Identification of central nervous system neurons

PII S0361-9230(01)00674-8Identification of central nervous system neurons innervating the respiratory muscles of the mouse:A transneuronal tracing studySusana P.Gayta ´n,1*Rosario Pa ´saro,1Patrice Coulon,2Michelle Bevengut 3and Ge ´rard Hilaire 31Department of Animal Physiology and Biology,University of Sevilla,Sevilla,Spain;2FRE CNRS,Neurocybernetique Cellulaire,Marseille,France;and 3FRE CNRS,Development and Pathology of Movement,Marseille,FranceABSTRACT:In recent years,the central control of breathing in mammals has been the subject of numerous studies.The aim of the present one was to characterize the neuronal network pro-jecting to the main respiratory motoneurons,in adult mice.To this end,the morphology and location of the respiratory mo-toneurons and their sequential connections with other neurons were revealed using a transneuronal tracing technique by means of the rabies virus infection.The injections of the rabies virus in the respiratory muscles resulted in labeling the mo-toneurons and their serially connected interneurons at multiple levels of the mouse central nervous system:spinal cord,pons and medulla,cerebellum,mesencephalon,diencephalon,and telencephalon.Most of these labeled areas have been previ-ously identified in the control of cardiorespiratory regulation,as well as in other autonomic functions.These anatomical data provide support for the integration of respiratory-related activ-ities in complex behavioral responses.Furthermore,these data suggest similarities in the evolution of central respiratory net-works in mammals.©2002Elsevier Science Inc.KEY WORDS:Control of respiration,Phrenic nucleus,Spinal cord,Hypothalamus,Ventrolateral reticular formation,Parabra-chial nucleus,Central gray,Thalamus,Cortex.INTRODUCTIONThe nervous control of respiration that has been mainly studied in vivo in adult mammals (rats,cats,dogs,rabbits)[2],is now studied in neonatal rodents [7,8].However,little information is available on the adult mouse [1,7,8].Substantial evidence in-dicates that central nervous system (CNS)regions involved in regulation of behavioral and emotional responses also play a role in the modulation of autonomic functions [2].From a comparative point of view,it is generally accepted that the respiratory control system possesses a similar anatomical orga-nization among different rodent species [2,11].However,dif-ferences between the rat and mouse respiratory system should be emphasized [7,8].First,the vagal control of the respiratory rhythm is stronger in the mouse than in the rat [7].Second,respiratory bursts mainly occur in phrenic roots in mouse prep-arations,whereas they occur in all the cervical rootlets in rat preparations [7].Finally,pontine control of the medullary re-spiratory rhythm generator may differ between the mouse and the rat.Respiratory bursts occur spontaneously in rat pontomed-ullary preparations,but the pons must be removed in the mouse preparation to obtain similar activity [7].We have studied the motor and premotor components of the anatomical network of respiratory control in mice by means of a transneuronal labeling technique using rabies virus [1,6].MATERIAL AND METHODSIn order to characterize the morphology and the location of the respiratory motoneurons and their serially connected chains of neurons,the Challenge Virus Standard of rabies virus was used.This tracer is amplified across the synapses [1]and has the ability to invade cell dendrites as well as somata,thereby labeling all the neuronal arborizations.Experiments were carried out in adult mice of the CH3/HeJ strain,at least 3months old,and weighing 36Ϯ2g.The animals were anesthetized by a peritoneal injection of sodium pentobarbital (30mg/kg)in accordance with the European Community Directive (86/609/EEC).Thereafter,2␮l of virus solution (2.5ϫ107PFU/␮l)[10]were injected through a 10-␮l Hamilton syringe inserted either in the left part of the diaphragm or in the left intercostal muscles.The surgical wound was sutured,and the animals were kept in a warm enclosure.After 3–5-day survival periods,the animals were deeply anaesthetized and transcardially perfused with paraformaldehyde (4%)in saline.To accurately define the spatial distribution of the cells,the brains were sectioned at 30␮m in either coronal (n ϭ2)or sagittal (n ϭ1)planes,by means of a freezing microtome.The virus was detected using immu-nohistochemistry with a mouse monoclonal antibody specific for the viral phosphoprotein as primary antibody,and a fluorescein (FITC)-conjugate (Jackson ImmunoResearch,West Grove,PA,USA)as secondary antibody to visualize the labeled neurons.The sections were mounted on gelatine-coated slides,air dried,and cover-slipped with DPX (Fluka Chemika,Switzerland).To delimit the CNS regions,every third section was counterstained using the Nissl method.The green fluorescent