Fig. 10); in contrast, in the GC, facial nucleus, and RTN, binding levels appeared constant between P4 and P30 and then decreased sharply between P30 and P60 (Fig. 10). The developmental changes in the caudal raphe´ were complex, with a significant decrease in the raphe´ pallidus with increasing age, and no change in the raphe´ obscurus (Fig. 10). In regards to 3H-8-OH-DPAT binding, there were no significant age-related changes except in the substantia gelatinosa of the spinal trigeminal nucleus, in which there was a decrease in binding between P30 and P60 ( p = 0.038) (Fig. 10). Thus, in the five nuclei with statistically significant changes in 3H-LSD binding, there were no concomitant changes in 3H-8-OH-DPAT binding, suggesting that the changes in 3H-LSD binding were due to changes in the developmental profile of the non-5-HT1A receptors labeled by 3H-LSD.

4. Discussion The 5-HT markers used in this study for 5-HT cells and receptor binding were chosen as anatomical markers of 5-HT-related function. Our goal was to assess potential changes in 5-HT neurotransmission over early postnatal development in the piglet through analyses of these markers. Previous studies indicate that different functions mediated by medullary 5-HT cells, such as chemosensitivity (Wang et al., 1998), as well as responses to medullary 5-HT inputs (Talley et al., 1997) develop postnatally, at least in part, suggesting postnatal changes in neurotransmission among these cells. This study characterizes the topography of the medullary 5-HT system in the piglet. We found that the cells of the medullary 5-HT system are in place around birth (P4). The overall distribution of 5-HT cells in the piglet medulla is consistent with that reported in other species, with the exception of a higher proportion (average is 76% of total) of 5-HT cells in extra-raphe´ nuclei (ventrolateral medulla) in the piglet compared with other species. Also of note, we observed small clusters of 5-HT cells along the ventral surface of the piglet medulla that have only been described in the rat. Although the positions of 5-HT cells were established by birth in the piglet, there were significant age-related changes in levels of 5-HT receptor binding, suggesting postnatal fine-tuning of this system. The implications of these findings are addressed in detail below. 4.1. Cell number, type and distribution in piglet compared with that in other species In general, the morphological subtypes and distribution of 5-HT cells in the medulla in the piglet are similar to those observed in the human, as well as in other mammalian

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species, specifically the cat (Jacobs et al., 1984), rabbit (Howe et al., 1983) and rat (Lidov and Molliver, 1982). The morphologies of 5-HT cells in the piglet, including the orientation of 5-HT dendrites with respect to the ventrodorsal and mediolateral dimensions, also are similar to those observed in other species, including humans. These comparative observations verify that this vital and phylogenetically ancient caudal 5-HT system is highly conserved across species. The similarity of 5-HT dendritic morphologies among species suggests conservation of overall connectivity and, by extrapolation, possibly function in the medullary system. Because of the importance of this system in modulating homeostatic functions and the necessity at birth of this system to serve independent, extra-uterine homeostasis, it is not surprising that mature cell number and morphology are well-established at birth in all species studied. The heterogeneous morphologies observed among medullary 5-HT cells may have functional relevance. For example, in vitro, 5-HT cells with a multipolar morphology (>3 neurites) are chemosensitive (Wang et al., 2001). The size of a cell’s soma and dendritic tree, and the number of basal dendrites emanating from the soma reflect the integrative capacity of a given neuron. The functional relevance of broad integration in this system remains to be determined. Our observation of 5-HT cells in the vicinity of blood vessels in the piglet, and even with apparent contact between the cell processes and the blood vessel walls, is consistent with similar observations in rats, and suggests that, as in other species, these 5-HT cells may function as chemoreceptors, via processes contacting and monitoring arterial blood gases. In rats 5-HT fibers abut the endothelial layer of blood vessels, as demonstrated ultrastructurally (Bradley et al., 2002). These cells are in ideal locations to sample blood gases and, through their connections with respiratory-related premotor nuclei in the brainstem, modulate respiration in response to changes in blood pH/carbon dioxide levels. It also has been proposed that neurons in the nucleus raphe´ obscurus help maintain adequate cerebral blood flow during sleep while there is simultaneous sleep state dependent reduction in somatic and visceral blood flow (Coote, 1982). Therefore, the contact of raphe neurons with blood vessels may be an anatomic substrate to sense both blood flow and carbon dioxide concentration, and thereby participate in maintenance of this sleep state-dependent process. We found in the piglet medulla that 5-HT cells were evenly distributed in the midline and lateral medulla, with roughly one quarter of the total number of 5-HT cells in any given section in the raphe´, and the other three-quarters equally distributed on either side in the extra-raphe´. This topography was consistent across the three ages included in this study, which suggests that the mature cell topography of the medullary 5-HT system is in place at birth. Lateralization of the 5-HT system primarily occurs in the rostral pons and midbrain (i.e., the rostral 5-HT system), presumably due to the increased number of forebrain targets of 5-HT cells with

