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Keywords:

  • nucleus ambiguus;
  • cholera toxin;
  • intrinsic laryngeal muscles;
  • brainstem;
  • larynx;
  • somatotopy;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Neurons innervating the intrinsic muscles of the larynx are located within the nucleus ambiguus but the precise distribution of the neurons for each muscle is still a matter for debate. The purpose of this study was to finely determine the position and the number of the neurons innervating the intrinsic laryngeal muscles cricothyroid, posterior cricoarytenoid, and thyroarytenoid in the rat. The study was carried out in a total of 28 Sprague Dawley rats. The B subunit of the cholera toxin was employed as a retrograde tracer to determine the locations, within the nucleus ambiguus, of the neurons of these intrinsic laryngeal muscles following intramuscular injection. The labelled neurons were found ipsilaterally in the nucleus ambiguus grouped in discrete populations with reproducible rostrocaudal and dorsoventral locations among the sample of animals. Neurons innervating the cricothyroid muscle were located the most rostral of the three populations, neurons innervating the posterior cricoarytenoid were found more caudal, though there was a region of rostrocaudal overlap between these two populations. The most caudal were the neurons innervating the thyroarytenoid muscle, presenting a variable degree of overlap with the posterior cricoarytenoid muscle. The mean number (±SD) of labelled neurons was found to be 41 ± 9 for the cricothyroid, 39 ± 10 for the posterior cricoarytenoid and 33 ± 12 for the thyroarytenoid. Anat Rec, 296:470–479, 2013. © 2013 Wiley Periodicals, Inc.

The larynx receives its motor innervation from two branches of the vagus nerve, the superior laryngeal nerve (SLN), and the recurrent laryngeal nerve (RLN). It is generally accepted that the SLN innervates the cricothyroid muscle (CT) whereas RLN innervates the remaining intrinsic laryngeal muscles (Sasaki, 2006; McHanwell, 2008). The cell bodies of the neurons that innervate the intrinsic laryngeal muscles are located within the nucleus ambiguus in the brainstem. This is a long rostrocaudally oriented column of neurons lying in the ventrolateral portion of the medulla oblongata, extending between the motor nucleus of the facial nerve to, at least, the level of the pyramidal decussation (Ramon y Cajal, 1909; Lawn, 1966b). In addition to innervating the muscles of the larynx, the nucleus ambiguus also contains neurons that provide motor innervation to the oesophagus, and the pharynx (Lawn, 1966a, 1966b; Bieger and Hopkins, 1987). It is generally accepted that laryngeal neurons are located in the caudal part of the nucleus ambiguus, the so-called loose and semicompact formations (Bieger and Hopkins, 1987) but some differences on the distribution of the neurons that innervate each individual intrinsic laryngeal muscle remain a matter of debate.

First, a number of studies describe laryngeal neurons as being located as a discrete single column of neurons extending along the rostrocaudal axis of the nucleus ambiguus in the brainstem (Szentagothai, 1943; Kalia and Mesulam, 1980; Hinrichsen and Ryan, 1981; Pásaro et al., 1981, 1983; Yoshida et al., 1982, 1984, 1985, 1998; Davis and Nail, 1984; Basterra et al., 1987; Okubo et al., 1987; Portillo and Pásaro, 1988; Van Daele and Cassell, 2009; Weissbrod et al., 2011; Pascual-Font et al., 2011). Other studies describe laryngeal neurons as being clustered along the nucleus ambiguus into several defined columns each columns being separated by gaps where no laryngeal neurons are present (Lawn, 1966a, 1966b; Gacek, 1975; Wetzel et al., 1980; Hisa et al., 1984; Bieger and Hopkins, 1987; Okubo et al., 1987; Patrickson et al., 1991; Núñez-Abades et al., 1992; Hirasugi et al., 2007). Moreover, while the majority of studies have focused on the rostrocaudal organization of laryngeal neurons in the nucleus ambiguus other authors have claimed patterns of dorsoventral (Gacek, 1975; Yoshida et al., 1985, 1998) or mediolateral (Flint et al., 1991) somatotopy within the nucleus ambiguus.

