Connections of the rostral ventral respiratory neuronal cell group: an anterograde and retrograde tracing study in the rat

Brain Research Bulletin, Vol. 47, No. 6, pp. 625– 642, 1998 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter

PII S0361-9230(98)00125-7

Connections of the rostral ventral respiratory neuronal cell group: An anterograde and retrograde tracing study in the rat Susana P. Gayta´n and Rosario Pa´saro* Department of Animal Physiology and Biology, University of Sevilla, Sevilla, Spain [Received 10 March 1998; Revised 15 July 1998; Accepted 28 July 1998] ABSTRACT: The connections of the rostral ventral respiratory cell group (VRG) were retrogradely and anterogradely determined after discrete injections of a mixture of the fluorescent tracers Fast Blue (FB) and Fluoro Ruby (FR) into the physiologically identified rostral inspiratory cell group. Retrogradely FBlabeled neurons and/or anterogradely FR-labeled fibers and terminal fields were located bilaterally in a variety of brain areas. Both retrograde and anterograde labelings were mainly found in: 1) the deep cerebellar nuclei; 2) the lateral lemniscus and paralemniscal nuclei, deep gray, and white intermediate layers of the superior colliculus, tegmental (laterodorsal and microcellular) nuclei, and central gray; and 3) the septohypothalamic nucleus, and lateral and posterior hypothalamic areas. The FR-labeled terminal-like elements were found in: 1) Crus 2 of the ansiform lobule, and the simple, 2, and 3 cerebellar lobules; 2) the subcoeruleus, deep mesencephalic, and Edinger-Westphal nuclei; and 3) the premammillary, lateral, and medial mammillary nuclei, retrochiasmatic part of the supraoptic nucleus, and the zona incerta. The FB-labeled neurons were found in: 1) the parapedunculopontine tegmental and cuneiform nuclei, caudal linear nucleus of the raphe, and adjacent area of the cerebral peduncle; 2) the thalamic posterior nuclear group and subparafascicular, parafascicular, and gelatinosus thalamic nuclei; 3) the parastrial amygdaloid and subthalamic nuclei; and 4) the olfactory tubercle, granular, and agranular insular cortex, parietal and lateral orbital cortices. The connections of the rostral VRG with several cerebellar, midbrain, diencephalic, and telencephalic regions could provide an anatomical substrate for a role of these regions in the control of respiratory-related functions. © 1999 Elsevier Science Inc.

subgroups, the rostral VRG (the intermediate part of the VRG) [9], located near the nucleus ambiguus, contains mostly inspiratory neurons. The rostral VRG in mammals is heterogeneous in composition, constituted by nucleus ambiguus motoneurons, propriobulbar, and bulbospinal respiratory neurons, and vagal and glossopharyngeal preganglionic parasympathetic neurons [8,9,21,23, 37–39,45]. The rostral VRG is the most conspicuous part of the VRG, given its higher number of neurons and density of afferent and efferent connections with other cardiorespiratory-related neurons of the pons and medulla compared to the other two subdivisions of the VRG [9,20,38,39]. Substantial evidence also exists that central nervous system (CNS) regions involved in the regulation of behavioral and emotional responses also play a role as modulators of autonomic functions. In this context, the cerebellum [4,25,34,58,61,68], as well as the central (periaqueductal) gray (CG) [5–7], have been shown to participate in cardiorespiratory-related functions. On the other hand, several CNS areas, such as the pedunculopontine and laterodorsal tegmental nuclei [22,36,47,71], hypothalamus [40,59, 72], thalamus [50], amygdala [1], and cortex [11,49,50] play a major role in the integration of autonomical control and behavioral responses. A detailed description of the afferent and efferent projections interconnecting the three different VRG neuronal cell groups with other pontomedullary nuclei has recently been provided in the rat [20,39]. However, to date, the organization of the central neural VRG connections has not been examined in a single comprehensive study. The aim of the present report was to map the afferent and efferent projections linking the rostral VRG neuronal cell group with different cerebellar, midbrain, diencephalic, and telencephalic nuclei. The final goal was to trace the neuronal network subserving the involvement of central structures in the regulation of respiratory-related functions. In the present work, two different fluorescent dyes were used simultaneously: Fast Blue (FB), which retrogradely labels the neuronal soma and proximal dendrites [30], and Fluoro Ruby (FR), an anterograde axonal tracer that results in a homogeneous labeling of the full extent of the axon and its collaterals, as well as in labeled varicosities or puncta in the terminal fields [54]. The connectivity of the cerebellar, midbrain,

KEY WORDS: Control of respiration, Central (periaqueductal) gray, Cerebral cortex, Deep cerebellar nuclei, Diencephalon.

INTRODUCTION The ventrolateral part of the medulla oblongata includes in the rat a region referred to as the ventral respiratory cell group (VRG), which contains structures necessary for respiratory rhythm generation [8,14,15,48,55,75] The neurons within the VRG can be classified according to a pattern of cell firing during respiration into three different subgroups: 1) the Bo¨tzinger complex, 2) the rostral VRG, and 3) the caudal VRG [10,14,15,75]. Of these

* Address for correspondence: Rosario Pa´saro, Departamento de Fisiologı´a y Biologı´a Animal, Facultad de Biologı´a, Avda. Reina Mercedes, 6, 41012-Sevilla. Fax: 34-54233480; E-mail: [email protected]