the evolutionary expansion of the cerebral cortex, but we observed an extensive lateralization in the piglet caudal 5HT system that is more pronounced than that observed in the rat (Lidov and Molliver, 1982), but may be similar to that observed in the rabbit (Howe et al., 1983) and cat (Jacobs et al., 1984). The proportion of cells in the caudal raphe´ versus extra-raphe´ nuclei in the piglet may represent a species difference but the implications of such a difference are unclear, due to the difficulty of comparing cell counts between species. The higher proportion of extra-raphe´ cells in the piglet may, however, represent underlying differences in function related to the spinal targets of these cells and the increasing complexity among higher vertebrates in the modulation of homeostatic control. In addition to the high percentage of the total 5-HT cell number outside the midline raphe´, the piglet has unique clusters of 5-HT cells near and at the lateral ventral surface. These cell clusters were in the parapyramidal region in some sections, but in others, were lateral to the PGCL. Another cluster of 5-HT cells was observed at the ventral aspect of the raphe´ pallidus. Similar clusters of 5-HT cells previously have been reported just lateral to the ventral medullary midline in the rat and postulated to be derived from the raphe´ pallidus (Lidov and Molliver, 1982), one of the regions shown previously by others to contain chemosensitive cells. It is suggested that these cells initially, phylogenetically and ontogenetically, are contiguous with the cluster of granular cells at the ventral aspect of the raphe´ pallidus and later are ‘‘pushed out’’ by the expanding inferior olive into the lateral ventral medulla (Lidov and Molliver, 1982). In the rat, Lidov and Molliver (1982) report that a small number of 5-HT cells remain between and within the pyramidal tract and the inferior olive, but they do not collectively name these cells. Based on our observations of these cell clusters in the piglet, and the striking morphological similarities between the lateral clusters and the ventral raphe´ pallidus cells, this seems a likely explanation of the source of these cells. Another possibility is that these 5-HT cells along the ventral medullary surface did not reach their final destination during their migration to the ventromedial medulla, presumably en route from the rhombic lip (Harkmark, 1954). One recognized limitation of this study is the use of immunhistochemically stained cells for both cell counting and estimates of cell size. Because we did not want to estimate total cell number in this system, but rather simply wanted the proportion of cells in the raphe´ versus the extra-raphe´ and along the rostrocaudal axis of the medulla, and a comparison of cell number relative to different age groups, we deemed it appropriate to count cells without using stereological techniques. Estimates of cell size only were made to compliment qualitative observations of cell morphology and were made with the understanding that variations in the quality and amount of DAB reaction product preclude precise measurements of cell size.

M.M. Niblock et al. / Autonomic Neuroscience: Basic and Clinical 110 (2004) 65–80