FITC-labeled neurons were visualized under a Zeiss Axiophot (Germany)fluorescence microscope,by means of a barrier filter system H (450–490-nm excitation wave-*Address for correspondence:Susana P.Gayta ´n,Departmento de Fisiologı´a y Biologia Animal,Facultad de Biologia,Avda.Reina Mercedes,6,41012-Sevilla,Spain.Fax:34-954233480.E-mail:sgaytan@us.esBrain Research Bulletin,Vol.57,Nos.3/4,pp.335–339,2002Copyright ©2002Elsevier Science Inc.Printed in the USA.All rights reserved0361-9230/02/$–see front matter335length)(Fig.1).The labeled neurons were charted onto anatomical outlines of brain sections (Figs.2,3).The terminology for CNS nuclei was based on the Paxinos and Watson [9]and Valverde [12]atlases.RESULTSLabeled neurons were found bilaterally throughout the CNS (x ៮ϭmean number of labeled cells in each slice and in each experiment).At the spinal level,phrenic (x ៮ϭ11;range,8–15)and intercostal (x ៮ϭ9;range,8–13)motoneurons,as well as interneurons (x ៮ϭ6;range,3–13)were labeled.The cells were organized in clusters of neurons in the ventral horn (Fig.1A)and were located inside the layers 6–9.However,in one of the experiments,cells were also found in the layers 1–3.At the pons and medulla levels (Figs.2,3),labeledneuronsFIG.1.Fluorescence photomicrographs,taken under a 450-nm excitation filter,of coronal sections of themouse brain.(A)Coronal section through the right ventral part of the spinal cord shows the labeled somas.The white arrow points to a phrenic motoneuron.(B)Coronal section of the left ventrolateral medulla shows labeled cells and fibers in the ambiguus nucleus and surrounding reticular formation,and the arrow points to a labeled ambiguus nucleus premotor neuron.(C)Labeled neurons within the medial cerebellar nucleus (arrows).Note the presence of labeled granule cells.(D)Fluorescence photomicrograph of labeled neurons (arrow)within the different parietal cortex layers (arrowheads).Bars:100␮m.Abbreviations:Amb,nucleus ambiguus;fu,funiculus of the spinal cord;Med,medial (fastigial)cerebellar nucleus;LRt,lateral reticular nucleus;Par,parietal cortex;py,pyramidal tract;Sp5C,spinal trigeminal nucleus,caudal part.336GAYTA´N ET AL.were observed bilaterally in different nuclei.The cells found in the nucleus of the solitary tract (x ៮ϭ9;range,2–19)showed the greatest density in its medial beled neurons were also located within the medial reticular formation (x ៮ϭ14;range,11–25)(Figs.1B,2),and in one experiment within the gelatinous layer of the caudal spinal trigeminal nucleus (x ៮ϭ9;range,7–13)(Fig.1B).Labeled cells were present in the retroambiguus (x ៮ϭ2;range 0–3)and ambiguus (x ៮ϭ8;range,2–14)nuclei (Figs.1B,2).Scattered labeled somata were also located within the lateralparagigantocellular nucleus (x ៮ϭ3;range,1–5),parapyramidal region (x ៮ϭ2;range,1–3),raphe obscurus nucleus (x ៮ϭ1;range,1–2),and A5area (x ៮ϭ2;range,1–3)(Fig.2).The greatest number (x ៮ϭ17;range,11–21)of labeled somata was found within the medial subdivision of the parabrachial/Ko ¨lliker –Fuse complex (Figs.2,3).In addition,neurons were present in the Barrington ’s nucleus (x ៮ϭ1;range,0–2),subcoeruleus nucleus (x ៮ϭ7;range,FIG.2.Schematic summary drawing of coronal sections of the mouse brain shows the distribution of labeled somata (dots,each dot represents 1labeled soma)after injection of the virus into the diaphragm and intercostal muscles.The data are from a representative single experiment (section thickness:30␮m).The coordinates indicate the distance on respect to the bregma taken as zero reference.Abbreviations:AP,area postrema;csc,commissure of the superior colliculus;DCIC,dorsal cortex of the inferior colliculus;DTg,dorsal tegmental nucleus;ECu,external cuneate nucleus;InG,intermediate gray layer of the superior colliculus;IRt,intermediate reticular nucleus;LC,locus coeruleus;LPGi,lateral paragigantocellular nucleus;PB,parabrachial nucleus;PnC,pontine tegmental nucleus,caudal part;ROb,raphe obscurus nucleus;SNR,substantia nigra,reticular part;Sol,nucleus of the solitary tract;Sp5I,spinal trigeminal nucleus,interpolar part.MOUSE CNS PROJECTIONS TO THE PHRENIC NUCLEUS 3373–9),locus coeruleus (x ៮ϭ2;range,0–6),lateral superior olive (x ៮ϭ2;range,1–4),and dorsal tegmental nucleus (x ៮ϭ2;range,1–3)(Figs.2,3).At the level of the cerebellum,labeled neurons were found within the medial cerebellar nucleus (x ៮ϭ9;range,2–19),and in one experiment,numerous granule cells were also labeled (x ៮ϭ16;range,13–38)(Figs.1C,2,3).At the midbrain level,labeled somata (x ៮ϭ14;range,8–26)were located in the periaqueductal central gray,arranged in a compact cluster near the aqueduct (Fig.2).Other labeled cells (x ៮ϭ2;range,1–6)were observed within the pedunculopontine tegmental nucleus (Fig.2).Scattered labeled neurons (1to 4)were also found within the deep mesencephalic nucleus and superior colliculus.At the hypothalamic level,a few