Second, some authors maintain that laryngeal neurons innervating individual laryngeal muscles are diffusely distributed along nucleus ambiguus while others maintain that a clear somatotopic organization can be seen. Most studies are in agreement in locating the neurons innervating the CT more rostral in the nucleus ambiguus than the remaining laryngeal muscles neurons (Szentagothai, 1943; Lawn, 1966a, 1966b; Hinrichsen and Ryan, 1981; Yoshida et al., 1982, 1984, 1985, 1998; Pásaro et al., 1983; Hisa et al., 1984; Davis and Nail, 1984; Basterra et al., 1987; Núñez-Abades et al., 1992; Hirasugi et al., 2007) in the semicompact formation of the nucleus ambiguus (Bieger and Hopkins, 1987). On the other hand, other published studies established that the neurons of the loose formation that innervate intrinsic laryngeal muscles of the larynx, but the CT, are intermingled within the nucleus ambiguus (Gacek, 1975; Hinrichsen and Ryan, 1981; Pásaro et al., 1983; Davis and Nail, 1984; Hisa et al., 1984; Basterra et al., 1987; Flint et al., 1991; Nahm et al., 1990, 1993).

The extent to which the somatotopic organization of laryngeal neurons in the nucleus ambiguus is conserved between species is also contested. Some authors maintain that the pattern is strongly conserved between species (Szentagothai, 1943; Yoshida et al., 1982, 1985, 1998; Okubo et al., 1987). However, because of the uncertainties in homology between laryngeal muscles in different species this conservation of somatotopic pattern is not always easy to establish. In the rat, for example, the subject of this study, the morphology of the larynx is different to other species. In the rat the arytenoid muscle is absent and instead its function is carried out by the superior cricoarytenoid muscle, a muscle found only in the rat (Kobler et al., 1994; Inagi et al., 1998). This is one of the differences that could explain the reported differences in somatotopic organization of laryngeal neurons in the rat (Portillo and Pásaro, 1988; Hirasugi et al., 2007).

From this brief review it can be seen that there have been a number of studies that have attempted to determine the somatotopic organization of the neurons innervating the intrinsic muscles of the larynx in a wide range of species. These studies have produced a number of discrepant results. However, not only the anatomical differences between species are the source of these discrepancies, a second source of problems is likely to be technical. A variety of different techniques and surgical protocols have been used to determine the location of laryngeal neurons within the nucleus ambiguus. The small size of the larynx, especially in some of the commonly used laboratory species such as rat, presents significant technical challenges to carrying out procedures reproducibly. This is particularly the case with studies that employ retrograde tracers to identify neurons where preventing tracer spread from the small intrinsic muscles is a particular problem.

Thus, the aim of our study is to examine the precise location and the number of the neurons innervating three intrinsic laryngeal muscles in the rat: CT, posterior cricoarytenoid (PCA) and thyroarytenoid (TA), using a less problematic retrograde tracer such as cholera toxin and a larger sample size to acquire statistically more significant data.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

The present research was performed according to the regulations and laws for care and handling of animals in research of the European Union (2010/63/UE) and of Spain (Royal Decree 1201/2005), and Law 32/2007, and was approved by the Complutense University of Madrid Committee of Animal Experimentation. Protocols were carried out on 28 adult male Sprague-Dawley rats (Rattus norvegicus) of 300–325 g b/w. Animals were maintained in the central facilities of the Complutense University, and all the surgical procedures were developed, under aseptic conditions, in the animal operating room of that unit. During the first 2 days following surgery, the animals were treated with a standard analgesic protocol consisting of a dose of buprenorphine (0.05 mg kg−1) plus meloxicam (1.0 mg kg−1) administered every 8 h. For a further 2 days, the rats received a dose of ibuprofen (2.5 mg kg−1).