4.2. The developmental profile of 5-HT receptor binding in the piglet Our analysis of 3H-LSD binding to 5-HT1A-D and 5-HT2 receptors, but not 3H-8-OH-DPAT binding to 5-HT1A receptors, in the piglet demonstrate significant age-related changes in both raphe´ and extra-raphe´ components of the medullary 5-HT system. In contrast, the only developmental changes in the medullary nuclei not containing 5-HT cell bodies are in the facial nucleus (i.e., a decrease in 3H-LSD binding with age) and spinal trigeminal nucleus (i.e., a decrease in 3H-8-OH-DPAT binding with age). These observations are in contrast to studies in the rat that show a significant decrease in 5-HT1A receptor binding to 3H-8OH-DPAT in the hypoglossal nucleus following a peak at postnatal day 7 (Talley et al., 1997). Although in the piglet there was a trend (with borderline statistical significance) in the hypoglossal nucleus towards a decrease in 3H-LSD binding to 5-HT1A – D and 5-HT2 receptors with age, no similar trend was seen with 3H-8-OH-DPAT binding. Thus, in the piglet, the most prominent changes in 5-HT receptor binding occur in the component nuclei of the medullary 5HT system itself, underscoring the idea that the early postnatal period is a critical period during which rapid and dramatic changes occur that refine the system. The nuclei with significant changes included the PGCL, GC, and raphe´ pallidus. Within the GC, the binding was stable until 30 days and decreased sharply thereafter; binding gradually decreased from P4 to P60 in the raphe´ pallidus and PGCL. Therefore, while the 5-HT cells are in place at birth in the piglet, fine-tuning of neurotransmission occurs postnatally, as reflected in alterations in the number and/or affinity of predominately non-5-HT1A receptors in specific nuclei.

5. Conclusions This study maps the distribution of 5-HT cell bodies and 5-HT1A – D and 5-HT2 receptor binding in the piglet medullary 5-HT system and compares these data with data reported for other vertebrate species. Our data highlight anatomical and neurochemical species differences in the piglet that may be important to interspecies interpretations and comparisons of homeostatic mechanisms mediated by the medullary 5-HT system. In addition to their comparative anatomical value, these data provide a basis for analyzing physiology and dysfunction in the piglet following manipulations that target the 5-HT system in an attempt to model human developmental disorders, including SIDS.

Acknowledgements The authors would like to thank Dr. Eugene E. Nattie for comments during manuscript preparation. The authors also thank Hong Gao, Karen Dewey, Stephen Dewey, and Chad

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Jarvis for excellent technical assistance. We appreciate the assistance of Dr. Iyue Sung in the statistical analyses of the 5HT receptor binding data. This work was supported by: the Deborah Evelyn Barrett Fellowship in SIDS Research (MMN), SIDS Alliance, CJ Murphy Foundation, NICHD PO1-F139254, Hearst Grant (DHMC Department of Pediatrics), and Children’s Hospital Mental Retardation Core Grant (P30-HD18655). References Azmitia, E.C., Gannon, P.J., 1986. The primate 5-HT system: a review of human and animal studies and a report on Macaca fascicularis. Adv. Neurol. 43, 407 – 468. Bernard, D.G., 1998. Cardiorespiratory responses to glutamate microinjected into the medullary raphe´. Respir. Physiol. 113, 11 – 21. Bernard, D.G., Li, A., Nattie, E.E., 1996. Evidence for central chemoreception in the midline raphe´. J. Appl. Physiol. 80, 108 – 115. Bjarkam, C.R., Sorensen, J., Geneser, F.A., 1997. Distribution and morphology of serotonin-immunoreactive neurons in the brainstem of the New Zealand white rabbit. J. Comp. Neurol. 380, 507 – 519. Bradley, S.R., Pieribone, V.A., Wang, W., Severson, C.A., Jacobs, R.A., Richerson, G.B., 2002. Chemosensitive 5-HT neurons are closely associated with large medullary arteries. Nat. Neurosci. 5, 401 – 402. Coates, E.L., Li, A., Nattie, E.E., 1993. Widespread sites of brain stem ventilatory chemoreceptors. J. Appl. Physiol. 75, 5 – 14. Coote, J.H., 1982. Respiratory and circulatory control during sleep. J. Exp. Biol. 100, 223 – 244. Curran, A.K., Chen, G., Darnall, R.A., Filiano, J.J., Li, A., Nattie, E.E., 2000. Lesion or muscimol in the rostral ventral medulla reduces ventilatory output and the CO2 response in decerebrate piglets. Respir. Physiol. 123, 23 – 37. Curran, A.K., Darnall, R.A., Filiano, J.J., Li, A., Nattie, E.E., 2001. Muscimol dialysis in the rostral ventral medulla reduced the CO2 response in awake and sleeping piglets. J. Appl. Physiol. 90, 971 – 980. Curran, A.K., Peraza, D., Elinsky, C.A., Leiter, J.C., 2002. Enhanced baroreflex-mediated inhibition of respiration after muscimol dialysis in the rostral ventral medulla. J. Appl. Physiol. 92, 2554 – 2564. Darnall, R.A., Curran, A.K., Filiano, J.J., Li, A., Nattie, E.E., 2001. The effects of a GABAA agonist in the rostral ventral medulla on sleep and breathing in newborn piglets. Sleep 24, 514 – 527. Dreshaj, I.A., Haxhiu, M.A., Martin, R.J., 1998. Role of the medullary raphe´ nuclei in the respiratory responses to CO2. Respir. Physiol. 111, 15 – 23. Ferguson, I.A., Hardman, C.D., Marotte, L.R., Salardini, A., Halasz, P., Vu, D., Waite, P.M.E., 1999. 5-HT neurons in the brainstem of the wallaby, Macropus eugenii. J. Comp. Neurol. 411, 535 – 549. Galland, B.C., Peebles, C.M., Bolton, P.G., Taylor, J.B., 1993. Sleep state organization in the developing piglet during exposure to different thermal stimuli. Sleep 7, 610 – 619. Gao, K., Mason, P., 2001. The discharge of a subset of 5-HT raphe´ magnus cells is influenced by baroreceptor input. Brain Res. 900, 306 – 313. Harkmark, W., 1954. Cell migration from the rhombic lip to the inferior olive, the nucleus raphe´ and the pons. A morphological and experimental investigation on chick embryos. J. Comp. Neurol. 100, 115 – 209. Haxhiu, M.A., Erokwu, B., Bhardwaj, V., Dreshaj, I.A., 1998. The role of the medullary raphe´ nuclei in regulation of cholinergic outflow to the airways. J. Auton. Nerv. Syst. 69, 64 – 71. Herman, J.K., O’Halloran, K.D., Bisgard, G.E., 2001. Effect of 8-OH 8OH-DPAT and ketanserin on the ventilatory acclimatization to hypoxia in awake goats. Respir. Physiol. 124 (2), 95 – 104 (Jan.). Hornung, J.P., Fritschy, J.M., 1988. 5-HT system in the brainstem of the marmoset: a combined immunocytochemical and three-dimensional reconstruction study. J. Comp. Neurol. 270, 471 – 487.

