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Cholinergic nerve development in fetal lung
DEVELOPMENTAL
BIOLOGY
66,541~546
Cholinergic
(1978)
Nerve
YASUHIDEMORIKAWA,PATRICIA Pediatric Surgical Massachusetts
Development
in Fetal
K. DONAHOE,AND
Research General
Laboratory, Division Hospital and Harvard
Received
January
of Pediatric Medical
6, 1978; accepted
Lung
W. HARDYHENDREN
Surgery and Department of Surgery, School, Boston, Massachusetts 02114
in revised
form
April
the
4, 1978
Choline& nerve development in rat fetal lung can be monitored effectively using acetylcholine esterase histochemical staining techniques on frozen sections. Neuroblasts were detected first by these techniques on fetal Day 13 in the indifferent mesenchyme surrounding the trachea. Subsequently, they migrated to the level of the third tracheal branch and matured progressively to ganglia. Neither nerve fibers, ganglia, nor nerve endings of cholinergic variety were detected further in the periphery of the lung. Neuroepitbelial cells with lightly staining cytoplasmic acetylcholine esterase-positive granules were fust seen on Day 17, after which they were found along the epithelial lining of the entire tracheobronchial tree. These epithelial cells may be potential intrapuhnonary chemoreceptors.
inatal and adult lungs were dissected for comparison. Fetal lungs were dissected in media at 16x using microsurgical techniques. The esophagus was removed with the specimen to compare ganglionic histology in each structure and to avoid trauma to the lung. Young fetuses were immediately immersed in a 10% form01 calcium solution (pH 7.2) (Baker, 1958). Older fetuses and adult lungs were infused via the trachea prior to dissection and then immersed in the same solution for 12 hr at 4’C. Specimens were then washed in gum sucrose and allowed to harden in fresh gum sucrose at 4’C for 1 to 3 days (Holt, 19611, after which they were cut into 8-pm sections on a Harris WRC cryostat at -25°C. Frozen sections were mounted on precooled 3% gelatinized slides and incubated in 10% form01 calcium at 4°C for 5 min to allow the gelatin to solidify. Acetylcholine esterase histochemical staining was done by the direct coloring method MATERIALS AND METHODS of Karnovsky and Roots (1964), using aceTimed pregnant female rats were ob- tylthiocholine iodine (Sigma) as a substrate tained from Holtzman Laboratories and fe- and incubating at 37°C for 1 hr. Buffered tuses were delivered by cesarean section at methyl green was used as a counter stain. 12 to 21 days post coitus. Fetal lung was Tetraisopropylphosphoramide (8 x lo-* m harvested from each day of pregnancy. Per- final concentration) was added during aceINTRODUCTION
Little is known about, the development of the autonomic nervous system in the embryonic lung, although much has been written about the autonomic innervation, of the adult and newborn lung in a variety of mammalian species. Physiologic studies have established that parasympathetic and sympathetic afferent. and efferent nerves mediate vegetative respiratory functions via vagus and sympathetic pathways. These respond to stretch, irritation, and chemical changes (Con, 02, pH) and initiate changes in frequency, depth, and regularity of breathing (Bartoli et al., 1974; Scheid et al., 1974). As part of a systematic morphologic study of the developing autonomic nervous system of the embryonic rat lung, this report describes the progressive development of the cholinergic nervous system of the lung during the latter half of the 22day gestation of this species.
541 0012-1606/78/~52-0541$02.00/0 Copyright 0 1978 by Academic
AU rights of reproduction
Press,
Inc.
in any form reserved.