Surgical Procedures and Muscle Tracing Protocol

Animals were anesthetized with an intraperitoneal injection of xylazine (Rompun, Bayer, Spain, 8 mg kg−1) plus ketamine (Imalgene, Merial, France, 90 mg kg−1) and maintained at 37°C on a rat heating pad connected to a temperature controller throughout the surgery and in the recovery period following anesthesia.

A midline skin incision was made ventral in the neck and the larynx was exposed after reflecting laterally the salivary glands and the infrahyoid muscles with a wire retractor. Once the larynx was exposed, the intrinsic laryngeal muscles were identified. The CT muscle was easily recognized in a ventral position in the caudal larynx. To reach the PCA muscle, it has to be uncovered by dissection of the inferior constrictor muscle and a slight rotation of the larynx with tweezers, taking care to avoid any accidental injury to the RLN. The TA muscle was exposed opening a window through the thyroid cartilage with the aid of straight corneal scissors.

Once each muscle was exposed, the tracer was manually injected with a Hamilton syringe coupled to a glass microtip.

All the rats were distributed into 6 groups (Table 1). In the first group the CT muscle was exposed and the tracer injected (n = 11 rats). In a second group an injection of tracer was made into the PCA muscle (n = 5 rats). In the third group the tracer was injected into the TA (n = 3 animals). In three further experimental groups injections were made into two different muscles: in two rats the CT and the TA were injected, in five rats in the PCA and the TA muscles, and in two rats in the CT and PCA muscles.

Table 1. Number of rats per experimental group
 GROUP IGROUP IIGROUP IIIGROUP IVGROUP VGROUP VI
    CT/TAPCA/TACT/PCA
 CTPCATACTPCATACTPCATACT TAPCA TACT PCA
  1. CT, cricothyroid muscle; PCA, posterior cricoarytenoid muscle; TA, thyroarytenoid muscle; AF488, cholera toxin conjugated to Alexa Fluor 488; AF594, cholera toxin conjugated to Alexa Fluor 594.

AF4888   4   31 14 11 1
AF 5943   1   01 11 41 1
TOTAL11   5   3252

Cholera toxin was used as a retrograde tracer. Two different tracers were employed: cholera toxin conjugated to Alexa Fluor 488 (CtB-AF488) and cholera toxin conjugated to Alexa Fluor 594 (CtB-AF594) (Invitrogen, CA). In each muscle was injected a volume of 0.5 µL of cholera toxin using a 1 mL Hamilton syringe. At least 2 min were invested in each injection. At the end of the procedure the surgical field was cleaned by saline and the wound closed in layers.

Staining the Labelled Neurons

Three days after the surgery rats were killed following established procedures employing a lethal dose of pentobarbital (200 mg kg−1) administered intraperitoneally. When all the rat's reflexes were absent, the rats were perfused through the left ventricle with saline (250–300 mL, 37°C) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 (400 mL, 4°C). Immediately after the perfusion, the brainstem and the larynx were removed out from each rat. The larynxes were observed under the stereomicroscope to assure that the injections were made in the desired muscle. No one sample was discarded due to misplaced injections during this series of experiments. Brainstems were postfixed 2 h in the same fixative solution, thoroughly rinsed in PB, and cryoprotected by immersion, overnight, in 15% w.v. sucrose in PB, followed by at 30% w.v. sucrose until they sank (2–3 days). Serial transverse sections of the brainstems, 50-µm thick, were obtained with a freezing microtome and serially collected in 24-well plates. The sections were incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Roche, Basel, Switzerland) for 15 min for the fluorescent identification of the nuclei. Finally, the sections were collected, mounted in slides and analyzed and also photographed with a fluorescence microscope (Nikon Eclipse 800M).