80

M.M. Niblock et al. / Autonomic Neuroscience: Basic and Clinical 110 (2004) 65–80

Howe, P.R., Moon, E., Dampney, R.A., 1983. Distribution of serotonin nerve cells in the rabbit brainstem. Neurosci. Lett. 38, 125 – 130. Ishimura, K., Takeuchi, Y., Fujiwara, K., Tominaga, M., Yoshioka, H., Sawada, T., 1988. Quantitative analysis of the distribution of serotonin-immunoreactive cell bodies in the mouse brain. Neurosci. Lett. 91, 265 – 270. Jacobs, B.J., Gannon, P.J., Azmitia, E.C., 1984. Atlas of 5-HT cell bodies in the cat brainstem: an immunocytochemical analysis. Brain Res. Bull. 13, 1 – 31. Kinney, H.C., Filiano, J.J., White, W.F., 2001. Medullary serotonergic network deficiency in the sudden infant death syndrome: review of a 15-year study of a single dataset. J. Neuropathol. Exp. Neurol. 60, 228 – 247. Knapp, D.J., Overstreet, D.H., Crews, F.T., 1998. Brain 5-HT1A receptor autoradiography and hypothermic responses in rats bred for differences in 8-OH-DPAT sensitivity. Brain Res. 782, 1 – 10. Lidov, H.G.W., Molliver, M.E., 1982. Immunohistochemical study of the development of 5-HT neurons in the rat CNS. Brain Res. Bull. 9, 559 – 604. Lindsey, B.G., Arata, A., Morris, K.F., Hernandez, Y.M., Shannon, R., 1988. Medullary raphe´ neurones and baroreceptor modulation of the respiratory motor pattern in the cat. J. Physiol. (Lond.) 512, 863 – 882. Mengod, G., Vilaro, M.T., Raurich, A., Lopez-Gimenez, J.F., Cortes, R., Palacios, J.M., 1996. 5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors. Histochem. J. 28, 747 – 758. Monti, J.M., Jantos, H., Monti, D., 2002. Increased REM sleep after intra-dorsal raphe nucleus injection of flesinoxan or 8-OHDPAT: prevention with WAY 100635. Eur. Neuropsychopharmacol. 12, 47 – 55. Morrison, S.F., 1999. RVLM and raphe´ differentially regulate sympathetic outflows to splanchnic and brown adipose tissue. Am. J. Phys. 276, R273 – R962. Morrison, S.F., Sved, A.F., Passerin, A.M., 1999. GABA-mediated inhibition of raphe´ pallidus neurons regulates sympathetic outflow to brown adipose tissue. Am. J. Physiol. 276, R290 – R297. Nattie, E.E., Li, A., 2001. CO2 dialysis in the medullary raphe´ of the rat increases ventilation in sleep. J. Appl. Physiol. 90, 1247 – 1257. Panigrahy, A., Filiano, J.J., Sleeper, L.A., Mandell, F., Valdes-Dapena, M., Krous, H.F., Rava, L.A., Foley, E., White, W.F., Kinney, H.C., 2000. Decreased 5-HT receptor binding in rhombic lip derived regions of the medulla oblongata in the sudden infant death syndrome. J. Neuropathol. Exp. Neurol. 59, 377 – 384.