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626 diencephalic, and telencephalic regions was explored bilaterally to determine the relative strength of the ipsilateral and contralateral projections. Preliminary results of these data have been presented elsewhere [18,19]. MATERIALS AND METHODS Experiments were performed on 18 adult female albino Wistar rats (weighing 250 – 400 g) following the N.I.H. Guide for the Care and Use of Laboratory Animals (N.I.H. Publication number 85-23, revised 1985). An experimental series (12 animals) was performed to determine the optimal survival time to obtain a brilliant labeling of the fluorochromes in the cerebral cortex. In the latter series and in the additional six animals, the injections of the tracers were made by the same procedure. Briefly, the animals were anesthetized with an intraperitoneal (i.p.) injection of a mixture of ketamine (50 mg/kg), diazepam (2.5 mg/kg), and atropine sulfate (0.5 mg/kg). For subsequent maintenance of the anesthesia, doses were administered i.m. when the animals demonstrated corneal or flexion withdrawal reflexes. Using sterile procedures, the animals were placed in an atraumatic headholder, the head ventroflexed 25° to the horizontal plane of the rat atlas of Paxinos and Watson [42], and an occipital craniotomy was performed to expose the dorsal surface of the medulla via the foramen magnum. During surgery, the rectal temperature was maintained at 37°C–38°C using heating pads. The deeply anesthetized animals maintained spontaneous breathing. Respiratory activity was monitored on an oscilloscope by means of two recording electrodes (insulated silver wire, 200 mm in diameter) inserted into the diaphragm from the abdominal side, indicating phases of the respiratory cycle. In all experiments, we first located the desired injection site (rostral VRG) at a given rostrocaudal distance (0.5–1.5 mm; Figs. 1A–D and 2A–F) from the calamus scriptorium (interaural 25.3 mm, Bregma 214.3 mm, c.f. obex, 42) as zero reference, by recording extracellular inspiratory multiunit activity with a glass micropipette, in agreement with previous descriptions [20]. The micropipette (30 –70 mm tip diameter, 4 – 8 MV) was filled with a solution of a mixture of 2% FB (EMS Polyloy) and 10% FR (D-1817, Molecular Probes) in 0.1 M phosphate buffer pH (PB) 7,4. Injections were made unilaterally by pressure pulses (Picospritzer, General Valve) applied to the micropipette at the site where inspiratory activity (Figs. 1A and 2A,D) was recorded along a dorsoventral extent of at least 150 mm. After injections, the pipettes were left in place for 15 min to prevent tracer leakage along the pipette tract. After withdrawal of the micropipette, the exposed medulla was protected with a silicone sheet, the surgical wound was sutured, and a prophylactic dose of ampicillin (10,000 units, i.m.) was administered. All animals recovered from surgery without any apparent functional alteration. In the experimental series prepared to titrate an optimal survival time for the tracers, the animals were injected with the previously-mentioned fluorochromes mixture and allowed to survive 7 (n 5 3), 10 (n 5 2), 13 (n 5 3), 15 (n 5 2), to 30 (n 5 2) days after the injection. An additional six animals were then injected with the fluorochrome mixture and allowed to survive for 12–14 days. At the completion of each experiment, all the animals were deeply anesthetized with sodium pentobarbital (50 mg/kg i.p.) and perfused transcardially with 10% formalin in PB. The brains were removed and soaked overnight in 10% sucrose in PB. To define accurately the spatial distribution of axonal trajectories, the brains were sectioned at 30 mm in either the coronal (n 5 13), sagittal (n 5 2), or horizontal (n 5 3) planes, using a freezing microtome. The sections were then mounted on gelatin-coated slides, air-dried, and coverslipped with DPX (Fluka Chemika). For

help in the delimitation of the CNS subregions, every third section was processed histochemically for the demonstration of acetylcholinesterase with the Karnovsky and Roots method [27] (Fig. 1C,D). Using a Zeiss Axiophot fluorescence microscope, the blue fluorescent FB-labeled neurons were visualized by means of a UV-H (360 nm excitation wavelength) barrier filter and the red fluorescent FR-labeled fibers and terminal-like elements by barrier filter system H (546 nm excitation wavelength). While examining the sections in fluorescence microscopy, the observed FR-labeled fibers and terminal-like elements and FB-labeled neurons were carefully charted onto standard brain sections, modified from the Paxinos and Watson atlas [42] (see Figs. 4 –10). Terminology for CNS nuclei was based on this atlas [42]. RESULTS After injections of 30 – 45 nl of a solution of FB and FR, the injected area was no wider than 250 –350 mm in diameter, and included the portion of the reticular formation that surrounds the nucleus ambiguus and part of the nucleus ambiguus itself [42] (Figs. 1B–D; 2B,C,E,F; 3A,B). The deposit of tracers did not spread into the lateral reticular nucleus. Each FB and FR injection site consisted of a very small central core of dense crystals of fluorochrome and an outer zone of fluorochrome-labeled glia (Figs. 1D; 2C,F; 3A,B). The features of retrograde and anterograde fluorescent labeling of these dyes were similar to those observed in a previous report [20]. The FB retrogradely labeled cells displayed intensely labeled somata and proximal dendrites (Figs. 3D,F; 7B,E,H; 8D; 10C). The FR-labeled axons were generally grouped in bundles (Figs. 3C,E; 7F; 9D). In contrast, the FR terminal fields were characterized by preterminal fibers with an irregular orientation, branching points and terminal-like varicosities or puncta (Fig. 7C). In several occasions, only a few fibers with several varicosities or puncta (,5) were observed in a nucleus (Fig. 9B). The FR-labelled axons and terminal-like elements and FB-labeled somata were found from medulla to cortex, 10 days after the fluorochrome injections; the intensity and density of FB and FRlabelings were consistent after 13–15 days survival. Less intensely FB-labeled cells were observed following 30 days survival; however, the FR-labeling did not fade out following this survival period. The FR-labeled fibers and terminal-like elements and FBlabeled cells were distributed bilaterally in the examined regions (Figs. 4 – 6). Cerebellum The FB-labeled neurons were observed bilaterally within the deep cerebellar nuclei: medial (fastigial, Med), interposed (Int), and lateral (dentate, Lat) nuclei. The ipsilateral labeled fibers and terminal-like elements were denser than the contralateral ones (Table 1, Fig. 4). Small-sized FB-labeled neurons were observed in the Med nucleus, arranged in two compact subgroups at the borders of the nucleus. This distribution was best observed in the sagittal sections. In the horizontal sections, FB-labeled neurons were also observed at the border of the Med nucleus, close to the ridge of small cells that form the Int dorsolateral hump (IntDL). A few labeled neurons (four to five per section) were also scattered ventral to the Int nucleus. A relative high number of FB-labeled neurons were observed in the Med dorsolateral protuberance (Table 1, Fig. 4). Furthermore, FB-labeled neurons were also observed at the level of the superior cerebellar peduncle (scp) (Fig. 7D). Large-sized FB-labeled neurons were observed in the anterior (IntA) and posterior (IntP) Int nuclei, separated by strands of small-sized FB-labeled neurons and FR-labeled fibers, best observed in the sagittal sections (Fig. 7A,E). The FB-labeled neurons were observed within the dorsolateral Int (IntDL) and Int dorso-

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FIG. 1. Location and electrophysiological identification of a FB and FR mixture injection in the VRG. (A) Extracellular inspiratory multi-unit activity recorded in the rostral ventral respiratory cell group (upper record) and diaphragm electromiography (EMG) showing the respiratory phase (lower record); (B) reconstruction of the extent of the fluorochrome injection on the coronal section photographed in C and D. The coordinate indicates the distance in mm on respect to the Bregma taken as zero reference. (C) Bright-field photomicrograph of the shaded area of the section drawn in B, histochemically defined by the Karnovsky and Root method for acetylcholinesterase [27], showing the location of nucleus ambiguus motoneurons (boxed area) and the fluorochrome injection (arrows). (D) Double exposure (bright-field and fluorescence) photomicrograph (360 nm excitation wavelength) of the shaded area drawn in B and photographed in C, showing the location of the fluorochrome injection (arrows); the boxed area encloses the nucleus ambiguus motoneurons labeled by acetylcholinesterase positivity. Bars: 500 mm. Amb, nucleus ambiguus; CVL, caudoventrolateral reticular nucleus; IO, inferior olive; LRt, lateral reticular nucleus; py, pyramidal tract; RVL, rostroventrolateral reticular nucleus; Sol, solitary tract nucleus; 5, spinal trigeminal tract; S, spinal trigeminal nucleus, interpolar part; 12, hypoglossal nucleus.