Neuron Counting and Positioning Within the Brainstem

The labelled neurons were always found ipsilateral to the injected muscle, with no evidence of contralateral projections. The counting of the stained neurons was always undertaken by two independent observers and, in each animal, that number was registered and the mean number of neurons (±S.D.) was calculated for each studied muscle.

For counting fluorescent neurons, we developed a variant of the method described by McHanwell and Biscoe (1981a, 1981b). The sections were examined at high magnification (40×) with the appropriate filter for detection of AF488, AF594, or DAPI fluorescence. The following criteria were used to identify cholera toxin-labelled fluorescent structures as a neuronal soma: the presence of a granular fluorescent product confined to the cytoplasm with a nucleus distinguishable as an area relatively clear of fluorescence. Once the neuronal perikaryon was recognized, and the nucleus was focused, the filter was changed to confirm the DAPI-labelled nucleus and to identify the nucleolus (Fig. 1).

image

Figure 1. Transversal section of the brainstem showing fluorescent-labelled neurons within nucleus ambiguus after injection of CtB-AF594 in the cricothyroid muscle. a, one group of laryngeal neurons located within nucleus ambiguus (scale bar: 500 μm). b, magnification of the box in a. The nuclei of the neurons appear in blue due to the DAPI staining, and the nucleolus can be identified. Neurons fitting these criteria can then be considered for both the counting and the positioning (scale bar: 100 μm). d: dorsal; l: lateral.

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The rostrocaudal location of the labelled neurons was measured and registered in reference to standard brainstem landmarks (without correction for shrinkage). Structures close to both the dorsal surface and the midline were selected (the latter to minimize variance due to asymmetrical sectioning). The landmarks chosen were the caudalmost extent of the dorsal cochlear nucleus, the rostral limit of the area postrema (AP), and the obex. The obex is a gross anatomical structure usually defined as the point on the midline of the dorsal surface of the medulla oblongata that marks the caudal angle of the fourth ventricle, where the central canal opens into the rhomboid fossa. However, in the rat, the AP fills the caudal angle of the fourth ventricle so we have considered the obex as the caudal end of the AP (Hamilton and Norgren, 1984; Paxinos and Watson, 2005). All the positional data presented in this study are expressed in micrometers and indicate a rostrocaudal measurement relative to the obex. The lateromedial and dorsoventral locations of the labelled neurons were also measured. The landmarks chosen in these cases were the AP, the basilar sulcus and the lateral edge of the brainstem. The position was established by Image J software.

The abbreviations used to designate brainstem structures were defined by Paxinos and Watson (2005). The terminology related to the nucleus ambiguus follows the topographical description made by Bieger and Hopkins (1987).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Injections of CtB in the CT, PCA, and TA muscles ofthe rat resulted in labelled neurons within the ipsilateral nucleus ambiguus, without any evidence of contralateral projections. All the labelled neurons were multipolar, with the 98% of them being stellate in morphology and the remaining ones fusiform. These results were the same independently of the side of the muscle traced. Labelled neurons or fibers were not found in any other location on the brainstem. There were no differences between the results obtained from animals traced either with CtB-AF488 or CtB-AF594. The analysis of the dorsoventral and lateromedial position of the neurons did not show any differences between the three different populations of neurons.

In control experiments (n = 2), in which the CtB was spread randomly over the larynx, only a few labelled neurons were located in cervical segments of the spinal cord; due to their location, those neurons correspond to extralaryngeal muscles.

Injections in the CT Muscle

Injections of CtB in the CT muscle resulted in labelled neurons within the semicompact formation of the nucleus ambiguus in all the rats studied (n = 13). The extent of the column, rostral to the obex, spanned between 2.4 and 1.4 mm (Fig. 2; Table 2). The 50% of the labelled neurons clustered in a rostrocaudal extension of 0.3 mm, in a segment between 2.1 and 1.8 mm (Fig. 2; Table 2). The mean number of labelled neurons was 41 (Table 2). The morphology of the labelled neurons was stellate, although some fusiform neurons were also observed (∼3%) (Figs. 1, 3, and 4). The results of the morphometry are shown in the Fig. 5.