Paxinos, G., Watson, C., 1986. The Rat Brain In Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney, Orlando. Portas, C.M., Thakkar, M., Rainnie, D., McCarley, R.W., 1996. Microdialysis perfusion of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in the dorsal raphe nucleus decreases serotonin release and increases rapid eye movement sleep in the freely moving cat. J. Neurosci. 16, 2820 – 2828. Portas, C.M., Bjorvatn, B., Ursin, R., 2000. Serotonin and the sleep/wake cycle: special emphasis on microdialysis studies. Prog. Neurobiol. 60, 13 – 35. Richerson, G.B., Wang, W., Tiwari, J., Bradley, S.R., 2001. Chemosensitivity of 5-HT neurons in the rostral ventral medulla. Respir. Physiol. 129, 175 – 189. Santarelli, L., Gobbi, G., Debs, P.C., Sibille, E.T., Blier, P., Hen, R., Heath, M.J., 2001. Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases 5HT function. Proc. Natl. Acad. Sci. U. S. A. 98, 1912 – 1917. Snider, R.S., Niemer, W.T., 1961. A Stereotaxic Atlas of the Cat Brain The University of Chicago Press, Chicago. Takeuchi, Y., Kimura, H., Sano, Y., 1982. Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat. Cell Tissue Res. 224, 247 – 267. Talley, E.M., Sadr, N.N., Bayliss, D.A., 1997. Postnatal development of 5HT innervation, 5-HT1A receptor expression, and 5-HT responses in rat motoneurons. J. Neurosci. 17, 4473 – 4485. Tillet, Y., 1987. Immunocytochemical localization of serotonin-containing neurons in the myelencephalon, brainstem, and diencephalon of the sheep. Neuroscience 23, 501 – 527. Tork, I., Hornung, J.P., 1990. Raphe nuclei and the 5-HT system. In: Paxinos, G. (Ed.), The Human Nervous System. Academic Press, San Diego, pp. 1001 – 1022. Wang, W., Pizzonia, J.H., Richerson, G.B., 1998. Chemosensitivity of rat medullary raphe´ neurons in primary tissue culture. J. Physiol. 511, 433 – 450. Wang, W., Tiwari, J.K., Bradley, S.R., Zaykin, R.V., Richerson, G.B., 2001. Acidosis-stimulated neurons of the medullary raphe´ are sertonergic. J. Neurophysiol. 85, 2224 – 2235. Ward, B.O., Wilkinson, L.S., Robbins, T.W., Everitt, B.J., 1999. Forebrain serotonin depletion facilitates the acquisition and performace of a conditional visual discrimination task in rats. Behav. Brain Res. 100, 51 – 65. Zec, N., Filiano, J.J., Panigrahy, A., White, W.F., Kinney, H.C., 1996. Developmental changes in [3H]-lysergic acid diethylamide (LSD) binding to serotonin receptors in the human brainstem. J. Neuropathol. Exp. Neurol. 55, 114 – 126.