medial crest (Table 1). A small group of FB-labeled neurons was found ventral to the IntDL, clearly segregated from those located at the level of the inferior cerebellar peduncle (icp) (Fig. 7G). In addition, FB-labeled neurons were observed in the Lat nucleus, loosely located within its ventromedial parvocellular portion (Figs. 3F; 4), as well as in the caudal pole of its dorsolateral magnocellular portion (Table 1). The FR-labeled fiber bundle efferent from the VRG was observed to run along the entire rostrocaudal extent of medulla and pons [20]. The labeled fiber bundles followed bilaterally the course of the ventral spinocerebellar tract (VSCT). A labeled fiber bundle coursed from the VSCT to the cerebellum (Fig. 7A,D). Few of the FR-labeled fibers were found in the dorsal portion of the medulla

and pons, traversing through the dorsal spinocerebellar tract. This latter group of fibers entered the cerebellum through the scp, inferior cerebellar peduncle (icp), and, to a lesser extent, the middle cerebellar peduncles (Fig. 7G,I). In the cerebellum, the FR-labeled fibers were observed to surround each of the deep cerebellar nuclei (Lat, Int, and Med) (Figs. 3E; 6B,C), and then followed two different pathways up to the cerebellar cortex. One of these fiber bundles coursed through the dorsal spinocerebellar tract and VSCT and the other through the cuneocerebellar tract. The FR-labeled fibers reaching the anterior lobe were also detected through lobules 1–3, giving off collaterals along the vermis and paravermis. The FR-labeled fibers were also seen in the posterior lobe, running through the simple lobule and Crus 2 of the ansiform

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FIG. 2. Location and electrophysiological identification of two different FB and FR mixture injections in the VRG. (A, D) Extracellular inspiratory multi-unit activity recorded in rostral VRG (upper records) and diaphragm electromiography (EMG) showing the respiratory phase (lower records). (B) reconstruction of the extent of the fluorochrome injection on the sagittal section photographed in C. The coordinate indicates the distance in mm lateral to the midline. (C) Fluorescence photomicrograph (546 nm excitation wavelength) of the shaded area of the section drawn in B showing the fluorochrome mixture injection (arrows). (E) reconstruction of the extent of the fluorochrome injection on the horizontal section photographed in F. The coordinate indicates the distance in mm on respect to the Bregma taken as zero reference. (F) Fluorescence photomicrograph (546 nm excitation wavelength) of the shaded area of the section drawn in E showing the fluorochrome mixture injection (arrows). Bars: 500 mm. CVL, caudoventrolateral reticular nucleus; LVe, lateral vestibular nucleus; LRt, lateral reticular nucleus; MdD, medullary reticular nucleus, dorsal part; ROb, raphe obscurus nucleus; RVL, rostroventrolateral reticular nucleus; Sol, solitary tract nucleus; p5, spintal trigeminal tract; 7, facial nucleus.

lobule of the pars lateralis, and to a lesser extent through Crus 1 (Figs. 6B,C; 7F). The FR-labeled fiber bundles crossed the midline through the uncinate peduncle and icp, connecting the lateral lobules and deep cerebellar nuclei of both sides. Therefore, they were observed to run near the preculminate and primary fissures

and lobule 8 (Fig. 6B,C). Another group of decussating fibers was observed at the level of lobule 1. In the deep cerebellar nuclei, the FR-labeled fibers and terminal-like structures were intermingled with the retrogradely FBlabeled cells and surrounding them, with a predominant ipsilateral

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FIG. 3. Fluorescence photomicrographs, taken under 546 nm excitation wavelength (A, C, E) and under 360 nm excitation wavelength (B, D, F), of horizontal sections. (A and B) Fluorescence photomicrographs of the shaded area of the section drawn in Fig. 2E showing the same fluorochrome mixture injection on the horizontal section in Fig. 2F. Bars: 150 mm. (C and D) Fluorescence photomicrographs of the contralateral VRG following the fluorochrome mixture injection photographed in A and B. Note in C the FR-labeled fibers and in D the FB-labeled neurons. The asterisks point at blood vessels taken as reference landmarks. (E) Fluorescence photomicrograph of FR-labeled fibers surrounding the lateral cerebellar nucleus on a horizontal section following the fluorochrome mixture injection photographed in A and B. (F) Fluorescence photomicrograph of FB-labeled neurons within the lateral cerebellar nucleus on a horizontal section following the fluorochrome mixture injection photographed in A and B. Bars: 500 mm. Lat, lateral (dentate) cerebellar nucleus; RVL, rostroventrolateral reticular nucleus; p5, spinal trigeminal tract.

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and Lat cerebellar nuclei (Table 2, Fig. 6B,C). Finally, labeled terminal-like elements were also found at the cerebellar lobules 2 and 3, simple lobule (Table 2, Fig. 7A,C,D,F), and Crus 1 and Crus 2 of the ansiform lobule. Midbrain

FIG. 4. Schematic summary drawing of coronal sections of the cerebellum, modified from the Paxinos and Watson atlas [42], showing the distribution of FB-labeled somata (squares, each square represents two labeled somata) following the FB and FR mixture injection into the rostral VRG. The data are from a representative single experiment (section thickness: 30 mm). The coordinates indicate the distance on respect to the Bregma taken as zero reference 1–10, cerebellar lobules; Crus 1–2, Crus 1–2 ansiform lobule; ICF, intercrural fissure; IntA, interposed cerebellar nucleus, anterior part; IntDL, interposed cerebellar nucleus, dorsolateral hump; IntDM, interposed cerebellar nucleus, dorsomedial; IntP, interposed cerebellar nucleus, posterior part; Lat, lateral (dentate) cerebellar nucleus; Med, medial (fastigial) cerebellar nucleus; MedDL, medial cerebellar nucleus, dorsolateral part; PCF, preculminate fissure; PF, paraflocculus; PSF, posterior superior fissure.

distribution. As a rule, dense fields of FR-labeled terminal-like elements overlapped FB-labeled somata. The FR-labeled fibers surrounded the Lat nuclei of both sides, giving off terminal-like elements within the Lat nuclei and at their periphery (Fig. 3E,F). The IntA nucleus showed the greatest density of terminal-like fibers, followed by the Med, IntP, and IntDL subnuclei (Fig. 6B,C). The FR-labeled terminal-like elements were observed within the Med dorsolateral protuberance, Med, IntA, IntP, IntDL,