image

Figure 2. Representative diagrams illustrating the distribution of the labelled neurons in several sections along the medulla oblongata. A, image of a dorsal view of the rat brainstem where the medulla oblongata has been marked with a square. B, schematic representation of the medulla oblongata squared in A. The colored column represents the location of the neuron populations which innervate each muscle as symbolized with the color codes below. C, dorsal view of a brainstem showing the rostrocaudal location of the labelled neurons in a representative experiment. The location of the labelled neurons after injection of tracer into CT and PCA are shown (each color spot represents one neuron). D–F, sequence of a set of transversal sections corresponding to the levels marked in C. Every traced neuron is represented by a color spot. G, dorsal view of a brainstem showing the rostrocaudal location of the labelled neurons in a representative experiment. The location of the labelled neurons after injection of tracer into PCA and TA are shown (each spot represents one neuron). H–J, transversal sections corresponding to the levels marked in G. Every traced neuron is represented by a color spot. In C and G the dashed line marks the obex level. Color code of the labelled neurons: blue, cricothyroid muscle (CT); green, posterior cricoarytenoid (PCA); yellow, thyroarytenoid (TA). 10 N, dorsal nucleus of the vagus; AmbL, loose formation of the nucleus ambiguus; AmbSC, semicompact formation of the nucleus ambiguus; c, caudal; l, left; r, right; ro, rostral; Sol/sol, solitary tract and nucleus of the solitary tract; sp5, spinal trigeminal tract. Numbers on the vertical axis indicate the distance in micrometers to the obex (scale bar: 1 mm).

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image

Figure 3. Labelled neurons with in the nucleus ambiguus after an injection of CtB-AF594 into cricothyroid muscle. a, two stellate-labelled neurons. b, one stellate-labelled neuron (left) and one fusiform-labelled neuron (center). d: dorsal; l: lateral. Scale bar: 100 µm.

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image

Figure 4. Photographs of transversal sections of the medulla oblongata showing labelled neurons after the injection of CtB-AF594 (red) or CtB-AF488 (green) in the intrinsic laryngeal muscles cricothyroid, a, posterior cricoarytenoid, b, thyroarytenoid, c, and both the cricothyroid and the posterior cricoarytenoid, d. The image in d demonstrates the overlap of the neuronal populations of the cricothyroid (red neuron) and the posterior cricoarytenoid (green neuron) muscles. d: dorsal; l: lateral. Scale bar: 100 µm.

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image

Figure 5. Histograms showing the morphometrical data of the labelled neurons after the injection of cricothyroid muscle (CT), posterior cricoarytenoid muscle (PCA) or thyroarytenoid muscle (TA) with CtB. Data are presented as mean ± SEM. Statistical evaluations were based on one way ANOVA test (*P < 0.001; ** P < 0.05).

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Table 2. Mean number and location of labelled neurons after CtB injection in the selected intrinsic laryngeal muscles. Data are expressed as mean ± SD
MUSCLENUMBER OFLOCATION OFLOCATION OF
 LABELLEDLABELLEDCLUSTERED
 NEURONSNEURONS (in pm)NEURONS (in pm)
  1. In the column “Location of neurons” the positive values represent rostral from the obex, whereas the negative one means caudal from the obex. Location of clustered neurons refers to the position of 50% of the labelled neurons of each population. c: caudal. r: rostral.