542
DEVELOPMENTAL BIOLOGY
VOLUME 65,1978
tylcholine esterase incubation to inhibit rina, smaller acetylcholine esterase-positive non-specific choline esterase activity. neuroblasts were found submucosally. On In addition to the frozen-section histo- Day 18 (Fig. lD), nerve fibers were seen chemical technique, fetal lung also was between the ganglia and the epithelium of fixed routinely and stained with hematoxthe trachea at the carina. Bare epithelial ylin and eosin for comparison. Perinatal cells (Fig. 3) had acetylcholine esterase-posand adult lungs were fixed routinely and itive granules in their cytoplasm. We called cells, after Lauwerstained with both H&E and silver using a these “neuroepithelial” modification of Bielshowsky’s impregnayns (1972). After Day 18, distal neuroblasts tion method (Mallory, 1961). further differentiated and assumed paramuscular and submucosal positions similar RESULTS to those of the carina. Nerve fibers at the Acetylcholine esterase activity was de- carina differentiated further and increased tected in the vagus nerve and in the mes- in number. Neither neuroblasts, ganglion enchyme surrounding the esophagus (Fig. cells, nor nerve fibers could be demon1A) on Day 12, but the peritracheal tissue strated in the periphery by these techfailed to stain. At Day 13, acetylcholine niques distal to the third branching point. esterase-positive neuroblasts were seen in However, small mesenchymal cells with the indifferent mesenchyme surrounding lightly stained acetylcholine esterase-posithe trachea for the first time, from the level tive granules could be seen in the distal of the carina to the first branch point of the peripheral perialveolar cells as early as Day trachea. At 14 days (Fig. lB), the indiffer13. These cells eventually differentiated ent mesenchyme surrounding the esopha- into smooth muscle in the adult preparagus began to differentiate into early smooth tion. muscle, and neuroblasts showed more inOnly after the ganglion cells assumed tense acetylcholine esterase activity, al- their characteristic morphology in the perthough their position was still diffuse. Tra- imuscular position at Day 15 could they be cheal neuroblasts at this stage could be detected by hematoxylin and eosin staining. detected from the carina (Fig. 2) to the These routine techniques could not detect second tracheal branch. Smooth muscle dif- the early neuroblasts as the acetylcholine ferentiated around the carina on Day 15, esterase histochemical techniques could. and diffuse neuroblasts stained more inAdult and perinatal lungs likewise were tensely. On Day 16, the acetylcholine ester- studied using acetylcholine esterase techase-positive neuroblasts localized outside of niques. Intense specific acetylcholine esterthe differentiating myoblasts. Cholinergic ase activity was found in the ganglia of the nerve fibers could not yet be detected at upper trachea, the carina, and the proximal any level. On Day 17 (Fig. lC), cartilage bronchi to the third tracheal branch. As in formed at the level of the carina and myo- the fetus, no activity could be detected blasts condensed to form recognizable more peripherally. Silver staining was smooth muscle. Cilia could be detected in abandoned after multiple modifications, bethe epithelial cells. Neuroblasts outside the cause nonspecific reticulum fibers stained smooth muscle differentiated into more so intensely. We found that accurate intermature ganglion cells characterized by clear pretation of the distal morphology was imnucleii with multiple nucleoli, with staining possible using modifications of Biellocalized to the cytoplasm in the perinushowsky’s technique. clear position. Differentiated ganglia could DISCUSSION be seen distinctly at the level of the third Acetylcholine esterase histochemical lotracheal branch below the carida. In addicalization techniques provided a satisfaction to the perimuscular ganglia at the ca-
BRIEF NOTES
FIG. 1. Acetylcholine esterase histochemistry of fetal rat trachea (T) showing positive staining in the vagus nerve (V). x 170. (B) Fourteen smooth muscle (Sm) and ganglia around the esophagus and neuroblasts Seventeen days: Neuroblasts outside the condensed smooth muscle of the (G). x 210. (D) Eighteen days: Postganglionic nerve fibers (ND travel to matured (G) and cartilage (Cl) has differentiated. X 380.
tory method with which to study the embryonic development of the parasympathetic nervous system in the rat lung, par-
543
and esophagus (Es). (A) Twelve days, days, demonstrating the presence of (Nb) around the trachea. x 170. (C) trachea have differentiated to ganglia the tracheal epithelium. Ganglia have
titularly if nonspecific choline esterase inhibitors were used. Parasympathetic ganglia were observed in trachea, carina, and
DEVELOPMENTAL BIOLOGY
VOLUME 65,1978
13 day 14 day 15 day FIG. 2. Migration of neuroblasts during embryonic development. Dots around bronchial buds indicate position of neuroblasts at various ages.