In all cases, FR-labeled fibers and terminal-like structures were intermingled with retrogradely FB-labeled neurons (Tables 1 and 2). As a rule, the FB-labeled neurons were more densely aggregated in the ipsilateral than in the contralateral side. In the ventral mesencephalon, FB-labeled neurons were found predominantly ipsilaterally in the ventrolateral and intermediate parts of the nucleus of the lateral lemniscus and paralemniscal area (Table 1, Fig. 5B). The FB-labeled somata formed a continuum with those located in the pons and medulla (not described in the present report), as a compact formation. A scarce number of multipolar FB-labeled neurons were also found within the deep white and gray layers and in the intermediate white layer of the superior colliculus, bilaterally (Table 1). The former neurons were more densely aggregated than those found in the intermediate white layer (Fig. 5B). The FB-labeled neurons were found bilaterally at the level of the mesencephalic tegmental nuclei (Table 1). The microcellular tegmental nucleus exhibited small-sized FB-labeled somata. Labeled neurons were also observed in the laterodorsal tegmental nucleus, in the parapedunculopontine tegmental nucleus, and in the oral part of the pontine reticular nucleus (Fig. 5B), as well as the adjacent area of the latter nucleus. The FB-labeled neurons were found in the cuneiform nucleus (Table 1, Fig. 5B). Scattered FB-labeled neurons were predominantly found in the caudal part of the linear nucleus of the raphe (Table 1). Scattered FB-labeled somata (two to four per section) were found bilaterally in the deep mesencephalic nucleus, intermediate zone, the substantia nigra, reticular and compact parts, the substantia innominata, and in the red nucleus, caudal, and magnocellular parts (Fig. 5B). On the other hand, FB-labeled somata were observed bilaterally at the border with the 3rd ventricle, within the different neuronal cell groups of the CG (Table 1, Fig. 5B). The labeled neurons were more numerous in the rostral and caudal areas than in the medial one, and were organized as a compact group in the caudal and rostral regions of CG and loosely in its medial zone. The neurons in the compact groups presented a larger soma size than the loosely organized ones (Fig. 8A–D). Smallsized FB-labeled somata were loosely distributed in the region surrounding the basal part of the cerebral peduncle, with an ipsilateral predominance (Table 1). Bundles of FR-labeled fibers were observed bilaterally in the adjacent areas of the collicular and the ventral and lateral tegmental tracts, the interpeduncular fossa, substantia nigra, and CG. Labeled fiber bundles were also observed through the superior and inferior colliculi commissures, the central nucleus of the inferior colliculus, the tegmental area, and the nucleus subcoeruleus, ventral part. A bundle of FR-labeled fibers was observed to traverse the pedunculopontine tegmental area and cuneiform nucleus, and through the tectospinal, medial lemniscal, and icp tracts (Fig. 6A). Compact FR-labeled fiber bundles were observed within the paralemniscal and lateroventral lemniscal nuclei. Sparse fiber bundles were observed to pass through the intercollicular nucleus, medial lemniscus, and different subregions of the raphe (Fig. 6A). FR-labeled fibers were observed in the deep mesencephalic nucleus, forming a thin compact bundle, giving off a few branches and a scarce number of “en passant” FR-labeled varicosities (Table 2). Finally, the fibers were followed along their ascending trajectory through the scp, and reached the Edinger-Westphal nucleus,

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631 where they crossed the midline, giving off “en passant” varicosities (Table 2, Fig. 6B). The FR-labeled fibers crossed the midline at different levels through the commissures of the superior and inferior colliculli (Figs. 6B,C; 9C,E). The FR-labeled terminal-like elements were observed bilaterally within the lateral lemniscus, deep gray layer, deep white layer, and intermediate white layer of the superior colliculus, and laterodorsal and microcellular tegmental nuclei. At the level of the CG, the FR-labeled fibers displayed terminal-like elements, usually surrounding the FB-labeled neurons (Table 2, Fig. 6A,B). Finally, terminallike elements were observed within the nucleus subcoeruleus, predominantly ipsilateral to the injection site (Table 2). Diencephalon Hypothalamic connectivity of the rostral VRG. The FB-labeled somata were observed in the septohypothalamic nucleus (Fig. 5A). The FB-labeled neurons were also observed in the lateral mammillary nucleus, and lateral hypothalamic area (Fig. 5A,B), where the neurons were loosely distributed and predominantly ipsilateral. Labeled neurons were also found bilaterally in the posterior hypothalamic area (Table 1). A few (two to four per section) smallsized FB-labeled neurons were scattered through the zona incerta, the hypothalamic supraoptic and retrochiasmatic nuclei, as well as the anterior hypothalamic area (Figs. 5; 8E,F). The FR-labeled fibers were less numerous in the diencephalon than in the mesencephalon areas. The FR-labeled fibers were observed bilaterally through the mammillary nuclei, lateral hypothalamic and posterior hypothalamic areas, and septohypothalamic nucleus. The FR-labeled fibers coursed through the zona incerta on their way to rostral decussations (Fig. 9C,D). The FR-labeled fibers gave off small branches and terminal-like elements in the premammillary, lateral mammillary, medial mammillary (medial and posterior parts), septohypothalamic, supraoptic nuclei, and posterior and lateral hypothalamic areas. In two of the experiments a few (,5 per section) FR-labeled terminal-like elements were observed in the dorsal area of the zona incerta (Table 2, Fig. 6A–C). Thalamic connectivity of the rostral VRG. The FB-labeled neurons were observed in the thalamic parafascicular nucleus, predominantly contralateral to the injection site. The FB-labeled neurons were also observed in the thalamic mediodorsal nucleus, with a slight ipsilateral predominance. The FB-labeled neurons were observed bilaterally in the thalamic posterior nuclear group, medial part, and subparafascicular nucleus. A scarce number of FB-labeled neurons were also observed in the nucleus gelatinosus (Table 1, Fig. 5A). The FR-labeled fibers and varicosities (,5 per section) were found in the subthalamic nucleus (Table 2). Finally,