Cricothyroid41 ± 9r: 2360 ± 40r: 2120 ±50
  c: 1440 ± 50c: 1820 ±40
Posterior cricoarytenoid39 ± 10r: 1990 ± 50r: 1670 ± 75
  c: 870 ± 60c: 1310 ± 75
Thyroarytenoid34 ± 12r: 760 ± 50r: 370 ± 40
  c: −350 ± 50c: 20 ± 30

Injections in the PCA Muscle

Injections of CtB in the PCA muscle (n = 10) resulted in one population of labelled neurons within the loose formation of the nucleus ambiguus, rostral to the obex, distributed in a rostrocaudal column from 2 to 0.8 mm. Half of the labelled neurons were clustered in a segment of 0.3 mm in length, from 1.6 to 1.3 mm (Fig. 2; Table 2). The mean number of labelled neurons was 39 (Table 2). The shape of the labelled neurons is stellate, similar to those labelled from the CT muscle, and fusiform neurons were also rare (∼2%; Fig. 4). The results of the morphometry are shown in the Fig. 5.

Injections in the TA Muscle

Injections of CtB in the TA muscle resulted in labelled neurons within the loose formation of the nucleus ambiguus in all the rats studied (n = 10). The pool of labelled neurons was the most caudal of the three neuronal populations identified, thus located caudal to those traced from the PCA muscle. The column, allocated around the obex, extended from 0.8 mm to −0.3 mm. The 50% of the labelled neurons were clustered in a column, 0.4 mm in length, located from 0.4 mm to the level of the obex (Fig. 2; Table 2). The mean number of labelled neurons was 33 (Table 2). All the traced neurons after injections in the TA muscle were stellate, fusiform cells were never observed (Fig. 4). The results of the morphometry are shown in the Fig. 5. Measurements of the labelled neurons showed that the TA neurons are the largest ones, being the smallest those neurons traced from the PCA muscle. Statistical analysis of the variables measured (area, major axis, and minor axis) demonstrated significant differences among the neurons traced from CT, PCA, and TA muscles both in area and major axis, whereas dealing with the minor axis the differences were significant between the TA muscle and both the CT and the PCA muscles (Fig. 5).

Ipsilateral Tracing Studies of Two Muscles

In 7 rats we injected two ipsilateral muscles in each animal using a different conjugated CtB in each muscle; these protocols yielded identical results to those obtained injecting just only one muscle but allowed us to directly determine the presence or not of an overlapping region between the neuron pools of both muscles. The results showed labelled neurons distributed within the nucleus ambiguus in two different columns, each of one occupying the same position observed in the single muscle injection experiments.

The injection of CT muscle and PCA muscle (n = 2), presented a region where the labelled neurons from both muscles overlapped, as forecasted by the previous individual muscle injection results (Fig. 4d). The overlapping area expanded between 2.0 and 1.7 mm (Fig. 2), which fairly corresponded with the measurements obtained from the single muscle injection experiments. The mean number of neurons located in the overlapping area was 7 for CT muscle and 13 for PCA muscle. If the injected muscles were the PCA and TA (n = 5) we found, in three out of five rats, a short region of overlap from 0.9 to 0.8 mm (Fig. 2). The mean number of labelled neurons allocated in those segments was 3 for both the PCA and the TA muscle.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Our results demonstrate that neurons that innervate the intrinsic laryngeal muscles CT, PCA, and TA, in the rat, are located in a continuous rostrocaudal column within the ipsilateral nucleus ambiguus, somatotopically arranged from the most rostral population, the CT neurons, to the most caudal population, the TA neurons. Each neuronal population has most of the half of their neurons clustered in a central region in the column, whereas the remaining ones are loosely distributed in the rostral and caudal poles, where small overlapping areas between adjacent neuron populations occur. This model is sustained by the fact that, in all the experiments, injection of CtB in the rat muscles CT, PCA and TA results exclusively in ipsilateral labelling of neuronal bodies within the nucleus ambiguus. Labelled neurons are clustered in populations corresponding to each injected muscle and rostrocaudally organized in columns extending over 1 mm in length, being the CT population the most rostral, followed caudalward by the PCA group, and, the most caudal, the set corresponding to the TA muscle. The constitution of each column is quite similar, with most of the half of the labelled cell bodies clustered in a rostrocaudal extension of 0.3–0.4 mm, whereas the remaining labelled somata are more loosely distributed within the rostral and caudal poles of the column. That arrangement gives continuity to the nucleus ambiguus column, since it allows that small territories of overlap, 0.3–0.1 mm in length, occurs between adjacent labelled neuronal populations. To our knowledge, this arrangement with most of the neurons grouped in a more compact core and the remaining ones scattered within the rostral and caudal poles has never been previously described.