the
FIG. 3. Neuroepithelial cells (NEB) with light cytoplasmic acetylcholine esterase activity are noted among the epithelial (Ep) cells of the IS-day fetal trachea. x 1000.
first, second, and third bronchial branches, but could not be demonstrated more distally by these techniques, in either embryonic, perinatal, or adult rat lung. Neuroblasts migrated distally, localized outside the smooth muscle, and then differentiated to mature ganglia, from which postsynaptic fibers extended to the epithelium. Small
ganglia formed in the submucosa, from which circumferential fibers encircled the trachea. Selected epithelial cells had fine cytoplasmic acetylcholine esterase-positive granules. Neuroblasts were first detected around the epithelium on Day 13, dispersed among the indifferent mesenchyme. At this stage
BRIEF
there was little to differentiate them morphologicahy from myoblasts or chondroblasts, except that they took the acetylcholine e&erase stain intensely. As ganglia differentiated after migration and assumed their distinct multinucleated pattern, the intense acetylcholine esterase activity increased markedly, as seen on Days 17 and 18 (Figs. 1C and D). At this stage their morphology was recognizable by routine histologic techniques. There appear to be two forms of acetylcholine esterase activity during embryonic development: intense activity specific to cholinergic neurogenic cells and less intense activity due to myogenic cells (Karnovsky, 1964). This low-intensity acetylcholine esterase activity was observed in the perialveolar cells which later developed into smooth muscle. We were unable to detect ganglia or cholinergic nerve fibers in the peripheral alveolar wall in either embryonic or adult lung using acetylcholine esterase techniques. These results support the observations of El-Bermani (1973). The more distal nerve fibers described by others (Hirsch et al., 1968; Meyrick et al., 1971; Hung et al., 1973; McLelland et al., 1972, 1974; Fillenz et al., 1972) may be vascular afferent or efferent fibers. Selected epithelial cells stained lightly for acetylcholine eaterase in their cytoplasmic granules. These cells are similar to the hypoxia-sensitive “neuroepithelial” cells described by Lauweryns et al. (1973) and may function as intrapulmonary chemoreceptors. In summary, the progressive development of the fetal parasympathetic nervous system can be monitored effectively using acetylcholine esterase histochemical staining techniques. These techniques provide the advantage of cholinergic specificity. Combined with electron microscopy and direct fluorescence of the adrenergic system, a fuller understanding of autonomic development can be attained. Neuroblasts migrate to the third branch point of the trachea and further differentiate into gan-
545
NOTES
glia. Beyond that point there appear to be no acetylcholine esterase-positive nerve libers, except for those associated with the pulmonary capillary vasculature. We were able to document the presence of neuroepithelial cells which stained for acetylcholine esterase. These potential chemoreceptor cells may be important in pathologic states characterized by autonomic lability, such as sudden infant death and infant apnea. We are grateful to Dr. Robert Crone and Dr. Jonathan Moss for their helpful suggestions. We also appreciate the technical assistance of Mrs. Carmelo Bondi and Mr. Thomas Manganaro and the editorial help of Ms. Bettejean Capistran. This investigation was supported by Grant CA17393-02 awarded by the