labeled neurons (1to 4)were found in the lateral and in the anterior hypothalamic areas.A sparse group of cells (x ៮ϭ5;range,2–9)was present in the zona incerta (Fig.3).Labeled somata were found within the subthalamic nucleus (x ៮ϭ8;range,5–13).At the thalamic level,labeled cells were observed within the laterodorsal (x ៮ϭ5;range,2–9),dorsomedial (x ៮ϭ3;range,1–5),and posterior (x ៮ϭ7;range,2–11)thalamic nuclei (Fig.3).The neurons found in the laterodorsal thalamic nuclei,were located at the ventral limit of the nucleus.Finally,labeled neurons were also present in the frontal (x ៮ϭ10;range,8–15)(Fig.3)and parietal (x ៮ϭ11;range,8–15)cortices (Figs.1D,3).DISCUSSIONThese preliminary results show labeled cells related to the control of breathing at different levels of the CNS.However,some of the labeled neurons may originate from virus infection through muscular afferents,because the dorsal roots were intact.Our findings show labeled phrenic and intercostal motoneurons and interneurons in the spinal cord.The latter may have an important role in respiratory integration.In mammals,it is postu-lated that the respiratory rhythm is generated by the oscillations of a neuronal network,which comprises different types of respiratory neurons,most of them being located in the reticular formation of the brainstem [2,3].Our data in the adult mouse are comparable to previous studies in the rat [3].Respiratory control-related neurons are present within the medial reticular formation and within the lateral tegmental field;although these projections may not be prominent in providing respiratory drive,they may be involved in the modulation of motoneuronal excitability,as suggested for other rodents [3].The neurons found within the medullary reticular formation,including the ventral respiratory group and the nucleus of the solitary tract,are comparable to the pattern of premotor neurons described in the rat.These neurons correspond totheFIG.3.Schematic summary drawing of sagittal sections shows the general distribution of labeled somata (dots,each dot represents 1labeled soma)after injections of the virus into the diaphragm and intercostal muscles.The data are from a representative single experiment (section thickness:30␮m).The coordinates indicate the distance lateral to the midline.Abbreviations:CPu,caudate putamen;IC,inferior colliculus;Gi,gigantocel-lular reticular nucleus;MD,mediodorsal thalamic nucleus;R,red nucleus;Tu,olfactory tubercle.338GAYTA´N ET AL.primary descending pathway,which provides the transmission of rhythmic respiratory drive to respiratory motoneurons[3].The neurons located within parabrachial/Ko¨lliker–Fuse complex sup-port the idea that this area is involved in the direct control of respiratory function via their projections to phrenic and intercostal motoneurons and premotor neurons[4,5,8]at the spinal level.So far,no evidence existed of premotor neurons located within nuclei outside the pons and medulla in mice.However,our results show otherwise.The labeled cells within the cerebellum could be part of the modulation of the respiratory outputs,via influences on medullary respiratory neurons[5].The link between the periaque-ductal central gray and the respiratory motoneurons could mediate respiratory-related functions,such as vocalization[5].The teg-mental pedunculopontine nucleus could participate in respiratory final respiratory motoneuronal output[5].In addition,labeled cells within the anterior and the lateral hypothalamic areas,as well as within the zona incerta,may correspond to second-order neurons implicated in autonomic functions,as described in rats[5].These regions are involved in the integration of vegetative and behavioral responses[5].Our experiments also have demonstrated labeled cells within the subthalamus and the laterodorsal,dorsomedial,and lateral posterior thalamic nuclei,which indicates that respiratory motoneurons receive information from structures involved in so-matosensorial and motor control as shown in rats by the existing connections between the thalamic and cardiorespiratory areas[5]. This connectivity may provide a pathway by which respiratory control-related neurons may gain access to the limbic system. Finally,the present study shows a moderate number of labeled neurons in the cerebral cortex,which may provide a cortical modulation of the respiratory motoneurons activities.In conclusion,these preliminary results showed that the Chal-lenge Virus Standard of rabies virus is a useful tool for labeling the respiratory network,which provides an anatomical basis for the circuitry underlying respiratory control.However,it will be nec-essary to use different virus infection times and dorsal rhizotomies to decipher step by step the anatomical organization and identify precisely the connections between the different labeled areas. 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