The position of the labelled neuronal populations fits that observed for the projections described in the rat for the SLN and RLN (Pascual-Font et al., 2011; Weissbrod et al., 2011) and are in agreement with some previous studies that show that in the rat the neurons innervating the intrinsic laryngeal muscles constitute a continuous column with a rostrocaudal somatotopic organization (Hinrichsen and Ryan, 1981; Portillo and Pásaro, 1988; Van Daele and Cassell, 2009), as it has also been described in other animal models (Szentagothai, 1943; Yoshida et al., 1982, 1985; Davis and Nail, 1984; Okubo et al., 1987). This fact can be consistent with the double innervation of the PCA muscle, both by the superior and the recurrent laryngeal nerves, proposed by some authors (Bieger and Hopkins, 1987; Kobler et al., 1994; Furusawa et al., 1996; Hydman and Mattson, 2008). Furthermore, the pattern we have described, the dense core with the loose poles, can explain those descriptions of discontinuity between the neuronal populations of the CT and PCA muscles (Bieger and Hopkins, 1987; Hirasugi et al., 2007), or those for the PCA and TA muscles (Hirasugi et al., 2007), or even the opposite description of long overlapping territories for the PCA and TA muscles (Hinrichsen and Ryan, 1981; Flint et al., 1991; Van Daele and Cassell, 2009). In other animal models these discrepancies have also been depicted, thus a gap between the pools of neurons belonging to the CT and PCA muscles has been shown in the rabbit (Lawn, 1966a; Okubo et al., 1987; Kitamura et al., 1987), and the dog (Hisa et al., 1984; Hisa and Sato, 1987), as well as the lengthy overlap of the neuronal populations of the PCA and the TA muscles both in the cat (Yoshida et al., 1982, 1998; Pásaro et al., 1983; Davis and Nail, 1984) and the monkey (Yoshida et al., 1985, 1987). Far beyond the interspecies variations, these differences could also be due to the “dense core loose poles” arrangement of the neuronal populations. We have not observed, in any of the rats studied, other somatotopic map than the rostrocaudal, thus nor the mediolateral described also in the rat (Flint et al., 1991), nor the dorsoventral depicted in the kitten (Gacek, 1975). We speculate that the use of CtB versus HRP can influence the presence of these differences since in the dorsoventral somatotopic map described for the nucleus ambiguus of the kitten two discrete neuronal populations appears for each injected muscle (Gacek, 1975), which is probably due to uncontrolled diffusion of HRP to neighbor structures (Hinrichsen and Ryan, 1981; Bieger and Hopkins, 1987; Patrickson et al., 1991; Furusawa et al., 1996). Our experiments strongly support the ipsilateral origin of the efferent ambigual projections in the rat (Hinrichsen and Ryan, 1981; Bieger and Hopkins, 1987; Patrickson et al., 1991; Furusawa et al., 1996; Pascual-Font et al., 2006a, 2006b, 2011) but not the bilateral innervation (Wetzel et al., 1980; Lobera et al., 1981; Pásaro et al., 1981; Van Daele and Cassell, 2009), and, once again, the unrestrained spreading of the tracer can be the reason that produces artifactual bilateral tracings. On the other hand, CtB has the advantage of the low risk of spurious labelling by uncontrolled spreading of the tracer to tissues adjacent to the injection site. To confirm that spurious labelling was not a problem in our study, control experiments were carried out releasing tracer randomly over the larynx and the trachea; in these experiments no labelled perikarya could be observed in the brainstem. We have established 3 days as survival period following the injection of CtB because it is an optimal time to a retrogradely labelling of neural soma (Vercelli et al., 2000). However, in previous laryngeal muscle CtB tracing experiments, animals were allowed to survive longer times, 5 days, but no differences were observed.