National Cancer Institute, DHEW. REFERENCES BAKER, J. R. (1958). “Principles of Biological Microtechnique, A Study of Fixation and Dyeing,” pp. 111-118. Methouens, London. BARTOLI, A., CROSS, B. A., Guz, A., JAIN, S. K., NOBLE, M., and TRENCHARD, D. (1974). The effect of carbon dioxide in the airways and alveoli on ventilation, A vagal reflex studied in the dog. J. Physiol. (London) 240,91-109. EL-BERMANI, A. I. (1973). Innervation of the rat lung. Acetylcholinesterase-containing nerves of the bronchial tree. Amer. J. Anat. 137: 19-29. FILLENZ, M., and WIDDICOMBE, J. G. (1972). Receptors of the lungs and airways. Zn “Handbook of Sensory Physiology” (E. Neil, ed.), Vol. 3, pp. 81-112. Springer, Berlin. HIRSCH, E. F., KAISER, C., BARNER, H. B., and COOPER, F. (1968). Innervation of the mammalian lung. I. The afferent receptors. Arch. Pathol. 85,
51-61. HOLT, S. J., and HICKS, R. M. (1961). Studies on formalin fixation for electron microscopy and cytochemical staining purposes. J. Biophys. Biochem. cytoz. l&31-45. HUNG, K., HERTWECK, M. D., HARDY, J. D., and LOOSLI, C. G. (1973). Electron microscopic observation of nerve endings in the alveolar walls of mouse lungs. Amer. Rev. Resp. Dis. 108, 328-333. KARNOVSKY, M. J. (1964). The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. J. Cell Biol. 23, 217-232. KARNOVSKY, M. J., and ROOTS, L. (1964). A “directcoloring” thiocholine method for cholinesterases. J. Histochem. Cytochem. 12,219-221. LAUWERYNS, J. M., and COKELAERE, M. (1973). Hypoxia-sensitive neuro-epithelial bodies intrapulmo-
546
DEVELOPMENTAL
BIOLOGY
nary secretory neuroreceptors, modulated by the central nervous system. 2. Zellforsch. 145,521-540. LALJWERYNS, J. M., COKELAERE, M., and THEUNYNCK, P. (1972). Neuro-epithelial bodies in the respiratory mucosa of various mammals. 2. Zellforsch. 136, 569-592. MALLORY, F. B. (1961). “Pathological Techniques,” pp. 158-160. Hafner, New York. MCLELLAND, J., COOK, R. D., and KING, A. S. (1972).
VOLUME
65, 1978
Nerves in the exchange area of the avian lung. Acta Amt. 83, 7-16. MEYRICK, B., and REID, L. (1971). Nerves in intraacinar alveoli: An electron microscopic study. Resp. Physiol. 11, 367-377. SCHEID, P., SLAMA, H., GATZ, R. N., and FEDDE, M. R. (1974). Intrapulmonary CO, receptors in the duck: III. Functional localization. Resp. Physiol. 22, 123-136.
BIOLOGY
66,541~546
Cholinergic
(1978)
Nerve
YASUHIDEMORIKAWA,PATRICIA Pediatric Surgical Massachusetts
Development
in Fetal
K. DONAHOE,AND
Research General
Laboratory, Division Hospital and Harvard
Received
January
of Pediatric Medical
6, 1978; accepted
Lung
W. HARDYHENDREN
Surgery and Department of Surgery, School, Boston, Massachusetts 02114
in revised
form
April
the
4, 1978
Choline& nerve development in rat fetal lung can be monitored effectively using acetylcholine esterase histochemical staining techniques on frozen sections. Neuroblasts were detected first by these techniques on fetal Day 13 in the indifferent mesenchyme surrounding the trachea. Subsequently, they migrated to the level of the third tracheal branch and matured progressively to ganglia. Neither nerve fibers, ganglia, nor nerve endings of cholinergic variety were detected further in the periphery of the lung. Neuroepitbelial cells with lightly staining cytoplasmic acetylcholine esterase-positive granules were fust seen on Day 17, after which they were found along the epithelial lining of the entire tracheobronchial tree. These epithelial cells may be potential intrapuhnonary chemoreceptors.
inatal and adult lungs were dissected for comparison. Fetal lungs were dissected in media at 16x using microsurgical techniques. The esophagus was removed with the specimen to compare ganglionic histology in each structure and to avoid trauma to the lung. Young fetuses were immediately immersed in a 10% form01 calcium solution (pH 7.2) (Baker, 1958). Older fetuses and adult lungs were infused via the trachea prior to dissection and then immersed in the same solution for 12 hr at 4’C. Specimens were then washed in gum sucrose and allowed to harden in fresh gum sucrose at 4’C for 1 to 3 days (Holt, 19611, after which they were cut into 8-pm sections on a Harris WRC cryostat at -25°C. Frozen sections were mounted on precooled 3% gelatinized slides and incubated in 10% form01 calcium at 4°C for 5 min to allow the gelatin to solidify. Acetylcholine esterase histochemical staining was done by the direct coloring method MATERIALS AND METHODS of Karnovsky and Roots (1964), using aceTimed pregnant female rats were ob- tylthiocholine iodine (Sigma) as a substrate tained from Holtzman Laboratories and fe- and incubating at 37°C for 1 hr. Buffered tuses were delivered by cesarean section at methyl green was used as a counter stain. 12 to 21 days post coitus. Fetal lung was Tetraisopropylphosphoramide (8 x lo-* m harvested from each day of pregnancy. Per- final concentration) was added during aceINTRODUCTION
Little is known about, the development of the autonomic nervous system in the embryonic lung, although much has been written about the autonomic innervation, of the adult and newborn lung in a variety of mammalian species. Physiologic studies have established that parasympathetic and sympathetic afferent. and efferent nerves mediate vegetative respiratory functions via vagus and sympathetic pathways. These respond to stretch, irritation, and chemical changes (Con, 02, pH) and initiate changes in frequency, depth, and regularity of breathing (Bartoli et al., 1974; Scheid et al., 1974). As part of a systematic morphologic study of the developing autonomic nervous system of the embryonic rat lung, this report describes the progressive development of the cholinergic nervous system of the lung during the latter half of the 22day gestation of this species.
541 0012-1606/78/~52-0541$02.00/0 Copyright 0 1978 by Academic
AU rights of reproduction
Press,
Inc.
in any form reserved.
542
DEVELOPMENTAL BIOLOGY
VOLUME 65,1978
tylcholine esterase incubation to inhibit rina, smaller acetylcholine esterase-positive non-specific choline esterase activity. neuroblasts were found submucosally. On In addition to the frozen-section histo- Day 18 (Fig. lD), nerve fibers were seen chemical technique, fetal lung also was between the ganglia and the epithelium of fixed routinely and stained with hematoxthe trachea at the carina. Bare epithelial ylin and eosin for comparison. Perinatal cells (Fig. 3) had acetylcholine esterase-posand adult lungs were fixed routinely and itive granules in their cytoplasm. We called cells, after Lauwerstained with both H&E and silver using a these “neuroepithelial” modification of Bielshowsky’s impregnayns (1972). After Day 18, distal neuroblasts tion method (Mallory, 1961). further differentiated and assumed paramuscular and submucosal positions similar RESULTS to those of the carina. Nerve fibers at the Acetylcholine esterase activity was de- carina differentiated further and increased tected in the vagus nerve and in the mes- in number. Neither neuroblasts, ganglion enchyme surrounding the esophagus (Fig. cells, nor nerve fibers could be demon1A) on Day 12, but the peritracheal tissue strated in the periphery by these techfailed to stain. At Day 13, acetylcholine niques distal to the third branching point. esterase-positive neuroblasts were seen in However, small mesenchymal cells with the indifferent mesenchyme surrounding lightly stained acetylcholine esterase-posithe trachea for the first time, from the level tive granules could be seen in the distal of the carina to the first branch point of the peripheral perialveolar cells as early as Day trachea. At 14 days (Fig. lB), the indiffer13. These cells eventually differentiated ent mesenchyme surrounding the esopha- into smooth muscle in the adult preparagus began to differentiate into early smooth tion. muscle, and neuroblasts showed more inOnly after the ganglion cells assumed tense acetylcholine esterase activity, al- their characteristic morphology in the perthough their position was still diffuse. Tra- imuscular position at Day 15 could they be cheal neuroblasts at this stage could be detected by hematoxylin and eosin staining. detected from the carina (Fig. 2) to the These routine techniques could not detect second tracheal branch. Smooth muscle dif- the early neuroblasts as the acetylcholine ferentiated around the carina on Day 15, esterase histochemical techniques could. and diffuse neuroblasts stained more inAdult and perinatal lungs likewise were tensely. On Day 16, the acetylcholine ester- studied using acetylcholine esterase techase-positive neuroblasts localized outside of niques. Intense specific acetylcholine esterthe differentiating myoblasts. Cholinergic ase activity was found in the ganglia of the nerve fibers could not yet be detected at upper trachea, the carina, and the proximal any level. On Day 17 (Fig. lC), cartilage bronchi to the third tracheal branch. As in formed at the level of the carina and myo- the fetus, no activity could be detected blasts condensed to form recognizable more peripherally. Silver staining was smooth muscle. Cilia could be detected in abandoned after multiple modifications, bethe epithelial cells. Neuroblasts outside the cause nonspecific reticulum fibers stained smooth muscle differentiated into more so intensely. We found that accurate intermature ganglion cells characterized by clear pretation of the distal morphology was imnucleii with multiple nucleoli, with staining possible using modifications of Biellocalized to the cytoplasm in the perinushowsky’s technique. clear position. Differentiated ganglia could DISCUSSION be seen distinctly at the level of the third Acetylcholine esterase histochemical lotracheal branch below the carida. In addicalization techniques provided a satisfaction to the perimuscular ganglia at the ca-
BRIEF NOTES
FIG. 1. Acetylcholine esterase histochemistry of fetal rat trachea (T) showing positive staining in the vagus nerve (V). x 170. (B) Fourteen smooth muscle (Sm) and ganglia around the esophagus and neuroblasts Seventeen days: Neuroblasts outside the condensed smooth muscle of the (G). x 210. (D) Eighteen days: Postganglionic nerve fibers (ND travel to matured (G) and cartilage (Cl) has differentiated. X 380.
tory method with which to study the embryonic development of the parasympathetic nervous system in the rat lung, par-
543
and esophagus (Es). (A) Twelve days, days, demonstrating the presence of (Nb) around the trachea. x 170. (C) trachea have differentiated to ganglia the tracheal epithelium. Ganglia have
titularly if nonspecific choline esterase inhibitors were used. Parasympathetic ganglia were observed in trachea, carina, and
DEVELOPMENTAL BIOLOGY
VOLUME 65,1978
13 day 14 day 15 day FIG. 2. Migration of neuroblasts during embryonic development. Dots around bronchial buds indicate position of neuroblasts at various ages.
the
FIG. 3. Neuroepithelial cells (NEB) with light cytoplasmic acetylcholine esterase activity are noted among the epithelial (Ep) cells of the IS-day fetal trachea. x 1000.
first, second, and third bronchial branches, but could not be demonstrated more distally by these techniques, in either embryonic, perinatal, or adult rat lung. Neuroblasts migrated distally, localized outside the smooth muscle, and then differentiated to mature ganglia, from which postsynaptic fibers extended to the epithelium. Small
ganglia formed in the submucosa, from which circumferential fibers encircled the trachea. Selected epithelial cells had fine cytoplasmic acetylcholine esterase-positive granules. Neuroblasts were first detected around the epithelium on Day 13, dispersed among the indifferent mesenchyme. At this stage
BRIEF
there was little to differentiate them morphologicahy from myoblasts or chondroblasts, except that they took the acetylcholine e&erase stain intensely. As ganglia differentiated after migration and assumed their distinct multinucleated pattern, the intense acetylcholine esterase activity increased markedly, as seen on Days 17 and 18 (Figs. 1C and D). At this stage their morphology was recognizable by routine histologic techniques. There appear to be two forms of acetylcholine esterase activity during embryonic development: intense activity specific to cholinergic neurogenic cells and less intense activity due to myogenic cells (Karnovsky, 1964). This low-intensity acetylcholine esterase activity was observed in the perialveolar cells which later developed into smooth muscle. We were unable to detect ganglia or cholinergic nerve fibers in the peripheral alveolar wall in either embryonic or adult lung using acetylcholine esterase techniques. These results support the observations of El-Bermani (1973). The more distal nerve fibers described by others (Hirsch et al., 1968; Meyrick et al., 1971; Hung et al., 1973; McLelland et al., 1972, 1974; Fillenz et al., 1972) may be vascular afferent or efferent fibers. Selected epithelial cells stained lightly for acetylcholine eaterase in their cytoplasmic granules. These cells are similar to the hypoxia-sensitive “neuroepithelial” cells described by Lauweryns et al. (1973) and may function as intrapulmonary chemoreceptors. In summary, the progressive development of the fetal parasympathetic nervous system can be monitored effectively using acetylcholine esterase histochemical staining techniques. These techniques provide the advantage of cholinergic specificity. Combined with electron microscopy and direct fluorescence of the adrenergic system, a fuller understanding of autonomic development can be attained. Neuroblasts migrate to the third branch point of the trachea and further differentiate into gan-
545
NOTES
glia. Beyond that point there appear to be no acetylcholine esterase-positive nerve libers, except for those associated with the pulmonary capillary vasculature. We were able to document the presence of neuroepithelial cells which stained for acetylcholine esterase. These potential chemoreceptor cells may be important in pathologic states characterized by autonomic lability, such as sudden infant death and infant apnea. We are grateful to Dr. Robert Crone and Dr. Jonathan Moss for their helpful suggestions. We also appreciate the technical assistance of Mrs. Carmelo Bondi and Mr. Thomas Manganaro and the editorial help of Ms. Bettejean Capistran. This investigation was supported by Grant CA17393-02 awarded by the National Cancer Institute, DHEW. REFERENCES BAKER, J. R. (1958). “Principles of Biological Microtechnique, A Study of Fixation and Dyeing,” pp. 111-118. Methouens, London. BARTOLI, A., CROSS, B. A., Guz, A., JAIN, S. K., NOBLE, M., and TRENCHARD, D. (1974). The effect of carbon dioxide in the airways and alveoli on ventilation, A vagal reflex studied in the dog. J. Physiol. (London) 240,91-109. EL-BERMANI, A. I. (1973). Innervation of the rat lung. Acetylcholinesterase-containing nerves of the bronchial tree. Amer. J. Anat. 137: 19-29. FILLENZ, M., and WIDDICOMBE, J. G. (1972). Receptors of the lungs and airways. Zn “Handbook of Sensory Physiology” (E. Neil, ed.), Vol. 3, pp. 81-112. Springer, Berlin. HIRSCH, E. F., KAISER, C., BARNER, H. B., and COOPER, F. (1968). Innervation of the mammalian lung. I. The afferent receptors. Arch. Pathol. 85,
51-61. HOLT, S. J., and HICKS, R. M. (1961). Studies on formalin fixation for electron microscopy and cytochemical staining purposes. J. Biophys. Biochem. cytoz. l&31-45. HUNG, K., HERTWECK, M. D., HARDY, J. D., and LOOSLI, C. G. (1973). Electron microscopic observation of nerve endings in the alveolar walls of mouse lungs. Amer. Rev. Resp. Dis. 108, 328-333. KARNOVSKY, M. J. (1964). The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. J. Cell Biol. 23, 217-232. KARNOVSKY, M. J., and ROOTS, L. (1964). A “directcoloring” thiocholine method for cholinesterases. J. Histochem. Cytochem. 12,219-221. LAUWERYNS, J. M., and COKELAERE, M. (1973). Hypoxia-sensitive neuro-epithelial bodies intrapulmo-
546
DEVELOPMENTAL
BIOLOGY
nary secretory neuroreceptors, modulated by the central nervous system. 2. Zellforsch. 145,521-540. LALJWERYNS, J. M., COKELAERE, M., and THEUNYNCK, P. (1972). Neuro-epithelial bodies in the respiratory mucosa of various mammals. 2. Zellforsch. 136, 569-592. MALLORY, F. B. (1961). “Pathological Techniques,” pp. 158-160. Hafner, New York. MCLELLAND, J., COOK, R. D., and KING, A. S. (1972).
VOLUME
65, 1978
Nerves in the exchange area of the avian lung. Acta Amt. 83, 7-16. MEYRICK, B., and REID, L. (1971). Nerves in intraacinar alveoli: An electron microscopic study. Resp. Physiol. 11, 367-377. SCHEID, P., SLAMA, H., GATZ, R. N., and FEDDE, M. R. (1974). Intrapulmonary CO, receptors in the duck: III. Functional localization. Resp. Physiol. 22, 123-136.