FIG. 5. (A–B) Schematic summary drawing of coronal sections, modified from the Paxinos and Watson atlas [42], showing the general distribution of FB-labeled somata (squares, each square represents two labeled somata) following the FB and FR mixture injection into the rostral VRG. The data are from a representative single experiment (section thickness: 30 mm). The coordinates indicate the distance on respect to the Bregma taken as zero reference. aca, anterior commissure; AcbC, accumbens nucleus; AI, agranular insular cortex; APir, amygdalopiriform transition area; APTV, anterior pretectal nucleus, ventral part; CA1–3, fields CA1–3 of Ammon’s horn; CG, central (periaqueductal) gray; Cg2, cingulate cortex, area 2; cp, cerebral peduncle, basal part; CPu, caudate putamen (striatum); DG, dentate gyrus; dhc, dorsal hippocampal commissure; D, nucleus of Darkschewitsch; DLG, dorsal lateral geniculate nucleus; DpMe, deep mesen-

cephalic nucleus; DR, dorsal raphe nucleus; ECIC, external cortex of the inferior colliculus; Ent, entorhinal cortex; EP, entopeduncular nucleus; f, fornix; G, gelatinosus thalamic nucleus; GI, granular insular cortex; HiF, hippocampal fissure; IF, interfascicular nucleus; IL, infralimbic cortex; InCo, intercollicular nucleus; InWh, intermediate white layer of the superior colliculus; LA, lateroanterior hypothalamic nucleus; LH, lateral hypothalamic area; LO, lateral orbital cortex; LSI, lateral septal nucleus; ml, medial lemniscus; MnR, median raphe nucleus; ox, optic chiasm; Par 1–3, parietal cortex, area 1–3; PaS, parasubiculum; PH, posterior hypothalamic area; PnO, pontine reticular nucleus, oral part; Po, posterior thalamic nuclear group; PRh, perirhinal cortex; PT, paratenial thalamic nucleus; py, pyramidal tract; Re, reuniens thalamic nucleus; RI, rostral interstitial nucleus of the medial longitudinal fasciculus; S, subiculum; scp, superior cerebellar peduncle; SNR, substantia nigra; SOR, supraoptic nucleus, retrochiasmatic part; TC, tuber cinereum area; TT, tenia tecta; Tu, olfactory tubercle; VDB, nucleus of the vertical limb of the diagonal band; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; xscp, decussation of the superior cerebellar peduncle; ZI, zona incerta.

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FIG. 6. (A–C) Schematic summary drawing of horizontal sections, modified from the Paxinos and Watson atlas [42], showing the general distribution of FR-labeled fibers (thin lines) and terminal-like elements (dots) following the FB and FR mixture injection into the rostral VRG. The sections were represented along a ventral– dorsal (top– bottom) axis and the data are from a representative single experiment (section thickness: 30 mm). The coordinates indicate the distance on respect to the Bregma taken as zero reference. ACo, anterior cortical amygdaloid nucleus; AHi, amygdalohippocampal area; APir, amygdalopiriform transition area; APT, anterior pretectal nucleus; CA1–3, fields CA1–3 of Ammon’s horn; CIC, central nucleus of the inferior colliculus; CLi, caudal linear nucleus of the raphe; Cop, copula pyramis; cp, cerebral peduncle, basal part; Crus 1–2, Crus 1–2 ansiform lobule; ctg, central tegmental tract; Cu, cuneate nucleus; DG, dentate gyrus; dhc, dorsal hippocampal commissure; Dk, nucleus of Darkschewitsch; DLG, dorsal lateral geniculate nucleus; DR, dorsal raphe nucleus; Ent, entorhinal cortex; EP, entopeduncular nucleus; EW, Edinger-Westphal nucleus; f, fornix; FF, fields of Forel; fmj, forceps major of the corpus callosum; HiF, hippocampal fissure; ic, internal capsule; ICF, intercrural fissure; IF, interfascicular nucleus; InCo, intercollicular nucleus; IPR, interpeduncular nucleus, rostral subnucleus; LA, lateroanterior hypothalamic nucleus; La, lateral amygdaloid nucleus; Lat, lateral (dentate) cerebellar nucleus; LLF, lateral lemniscal fields; LOT, nucleus of the lateral olfactory tract; MDC, mediodorsal thalamic nucleus, central part; MGV, medial geniculate nucleus, ventral part; ml, medial lemniscus; MnR, median raphe nucleus; Mo5, motor trigeminal nucleus; Op, optic nerve layer of the superior colliculus; ox, optic chiasm; PaS, parasubiculum; PF, paraflocculus; PH, posterior hypothalamic area; PM, paramedian fissure; PnO, pontine reticular nucleus, oral part; Po, posterior thalamic nuclear group; PrF, primary fissure; PRh, perirhinal cortex; PSF, posterior superior fissure; R, red nucleus; RI, rostral interstitial of the medial longitudinal fasciculus; S, subiculum; SC, superior colliculus; scp, superior cerebellar peduncle; Sim, simple lobule; sm, stria medullaris of the thalamus; SNR, substantia nigra, reticular part; SubCV, nucleus subcoeruleus, ventral part; SubI, subincertal nucleus; TC, tuber cinereum area; Te1, temporal cortex, area 1; ts, tectospinal tract; VCP, ventral cochlear nucleus, posterior part; VPM, ventral posteromedial thalamic nucleus; ZI, zona incerta. 1–10, cerebellar lobules; 3n, oculomotor nerve or its root; 3V, 3rd ventricle; 7, facial nucleus; 12, hypoglossal nucleus.

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TABLE 1 FAST BLUE-LABELED NEURONS FOUND AFTER THE INJECTION OF THE FLUOROCHROME INTO THE ROSTRAL VENTRAL RESPIRATORY CELL GROUP OF THE RAT Density Structures Containing FB-Labeled Neurons

Cerebellum Medial (fastigial) cerebellar nucleus Medial cerebellar nucleus, dorsolateral protuberance Interposed cerebellar nucleus, anterior part Interposed cerebellar nucleus, posterior part Interposed cerebellar nucleus, dosolateral hump Interposed cerebellar nucleus, dorsomedial crest Lateral (dentate) cerebellar nucleus Nuclei of the pons and midbrain Lateral lemniscus Paralemniscal area Deep gray layer of the superior colliculus Deep white layer of the superior colliculus Intermediate white layer of the superior colliculus Laterodorsal tegmental nucleus Microcellular tegmental nucleus Parapedunculopontine tegmental nucleus Pontine reticular nucleus, oral part Caudal linear nucleus of the raphe Cuneiform nucleus Caudal central (periaqueductal) gray Medial central (periaqueductal) gray Rostral central (periaqueductal) gray Surrounding area of the cerebral peduncle, basal part Diencephalon Septohypothalamic nucleus Lateral mammillary nucleus Lateral hypothalamic area Posterior hypothalamic area Parafascicular thalamic nucleus Mediodorsal thalamic nucleus Posterior thalamic nuclear group, medial part Subparafascicular thalamic nucleus Gelatinosus thalamic nucleus Telencephalon Parastrial nucleus Olfactory tubercle Granular insular cortex Agranular insular cortex Parietal cortex, area 1 Lateral orbital cortex a

Ipsi

Contra

23a (5–27)b

6 (3–12)

12 (2–16)

3 (0–4)

11 (2–15)

2 (1–8)

9 (3–12)

3 (0–4)

2 (0–5)

1 (0–2)

8 (2–16) 13 (2–18)

3 (1–4) 4 (0–5)

15 (6–23) 26 (21–30)

3 (2–8) 10 (8–19)

2 (0–6)

4 (0–8)

2 (0–5)

4 (0–9)

1 (1–2) 16 (4–32) 23 (12–30)

4 (1–5) 17 (5–31) 11 (2–23)

4 (1–5) 2 (0–3)

6 (1–8) 4 (4–8)

12 (4–18) 6 (2–10) 4 (1–5)

sparsely distributed small-sized FB-labeled neurons (1–3) were observed in the gustatory, paratenial, reticular, and reuniens nuclei (Fig. 5A). Loosely arranged FR-labeled fibers reached the thalamus through the surpramammillary and tegmental decussationes (Fig. 9E). Only FR-labeled fibers of passage were found in the thalamic reticular nucleus (Fig. 6C). Less than five FR-labeled varicosities per section was observed in the thalamic parafascicular nucleus and mammillothalamic tract. Telencephalon Amygdaloid connectivity of the rostral VRG. The FB-labeled cells were observed in the parastrial nucleus, at the medial pole of the ventral subdivision of the bed nucleus of the stria terminalis; these neurons were predominantly contralateral (Table 1). The FB-labeled neurons (one to two in some sections) were also observed within the bed nucleus of the stria terminalis, medial division, posterior medial, and ventral subdivisions of the globus pallidus. The FR-labeled fibers and varicosities (,5 per section) were found in the amygdalohippocampal area (which could only be observed in the horizontal sections, Figs. 6A; 9B) (Table 2). Scattered FR-labeled fibers were also observed to traverse the medial, posteroventral, and posteromedial cortical amygdaloid nuclei. The FR-labeled varicosities (,5 per section) were observed through the bed nucleus of the stria terminalis, medial division posteromedial part, and the fornix (Fig. 6A). Source of afferents to the rostral VRG. A high number of small-sized FB-labeled neurons were observed in the olfactory tubercle, predominantly ipsilateral (Table 1, Fig. 10D,E). The FB-labeled neurons were also observed in the parietal cortex, predominantly contralateral. FB-labeled neurons were observed at the area 2, adjacent to area 1 (Table 1, Fig. 5A). On the one hand, FB-labeled somata were densely packed in the insular cortex, predominantly ipsilateral in the granular and bilateral in the agranular insular cortex (Table 1, Figs. 5A and 10A–C). On the other hand, a group of FB-labeled somata was also observed in the lateral orbital cortex, predominantly ipsilateral to the injection site (Table 1). Additionally, FB-labeled neurons (1– 4) were observed in the frontal cortex.

35 (12–40)

32 (8–35)

7 (4–8)

5 (3–7)

16 (13–21)

17 (14–22)

DISCUSSION

5 (0–6)

11 (4–23)

26 (21–32) 15 (10–17) 10 (4–12) 6 (2–7) 4 (2–5) 8 (1–12)

13 (11–17) 5 (2–10) 5 (2–10) 14 (4–22) 11 (8–19) 3 (1–4)

6 (1–7) 11 (6–14) 2 (0–3)

6 (2–7) 10 (5–16) 4 (0–6)

5 (3–6) 20 (12–22) 9 (8–10) 11 (13–19) 1 (1–3) 6 (2–8)

13 (6–17) 2 (0–3) 1 (0–2) 12 (9–17) 22 (19–28) 1 (0–1)

The present study that extends previous studies concerning the VRG connectivity within the pons and medulla [20,39], based on double labeling with FB and FR demonstrated simultaneously the pattern of distribution of the VRG afferent and efferent projections in the cerebellum, midbrain, diencephalon, and telencephalon in the rat. The circuitry identified here may provide a structural basis for the suprapontine modulation of respiratory-related functions such as vocalization and swallowing. It is known that the rostral VRG area includes motoneurons that control the upper airways, intermingled with neurons that influence respiration and other autonomic functions. Separating these populations from each other would be a very different task demanding other experimental approaches. The problem of fibers of passage is crucial in the present experiments. Some of the control animals, where the injection area was located ventrolaterally or ventromedially to the VRG, presented a different pattern of labeling. For example, after injections of an anterograde marker into a region located ventromedially to the VRG, FR-labeled fibers and varicosities were found in the nucleus of the solitary tract and area postrema. These results agree with previous data [16]. These data constitute a validation of the

The mean number of labelled neurons from 7 rats; b range.

634

´ N AND PA ´ SARO GAYTA

FIG. 7. (A, D, G) Drawings of sagittal sections of the cerebellum showing the distribution of FR-labeled fibers (lines) and terminal like elements (dots), and FB-labeled neurons (squares, each square represents two labeled somata) following the FB and FR mixture injection into the rostral VRG. (B, E, H) Fluorescence photomicrographs (546 nm excitation wavelength) of blue fluorescent FB-labeled somata on the light shaded areas of the sections drawn in A, D, and G. Bars: 125 mm. (C, F, I) Fluorescence photomicrographs (360 nm excitation wavelength) of red-fluorescent FR-labeled fibers and terminal like elements (arrows) on the dark shaded areas of the sections drawn in A, D, and G. Bars: 500 mm. The coordinates indicate the distance in mm lateral to the midline. Cop, copula pyramis; ICF, intercrural fissure; IntA, interposed cerebellar nucleus, anterior part; IntDL, interposed cerebellar nucleus, dorsolateral hump; IntP, interposed cerebellar nucleus, posterior part; LatPC, lateral cerebellar nucleus, parvocellular; PPF, prepyramidal fissure; Pr, primary fissure; Sim, simple lobule.

specificity of the location of the injection. However, the presence of spurious anterograde or retrograde labeling due to uptake of fibers of passage cannot be completely discarded with the present methodology. Cerebellum In a recent study, it has been demonstrated that the Med nucleus can modulate the respiratory output via influences on medullary-

respiratory neurons [70]. The present results pointed out an anatomical substrate for this influence. In the present study, neurons within the Int nuclei that project to the rostral VRG neuronal cell group have been identified. Furthermore, descending Lat nucleus projections terminate in the rostral VRG neuronal cell group (Fig. 11). A similar but more widespread terminal field of Lat nucleus efferents was demonstrated earlier by autoradiography in the rat [12].

ROSTRAL VRG PROJECTIONS IN THE RAT

635

TABLE 2

Midbrain

FLUORO RUBY-LABELED TERMINAL-LIKE ELEMENTS FOUND AFTER THE INJECTION OF THE FLUOROCHROME INTO THE ROSTRAL VENTRAL RESPIRATORY CELL GROUP OF THE RAT FR-Labeled Terminal-like Elements Labeled Nuclei

Cerebellum Medial (fastigial) cerebellar nucleus Medial cerebellar nucleus, dorsolateral protuberance Interposed cerebellar nucleus, anterior part Interposed cerebellar nucleus, posterior part Interposed cerebellar nucleus, dosolateral hump Lateral dentate nucleus Simple cerebellar lobule Crus 2 of the ansiform lobule Cerebellar lobule 2 Cerebellar lobule 3 Nuclei of the pons and midbrain Lateral lemniscus Deep gray layer of the superior colliculus Deep white layer of the superior colliculus Intermediate white layer of the superior colliculus Deep mesencephalic nucleus Laterodorsal tegmental nucleus Microcellular tegmental nucleus Edinger-Westphal nucleus Caudal central (periaqueductal) gray Medial central (periaqueductal) gray Rostral central (periaqueductal) gray Nucleus subcoeruleus, ventral part Diencephalon Septohypothalamic nucleus Lateral hypothalamic area Posterior hypothalamic area Premammillary nucleus, ventral part Lateral mammillary nucleus Medial mammillary nucleus, medial part Medial mammillary nucleus, posterior part Zona incerta Supraoptic nucleus, retrochiasmatic (diffuse) part Subthalamic nucleus

Ipsi

Contra

**

*

* ** ** ** * * * * *

* * * * * */2 */2 */2 */2

* * * * * * * * * * * **

* * * * * * * * * * * *

* * * * * * * * * *

* * * * * * * * * *

a The anterogradely labeled fibers and terminal-like elements were counted, in samples of 0.1 mm2 per section; the semiquantitative estimate refers to the mean number from seven rats; p/2, ,5 labeled terminal-like elements, in some of the experiments or in some of the sections; p, 5–29 labeled terminal-like elements; pp, 30 –50 labeled terminal-like elements.

Experiments made by electrolytic lesions in the lateral reticular nucleus [35] involving the nucleus ambiguus [37] and area A1 [14], partly overlapping our injection area, also showed efferents reaching the cerebellum. Other studies demonstrated the presence of fibers destined to lobules 1–5, and scanty terminals in the most medial part of Crus 1 [35] in the ipsilateral cerebellar cortex, arising from the nucleus ambiguus and periambigual area [39]. Furthermore, horseradish peroxidase (HRP) injections made into the anterior lobe, the nodulus, the flocculus, and the Med nucleus, labeled large or medium-sized cells bilaterally around the nucleus ambiguus [29].

The CG is known to play a role in vocalization and swallowing as well as other functions [32,74]. Although the principal source of projections to the CG arises from the midbrain and diencephalon, some studies have demonstrated inputs originating in the brain stem [6,7]. Furthermore, it has been shown by means of anterograde tracing with Phaseolus vulgaris leucoagglutinin at different subregions of CG project to the ventrolateral medulla including the VRG [7,63]. Recent studies have also suggested that the CG neurons related to different functions may be organized in different overlapping longitudinal columns [7]. In the present study, we have found that CG neurons projecting to the VRG neuronal cell group were distributed into three different groups, pointing out an organization of the CG medullary efferent projections. The reciprocal link between the CG and the rostral VRG could mediate the coordination of respiratory, laryngeal, and orofacial activity for vocalization and other respiratory-related functions (Fig. 11). Our results showed that the tegmental nuclei, pedunculopontine, and laterodorsal are bilaterally connected with the VRG, consistent with previous descriptions [50,69]. The microcellular tegmental nucleus is connected with the interpeduncular and cuneiform nuclei [41]. Recent studies also described bilateral neuronal connections between the cuneiform nucleus and the ventrolateral medulla [31]. On the one hand, the reciprocal connectivities between the rostral VRG and the cuneiform and deep mesencephalic nuclei pointed out by the present study suggest that the respiratory-related neurons of the VRG may be modulated in different states of arousal and/or selective attention [53,65] (Fig. 11). On the other hand, the raphe nuclei, forming part of the serotonergic system [33,64], have been related to cardiorespiratory and other autonomic functions. The present finding of a projection from the caudal linear nucleus of the raphe to the VRG (Fig. 11) links this nucleus with respiratory-related neurons. The EdingerWestphal nucleus has been described as the origin of parasympathetic preganglionic neurons [28]. The present results have shown a VRG projection to the Edinger-Westphal nucleus, probably deriving from the periambigual parasympathetic neurons [37]. It has been shown in the cat that the inferior colliculi receive inputs from different brain stem nuclei, and these fibers are grouped in a tract crossing through the lateral lemniscus [46]. However, there is not conclusive evidence of such organization in the rat. Contributing to this issue, we have found a reciprocal connection between the inferior colliculi and the rostral VRG neuronal cell group. Furthermore, the superior colliculi are interconnected with the rostral VRG [44]. This pattern of organization could imply that neurons involved in the defense reaction could also integrate respiratory-related activities (Fig. 11), as well as visual stimuli. Finally, the nucleus subcoeruleus, a noradrenergic cell group, is considered to have a role in the modulation of pain and modification of motor behaviors such as locomotion [60,66]. The present finding that rostral VRG neuronal cell group projects to the nucleus subcoeruleus suggests that the latter nucleus could also integrate respiratory information (Fig. 11). Diencephalon The present findings showed that the septohypothalamic nucleus projects to and receives afferents from the VRG (Fig. 11). It is known that the mammillary body receives inputs from the brain stem [66]. The finding of rostral VRG efferent projections to the mammillary nuclei is consistent with previous descriptions. Furthermore, the lateral mammillary nucleus is reciprocally connected with the rostral VRG (Fig. 11). From the present results it could be pointed out that the septohypothalamic nucleus and mammillary

636

´ N AND PA ´ SARO GAYTA

FIG. 8. (A, C, E) Drawings of horizontal (A and E) and coronal (C) sections through the midbrain showing the distribution of FB-labeled neurons (squares, each square represents two labeled somata), following the FB injection into the rostral VRG. (B, D, F) Fluorescence photomicrographs (546 nm excitation wavelength) of blue fluorescent FB-labeled somata on the shaded areas of the sections drawn in A, C, and E. (A–D) FB-labeled neurons in the periaqueductal gray. The white squares point at two different somata sizes of the FB-labeled neurons. (E and F) FB-labeled neurons in the posterior hypothalamic area. Coordinates: as in Fig. 3. Bars: 500 mm. CG, central (periaqueductal) gray; ECIC, external cortex of the inferior colliculus; f, fornix; MD, mediodorsal thalamic nucleus; MGM, medial geniculate nucleus, medial part; PH, posterior hypothalamic area; PRh, perirhinal cortex; 3V, 3rd ventricle.

ROSTRAL VRG PROJECTIONS IN THE RAT

FIG. 9. (A and C) Drawing of horizontal sections showing the distribution of red fluorescent FR-labeled fibers and terminal like elements, following the FR injection into the rostral VRG. (B, D, E) Fluorescence photomicrographs (360 nm excitation wavelength) of red fluorescent FR-labeled fibers and terminal like elements (arrows) on the shaded areas of the sections drawn in A and C; (B) FR-labeled fibers and terminal like elements in the amygdalohippocampal area; (D) FR-labeled fibers traversing the zona incerta; (E) FR-labeled fibers crossing the midline at the level of the decussation of the superior cerebellar peduncle. Bars: 250 mm. Coordinates: as in Fig. 3. AHi, amygdalohippocampal area; APir, amygdalopiriform transition area; ic, internal capsule; LA, lateroanterior hypothalamic nucleus; R, red nucleus; xscp, decussation of the superior cerebellar peduncle; ZI, zona incerta.

637

´ N AND PA ´ SARO GAYTA

638

FIG. 10. (A and D) Drawings of horizontal sections showing the distribution of FB-labeled neurons (squares, each square represents two labeled somata), following the FB injection into the rostral VRG. (B, C, E) Fluorescence photomicrographs (546 nm excitation wavelength) of blue fluorescent FB-labeled somata on the shaded areas of the sections drawn in A and C. (B and C) FB-labeled neurons in the granular insular cortex, C is a higher magnification of the boxed area in B. The arrows indicate the same FB-labeled neurons. (E) FB-labeled neurons in the olfactory tubercle. Coordinates: as in Fig. 3. Bars: 500 mm. AcbC, accumbens nucleus, core; CPu, caudate putamen (striatum); GI, granular insular cortex; Par1, parietal cortex, area 1; Po, posterior thalamic nuclear group; TT, tenia tecta; Tu, olfactory tubercle.

body participate in accessory control of respiratory-related activities. Neurons of the lateral hypothalamic area send projections to widespread regions in the brain stem, which are mostly reciprocal.

These target regions include nuclei that relay visceral sensory information to the diencephalon and to the brain stem, such as the nucleus of the solitary tract, parabrachial nucleus [51,57,62], and other nuclei involved in somatomotor control mechanisms.

ROSTRAL VRG PROJECTIONS IN THE RAT

639

FIG. 11. Diagram showing the sources of afferents and targets of efferent projections of the rostral VRG.

The posterior nucleus of the hypothalamus projects to the brain stem, including the ventrolateral medulla: nucleus reticularis parvocellularis and the rostral ventrolateral medullary region [26,67]. The posterior hypothalamic region has been linked to the control of breathing, cardiovascular activity, locomotion, antinociception, and arousal/wakefulness [40,50,53,57]. In this context, the present described projections pointed out a role for the rostral VRG (Fig. 11) as final common pathway between the diencephalon/lower

brain stem and spinal cord systems in the control of respiratoryrelated functions [13]. The supraoptic nucleus plays an important role in neurovegetative control. The supraoptic nucleus receives abundant inputs from the A1 group of the ventrolateral medulla [52], and the present results indicate that it is also a target of rostral VRG efferents. Therefore, these data indicate that VRG connection to several hypothalamic nuclei may integrate respiratory commands (Fig. 11).

640 The thalamic intralaminar nuclei constitute a diverse group of structures that receive inputs from the brain stem reticular formation [17]. The rostral VRG receives projections from the mediodorsal nucleus and the agranular insular cortex (Fig. 11). On the one hand, the connection between the thalamic parafascicular and subparafascicular nuclei and the rostral VRG shown in the present study may provide an anatomical basis for the thalamic control of respiratory-related neurons. Furthermore, the VRG could integrate thalamic and cortical inputs. On the other hand, the rostral part of the thalamic posterior nuclear group possesses different connections and functions than its caudal part [43]. It is noteworthy that neurons projecting to the rostral VRG were found in the present study to be confined to the rostral part of the thalamic posterior nuclear group. The present results have also shown that the rostral VRG projects to the amygdalohippocampal area, pointing out that the amygdala may also integrate respiratory inputs as part of its integrative role of autonomic functions [1]. Furthermore, the subthalamic nucleus is a key structure in the basal ganglia circuitry that influences motor activities [24]. To date, projections from the subthalamic nucleus to the brain stem reticular formation have not been well documented. The present data showed that the subthalamic and parastrial nuclei project to the rostral VRG (Fig. 11). These connections indicate that the rostral VRG is interconnected with higher structures involved in motor activities, which in turn would require some degree of coordination of respiratory movements. Telencephalon The olfactory tubercle mediates autonomic functions through a polysynaptic pathway connecting it to the nucleus ambiguus, periambigual area, and ventrolateral medulla, providing a link for the control of sympathetic and parasympathetic coordination [56]. In the present results, a direct projection has also been demonstrated to the rostral VRG, that might participate in the modulation of olfactory behaviors, such as sniffing and sneezing. The rat prefrontal cortex includes the agranular insular and frontal cortices, which are primarily involved in motor functions [2,73]. In the present results, connections between the frontal cortex and the rostral VRG have been demonstrated (Fig. 11), which could imply a direct cortical control of respiratory-related neurons. In fact, the general visceral areas of the insular cortex, granular and agranular, possess neurons responding to respiratory stimulation and they are somatotopically organized [49]. Recent studies have shown the existence of direct pathways from cortical motor areas towards the main cardiovascular medullary nuclei: the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, and the rostral ventrolateral medulla. These areas probably program cardiovascular adjustments, preparatory, or concomitant to the control of striated muscles [3]. Our results could imply a similar pathway programming respiratory adjustments. It is noteworthy that the preponderance of visceral sensory afferents to the insular cortex terminates in the granular and dysgranular fields, whereas the prominent efferent projections from the insular cortex to autonomic structures arise from the agranular field. The present results also show bilateral projections from the agranular insular cortex to the rostral VRG, in agreement with previous data (Fig. 11) [2]. The parietal areas receive projections from cutaneous mechanoreceptors, propioceptive sensory information, and have been involved in the control of autonomic functions [73]. In the present study, the Par 1 and Par 2 areas efferent projections to the rostral VRG could imply that they are also involved in the control of

´ N AND PA ´ SARO GAYTA motor activities such as breathing, vocalization, and swallowing (Fig. 11). CONCLUSION We have previously proposed that the rostral VRG plays an integrative role in respiratory control, similar to that exerted by the solitary tract nuclei in cardiovascular control. This assumption, based on the demonstration of VRG connections with pontomedullary, cardiovascular, and premotor pathways to the spinal cord [20,38,39], is further supported by the present findings. In the present study, rostral VRG has been found to be interconnected with different cerebellar, midbrain, diencephalic, and telencephalic areas related to autonomic functions. These results provide an anatomical basis for the complex circuitry underlying autonomic control. These connections also provide an anatomical support for the integration of respiratory-related activities in complex behavioral responses. ACKNOWLEDGEMENTS

We are indebted to Dr. Juan Ribas for his critical and helpful comments on this manuscript. This work was supported by grants from the D.G.I. C.Y.T. PB94-1443 and the Junta de Andalucı´a.

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37.

38. 39. 40. 41.

42. 43. 44.

45. 46. 47.

48. 49.

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