To locate the labelled neurons we have followed the definition of the obex given by Hamilton and Norgren (1984), meanwhile other authors refer to the obex as the level where central canal opens into the fourth ventricle (Portillo and Pásaro, 1988; Hirasugi et al., 2007); as this landmark is 0.8 mm rostral to the true obex in the rat, this difference is carried to the measured positions of the labelled neurons along the nucleus ambiguus. In addition to the later methodological consideration, we have developed and applied a correction factor for the obliquity of the histological sections, a useful tool which allows gaining in precision in the location of the laryngeal neurons as we have observed in the reduction in the standard deviation appreciated after applying the correction.

Labelled neurons are mainly stellate in shape but, for the CT and PCA populations, some fusiform somata were observed. The largest labelled neurons correspond to the TA muscle, followed in size by those for the CT muscle, and being the smallest those for the PCA muscle. It is generally accepted that there are differences in somata size within the nucleus ambiguus (Lawn, 1966a, 1966b; Hinrichsen and Ryan, 1981; Pásaro et al., 1983; Portillo and Pásaro, 1988; Patrickson et al., 1991; Saxon et al., 1996; Hayakawa et al., 1999) and most of the authors agree that the TA neurons are the largest and the PCA neurons are the smallest (Davis and Nail, 1984; Portillo and Pásaro, 1988; Hirasugi et al., 2007). This variability in size has been related with the roles played by these neurons in laryngeal physiology, thus the larger size of the TA neurons is associated to the rapid adductor activity during breathing cycle, meanwhile the slower speed of contraction of the CT and PCA muscles could be linked to the smaller size of their innervating neurons (Hinrichsen and Ryan, 1981; Davis and Nail, 1984; Patrickson et al., 1991; Yoshida et al., 1998). Hayakawa et al. (1999) identified two types of neurons innervating the PCA muscle; the largest ones probably receiving excitatory inputs and the small ones receiving inhibitory inputs. We have not identified these two different sizes in the neurons labelled from the PCA muscle.

The main limitation in our study is due to the technical approach. It is not possible to guarantee that all the synaptic terminals innervating one muscle can uptake the tracer, and this means that surely some neurons are not labelled and, hence, not taken into account when the total number of neurons innervating one muscle was measured. When CtB is injected into one muscle it bounds to GM1 ganglioside embedded in the lipid matrix of the synaptic membrane, later incorporated inside the neuron, and finally retrogradely transported to the soma (Vercelli et al., 2000; Conte et al., 2009). Although we cannot expect that in all the terminals these processes will be fulfilled, the consistency in the measurements of the number of labelled neurons along the processed animals make us confident that the quantity of identified cells is closer to the real number of neurons innervating one muscle. In addition, the number of identified neurons for the PCA and the TA muscles fit well with the number of myelinated axons found in the recurrent laryngeal nerve of the rat (Pascual-Font et al., 2006c), and with the data from other quantitative studies in the rat (Hinrichsen and Ryan, 1981; Portillo and Pásaro, 1988). On the other hand, and regarding to the morphometrical analysis, the sizes obtained in the present work, performed by fluorescent tracers, are in the range of those obtained in previous morphometrical analysis developed in bright-field microscopy (Wetzel et al., 1980; Hinrichsen and Ryan, 1981; Bieger and Hopkins, 1987; Portillo and Pásaro, 1988; Patrickson et al., 1991).

In conclusion, our report precisely locates the neurons innervating significant intrinsic laryngeal muscles and, additionally, establishes a well defined model that permits to develop studies on the reorganization of the laryngeal somatotopy both when the laryngeal nerves are injured and allowed to reinnervate the larynx.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors are grateful to Dr. Marc Rodriguez- Niedenführ for his critical review of the manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED