Laryngeal afferent stimulation enhances fos immunoreactivity in periaqueductal gray in the cat

Laryngeal Afferent Stimulation EnhancesFos Immunoreactivity in Periaqueductal

Gray in the Cat

R. AMBALAVANAR,1* Y. TANAKA,1,2 M. DAMIRJIAN,1 AND C.L. LUDLOW1

1Voice and Speech Section, NIDCD, NIH, Bethesda, Maryland, Maryland 208922Department of Otolaryngology, Head and Neck Surgery, School of Medicine, Kurume

University, 67 Asahi-Machi, Kurume City 830, Japan

ABSTRACTThe main functions of the larynx are protection of the airways, respiration, and

vocalization. Previous studies have suggested a link between the mechanisms controllingvocalization and afferent feedback from the larynx. We inquired whether stimulation of thelaryngeal afferents that run in the internal branch of the superior laryngeal nerve (ISLN)activates neurons of the periaqueductal gray (PAG), a midbrain region implicated invocalization. We counted the number of neurons expressing Fos, the protein product of theimmediate early gene c-fos, in the PAG. The counts were done both in experimental cats afterelectrical stimulation of the ISLN and nonstimulated controls. We also investigated thepossible presence of nitric oxide synthase, an enzyme that synthesizes nitric oxide, in PAGneurons that respond to laryngeal afferent stimulation by double labeling for reducednicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and Fos. Fos expressionwas significantly greater (P # 0.00714) in the lateral and dorsolateral regions of the PAG inthe experimental group than in the controls. The Fos-immunoreactive neurons did not containNADPH-diaphorase, a marker for nitric oxide synthase. Our study suggests that laryngealafferent stimulation activates neurons in discrete longitudinal columns of the PAG includingthe regions that have previously been shown to be involved in vocalization, and that theseneurons do not contain nitric oxide synthase. J. Comp. Neurol. 409:411–423, 1999.Published 1999 Wiley-Liss, Inc.†

Indexing terms: vocalization; adductor reflex; immunohistochemistry; superior laryngeal nerve;

electrical stimulation

The periaqueductal gray (PAG) is a functionally complexstructure surrounding the cerebral aqueduct in the mam-malian midbrain and is involved in antinociception (Liebe-skind et al., 1973), regulation of the cardiovascular system(Skultety, 1962; Depaulis et al., 1988; Carrive et al., 1988),defense reactions (Bandler et al., 1985a,b), reproductivebehavior (Sakuma and Pfaff, 1979a,b), and respiration(Kabat, 1936; Sessle et al., 1981; Davis et al., 1993a,b).Natural sounding vocalization can be elicited by electricalstimulation of the PAG and the adjacent tegmentum inmany vertebrates including the chimpanzee (Brown 1915),monkey (Magoun et al., 1937; Jurgens and Pratt, 1979a,b),cat (Kelly et al., 1946), rat (Waldbillig, 1975), guinea pig(Martin, 1976), and bat (Suga and Yajima, 1988). Damageto the PAG disrupts both spontaneous vocalization andvocalization evoked from other brain sites, demonstratingthat the PAG is essential for vocalization (Kelly et al.,1946; Skultety, 1962; Randall, 1964; Jurgens and Pratt,1979a,b).

Bandler and others (Bandler et al., 1991; Bandler andShipley, 1994; Beitz, 1994) have proposed that a columnarorganization across the rostrocaudal levels provides func-tional specificity in the PAG. Chemical stimulation of thelateral column of the PAG by microinjections of excitatoryamino acid (EAA) that selectively excites neuronal cellbodies, elicited natural sounding vocalization in cats (Ban-dler, 1982). Similar studies have shown species-specificvocalizations with stimulation in the lateral column of thePAG at the intermediate and caudal levels in the monkey(Jurgens and Richter, 1986; Jurgens and Lu, 1993; Lu and

Grant sponsor: NIDCD intramural research program; Grant number:653.

*Correspondence to: Dr. R. Ambalavanar, VSS, NIDCD, 10 Center DriveMSC 1416, Bethesda, MD 20892–1416. E-mail: [email protected]

Received 18 March 1997; Revised 9 December 1998; Accepted 4 February1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 409:411–423 (1999)

PUBLISHED 1999 WILEY-LISS, INC. †This article is a USgovernment work and, as such, is in the public domain in theUnited States of America.

Jurgens, 1993), cat (Carrive et al., 1988; Bandler andCarrive, 1988; Davis et al., 1993b; Zhang et al., 1994), andrat (Depaulis et al., 1992). Chemical stimulation of thedorsolateral columnar region of the PAG in the cat, how-ever, evokes a nonvoiced hissing predominantly at therostral level, whereas stimulation of the lateral region atthe intermediate levels evokes voiced types of vocalizationsuch as howls, mews, or growls (Zhang et al., 1994).

The larynx performs vocalization and protects the air-ways. Both mechanical and chemical stimuli produceafferent discharges in the superior laryngeal nerve (SLN;Sant’Ambrogio et al., 1983, 1995; Hwang et al., 1984). Avariety of tactile and proprioceptive stimuli occur duringvocalization. Physiological studies have shown that theafferent input from the upper airways affects the timing ofthe vocalization motor pattern during PAG stimulation inthe cat (Davis et al., 1993b; Sakamoto et al., 1993; Zhanget al., 1994; Shiba et al., 1995) and voice quality in squirrelmonkey (Thoms and Jurgens, 1981; Jurgens and Kirz-inger, 1985). Although the above studies suggest that thelaryngeal afferent feedback via the PAG may play a role inthe modulation of laryngeal muscle activity during vocal-ization, the neuroanatomical evidence of this control islacking.

There are no reports of primary laryngeal afferentsterminating in the PAG to affect this afferent feedbackcontrol. The nucleus tractus solitarii (NTS) of the dorsome-dial medulla is the central termination area for thelaryngeal afferent axons that run in the internal branch ofthe SLN (ISLN) in the cat (Kalia and Mesulam, 1980;Nomura and Mizuno, 1983) and rat (Patrickson et al.,1991; Mrini and Jean, 1995). It is possible that laryngealafferent information reaches the PAG by polysynapticconnections through the NTS. Based on neuroanatomicalstudies, the NTS is reciprocally connected with the PAG inthe cat (Yoshida et al., 1985; Bandler and Tork, 1987), rat(Herbert and Saper, 1992; Cameron et al., 1995), andrabbit (Mellor and Dennis, 1986). The tracers used in theprevious studies, such as horseradish peroxidase (Kaliaand Mesulam, 1980; Nomura and Mizuno, 1983; Mrini andJean, 1995), cannot be used to localize second or higherorder neurons receiving laryngeal afferent input. Theprotein product of the immediate early gene c-fos isregarded as a marker for neuronal activation, and can mappolysynaptic pathways with single cell resolution in thecentral nervous system (Sagar et al., 1988). In the presentstudy, we used Fos immunocytochemistry to determinewhether neurons of the PAG are responsive to laryngealafferent stimulation, and if so, their distribution withinthe PAG.

The enzyme reduced nicotinamide adenine dinucleotidephosphate (NADPH)-diaphorase is a marker for nitricoxide synthase (NOS), an enzyme that synthesizes nitricoxide (NO), a transmitter or signaling molecule (Bredt andSnyder, 1992; Snyder, 1992). NADPH-diaphorase is selec-tively located within the dorsolateral PAG column of therat (Herbert and Saper, 1992; Onstott et al., 1993; Carriveand Paxinos, 1994) and in the ventrolateral regions (supra-oculomotor cap) in the rat, rabbit, cat, monkey, and human(Carrive and Paxinos, 1994). In addition, iontophoreticapplication of NO donors to the PAG neurons inhibitneuronal activity within the PAG (Lovick and Key, 1996).We inquired whether the neurons expressing Fos afterISLN stimulation are possibly inhibitory to the other PAGneurons by double labeling for NADPH-diaphorase and

Fos. Parts of this study have appeared in abstract form(Ambalavanar et al., 1996a,b).

MATERIALS AND METHODS

Animals and surgery

Twelve cats of either sex were divided into three groupsfor Fos studies: experimental (group 1, n 5 5), sham-operated controls (group 2, n 5 5), and anesthetic controls(group 3, n 5 2). Three additional cats (group 4, n 5 3)were used in a separate experiment to evaluate the effectof stimulation of the ISLN on respiration, blood pressure,heart rate, and on muscles other than the intrinsic laryn-geal muscles. The animals received humane care in compli-ance with the Guide for the Care and Use of LaboratoryAnimals, prepared by the National Academy of Sciencesand published by the National Institutes of Health (NIHPublication No. 86–23, revised 1985). Tissues from ten ofthese animals were used in a related study (Tanaka et al.,1996).

All the animals were initially anesthetized with ket-amine (25–30 mg/kg) and then with a-chloralose (40mg/kg). The cats in group 3 did not have any surgery. Thefollowing procedures were done for the cats in groups 1, 2,and 4. A tracheostomy canula was inserted as low aspossible into the trachea to avoid stimulation to thelarynx, and spontaneous breathing was maintained withoxygen (100% O2, 2 L/minute). A femoral artery wascanulated to monitor the blood pressure and blood gasesperiodically. The blood pH was maintained between 7.2and 7.4. The blood pCO2 and pO2 were maintained be-tween 30–50 mmHg and 400–500 mmHg, respectively.Blood pressure, heart, and respiratory rates (using anendotracheal CO2 monitor) were also monitored continu-ously. Recordings of blood pressure and heart rate (mea-sured with a 5 lead ECG monitor) are given in Figure 1Afor a cat from group 4.

Electrical stimulation

Two hooked wire electromyographic (EMG) electrodeswere inserted into both the right and left thyroarytenoid(TA) muscles of the cats in groups 1, 2, and 4 through thecricothyroid membrane. The three cats in group 4 also hadtwo hooked wire electrodes inserted into each of thefollowing muscles: the right and left cricothyroid, right andleft posterior cricoarytenoid (intrinsic laryngeal muscles),and the diaphragm, mylohyoid, genioglossus, and superiorconstrictor muscles. In all the cats except the ones in group3, the right ISLN was surgically exposed and secured in abipolar cuff electrode with a 2-mm inner diameter and2-mm spacing between the contact points. As the handlingof the animals and surgical procedures may also induceexpression of Fos, the cats were maintained resting quietlyfor 4 hours after the initial surgery. Following the restingperiod, the ISLN was stimulated only in cats from groups 1and 4 at intensity levels where the muscle responsebecame supramaximal and produced bilateral responsesin the TA muscles (0.6–0.9 Volts; Fig.1B). Stimuli were 0.2milliseconds in duration, and presented at 0.5 Hz for 60minutes. This low rate of ISLN stimulation, 0.5 Hz, waschosen to reduce any effects on respiration and cardiovascularfunction. In another study (Ludlow and Luschei 1997), nochange in respiratory cycle duration was observed at stimulusrates of 0.5 Hz or less. The control cats (group 2 and 3)were not stimulated but rested during this period.

412 R. AMBALAVANAR ET AL.

Tissue preparation

Thirty minutes after the completion of either stimula-tion (groups 1 and 4), or after resting (groups 2 and 3), thecats were deeply anesthetized with pentobarbital (100mg/kg, i.p) and perfused transcardially with 0.1 M phos-phate-buffered saline (PBS, pH 7.4) followed by a mixtureof 2% paraformaldehyde and 0.2% picric acid in 0.1 Mphosphate buffer (pH 7.4). The brains were removed,postfixed overnight in the same fixative, and cryoprotectedin 30% sucrose. Serial transverse sections (50-µm-thick) ofthe midbrain were cut through the PAG and collected inPBS in separate wells for Nissl staining, immunohisto-chemical processing of Fos, NADPH-diaphorase histochem-istry, and double labeling of Fos and NADPH-diaphorase.The Nissl-staining employed the standard cresyl-fast vio-let staining technique.

Fos immunohistochemistry

The sections were washed well in PBS with 0.3% TritonX-100 (PBST) and incubated in 0.1% H2O2 for 1 hour toinactivate the endogenous peroxidase activity. Following

an incubation in normal goat serum for 1 hour at roomtemperature, the sections were incubated in an antibodyagainst Fos (Oncogene Sci., Cambridge, MA, Ab-2, 1:1,000)for 48 hours at 4°C. The sections were washed well inPBST and incubated in biotinylated anti-rabbit antibodyovernight at 4°C, washed again, and incubated in avidin-biotin complex (ABC Elite kit, Vector Labs., Burlingame,CA) for 1 hour at room temperature. The bound antibodywas visualized as a dense blue-black reaction productusing buffered glucose oxidase, and diaminoibenzidinesolution (Shu et al., 1988). To test for evidence of nonspe-cific labeling, some sections were stained by substitutingeither PBS for the primary antibody or using a pread-sorbed antibody (1 µg peptide/ml diluted antibody). Thesecontrol sections did not show any labeling.

NADPH-diaphorase histochemistry

After washing in PBS, the sections were incubated in amedium containing 1 mg/ml reduced b-NADPH (Sigma,St. Louis, MO), 0.15 mg/ml Nitroblue tetrazolium (Sigma),and 0.3% Triton X-100 in 0.05 M Tris-HCl buffer (pH 7.6)

Fig. 1. A: Heart rate (HR) and arterial blood pressure (Art/s,systolic; MAP, mean arterial pressure; Art/d, diastolic) readings fromone cat before and during internal branch of the superior laryngealnerve (ISLN) stimulation at 0.5 Hz at a supramaximal response level(0.8 volt) with a 0.2-ms pulse. B: The average of 114 responses tostimuli at 0.5 Hz are shown in both ipsilateral and contralateralintrinsic laryngeal muscles during electrical stimulation of the ISLN.

The latency of the ipsilateral thyroarytenoid (ITA) muscle response isaround 10 milliseconds with multiple peaks showing a polysynapticresponse. Note a similar response in the contralateral thyroarytenoid(CTA) muscle. Responses can also be seen in the ipsilateral cricothy-roid (ICT) but are limited in the contralateral cricothyroid (CCT)muscle. Similar responses are seen in both ipsilateral and contralat-eral posterior cricoarytenoid muscles (IPCA and CPCA).

NEURONAL ACTIVATION IN THE PERIAQUEDUCTAL GRAY 413

for 30–45 minutes at 37°C (see Scherer-Singler et al.,1983). The NADPH-diaphorase-positive neurons showed apurple color reaction product. No staining was observedwhen NADPH was omitted from the incubation medium.

Double labeling

The sections collected for double labeling were firststained for NADPH-diaphorase and washed and immuno-

stained for Fos as described above. The Fos immunoreactiv-ity was visualized as brown reaction product with DABand H2O2. Photomicrographs were taken with Koda-chrome 25 day light film for color slides with didymiumand 80A filters. The color slides were made into a Kodakphoto CD from which the photographic plate (Fig. 2) wasmade by using Adobe Photoshop 4.0 on a Power Macintoshwith the labels overlayed by Corel 8.0 on a Power PC.

Fig. 2. Sections from an experimental cat from different rostrocau-dal levels (caudal, caudal intermediate, and rostral intermediate) ofthe periaqueductal gray (PAG; A–C) double-labeled for reduced nico-tinamide adenine dinucleotide phosphate (NADPH)-diaphorase andFos (approximate coordinates based on Berman’s atlas are given abovefor each section on A–C). The approximate boundaries of the PAGregions (dl, dorsolateral; dm, dorsomedial; l, lateral; vl, ventrolateral;and vm, ventromedial) are marked on A–C. NADPH-diaphorase-positive neurons and fibers are stained purple, whereas the Fos-likeimmunoreactive nuclei are brown. The dense bands of purple stainingat the dorsolateral position of the aqueduct (aq) indicate the dorsolat-eral columns of the PAG (A–C) and the bands ventral to the aqueduct

are the supraoculomotor caps (soc). The area within the rectangles ‘‘dl’’(dorsolateral) and ‘‘l’’ (lateral region) in A are shown at a higher powerin D and E, respectively. Many brown Fos-immunoreactive nuclei canbe clearly seen (arrows) amongst the densely stained purple NADPH-diaphorase-stained neurons and fibers in the dorsolateral (D,F) andlateral (E,G) regions. NADPH-diaphorase-positive neurons containclear, unstained nuclei in the dorsolateral (F) and lateral (G) regions.The Fos-like immunoreactive nuclei are distributed in close proximityto the NADPH-diaphorase-positive neuronal cell bodies and fibers(F,G). III, oculomotor nucleus. Scale bar 5 3 mm in A–C; 600 µm in Dand E; 100 µm in F and G.


Quantification of Fos-immunopositive nuclei

To quantify the Fos-like immunoreactive nuclei (FLI),the PAG was divided into four rostrocaudal levels usingthe coordinates of Berman’s atlas (Berman, 1968), i.e.,caudal (P1.5– P0.2), caudal intermediate (A 0.6–A 1.6),rostral intermediate (A 2.5–A 3.3), and rostral (A 4.1–6.4).Within each level, the PAG was subdivided into six re-gions: dorsomedial, dorsolateral, lateral, ventrolateral,ventromedial including dorsal raphe and juxta-aqueductal(see Fig. 2). The region of the PAG immediately surround-ing the cerebral aqueduct with the smallest neurons andthe lowest neuronal density, is the juxta-aqueductal col-umn in the rat (Beitz, 1985; Herbert and Saper, 1992) andin the cat (Hamilton 1973; Liu and Hamilton 1980). Itconsists of a moderate level of NOS containing fibers andprocesses throughout the rostrocaudal extent (Onstott etal., 1993). The juxta-aqueductal column was marked onNADPH-diaphorase-stained sections and was confirmedwith the help of Nissl-stained sections.

The boundaries of the other regions were carefullymarked on the basis of anatomical and neurochemicalobservations as described by Bandler and colleagues (Ban-dler and Depaulis, 1991; Bandler and Shipley, 1994). Thesize and shape of the regions vary along the rostrocaudalaxis (Bandler and Depaulis, 1991; Bandler and Shipley,1994). Sections stained for NADPH-diaphorase were usedto assist in the identification of the subregions of the PAG(Fig. 2A–C). The dorsolateral region was identified as aclearly NADPH-positive band. The NADPH-negative re-gion medial to the dorsolateral region was identified as thedorsomedial region. The NADPH-negative region belowthe dorsolateral region was identified as the lateral region.The region ventral and medial to the lateral region was theventromedial region at the rostral levels. Caudally, theventrolateral region was identified with the disappearanceof the supraoculomotor cap and the region medial to thatwas occupied by the ventromedial region including thedorsal raphe. The lateral wings of the dorsal raphe wereused as the ventral border of the ventrolateral region. Theborders marking different regions of the PAG at the fourrostrocaudal levels are shown in Figure 3.

The sections were examined at 3100 magnification andthe outlines of the sections and the locations of all stainednuclei were marked manually on a video computer-basedimage analysis system (Neurolucida, Microbrightfield, Inc.,Colchester, VT), without knowing whether the cats wereexperimental or control. All manually marked nuclei werethen counted automatically in separate regions. Countswithin each subdivision were totaled for three brainsections at least 250 µm apart in each rostrocaudaldivision to obtain an aggregate in each animal. An exampleof one section at each of the rostrocaudal levels from oneexperimental, one sham-operated control and one anesthe-tized control are shown in Figure 3.

Statistical analyses

To examine the effect of ISLN stimulation on FLIexpression across all the regions of the PAG, an initialsplit-plot analysis of variance (ANOVA) was conducted.The group effect (sham-operated control versus stimu-lated) was analyzed as a whole-plot effect, with individualanimals regarded as ‘‘plots.’’ Regional differences (juxta-aqueductal, dorsomedial, dorsolateral, lateral, ventrolat-

eral, and ventromedial) and their interaction with thegroup effect (Group by Region interactions) were alsoexamined.

The response variable was the square root of the totalFos count, summed over the right and left sides and overthe rostrocaudal levels. The square root transformationwas used to stabilize the variances of the counts and maketheir distribution more normal or bell-shaped. Statisticalanalysis on the raw FLI counts are not valid because of thestrong relationship between mean and variance in countdata. Preliminary analyses showed that the differencesbetween the right and left sides were not significant.

If the stimulation effect differed among regions (P #0.05), separate ANOVAs were conducted for each region toexamine group differences (stimulated versus sham-operated control), differences due to rostrocaudal levels(caudal, caudal intermediate, rostral, and rostral interme-diate), and interactions between the stimulation effect andlevel (Group by Level interactions). Because a total of 7analyses were conducted (6 regions including juxta-aqueductal, dorsomedial, dorsolateral, lateral, ventrolat-eral, and ventromedial, and the initial multifactorialANOVA), the criterion probability level for statisticalsignificance was Bonferroni-corrected to # 0.00714(0.05/7 5 0.00714).

RESULTS

Cardiorespiratory effects andelectromyographic (EMG) responses

The heart rate and blood pressure were not affected bythe stimulation in all 3 cats in group 4 (see Fig. 1A for datafrom one cat). As a result of stimulation, the respiratoryrate was reduced by 25% in one of the three cats examined,whereas the other two remained constant regardless ofsimulation. Electrical stimulation of the ISLN evoked anEMG response at a latency of approximately 10 millisec-onds in both the right and left TA muscles (Fig. 1B).Similar responses were also observed in the cricothyroidand posterior cricoarytenoid muscles (Fig. 1B).

Fos-like immunoreactivity within theperiaqueductal gray

Experimental group. Stimulation of the ISLN re-sulted in substantial expression of FLI in the lateralregions, particularly at the caudal two-thirds (P 1.5–A 2.5;Fig. 4A,C) of the PAG. At the level of the rostral dorsalraphe and the trochlear nucleus (A 0.6), the lateral regionshowed many FLI forming clear clusters bilaterally with afew FLI in the dorsolateral regions (see examples in Figs. 3and 4). The juxta-aqueductal region also expressed manyFLI in this group. Further, at caudal levels of the PAG, atthe level of the inferior colliculus and nucleus cuneiformis(P 0.9–P 1.5), many FLI were scattered in the ventrolateraland ventromedial regions including the dorsal raphe bilat-erally (Fig. 4F; see examples of one cat from each group inFig. 3). The ventrolateral, ventromedial, and the juxta-aqueductal regions showed fewer FLI in the rostral thanthe caudal levels.

Rostrally, at the level of oculomotor nucleus (A 2.5), FLIwere found bilaterally in the lateral, dorsolateral, andoccasionally in the dorsomedial regions. Further rostrally,at the level of Edinger-Westphal nucleus (A 4.1), occasionalFLI were found in these regions in the experimental cats.


Fig. 3. Computer video lucida drawings of sections (50-µm-thick)from one cat in each group, through four rostrocaudal levels (approxi-mately P 1.5, A 0.6, A 2.5, A 4.1 of the Berman’s atlas coordinates) of theperiaqueductal gray (PAG). Each dot indicates a single Fos-immunore-active neuron in a single section. The approximate boundaries of thePAG regions (dl, dorsolateral; dm, dorsomedial; l, lateral; vl, ventrolat-eral; and vm, ventromedial) are marked on the drawings. Left:Stimulation of the internal branch of the superior laryngeal nerve

(ISLN) resulted in extensive Fos expression in the lateral PAG. Thejuxta-aqueductal, ventrolateral, and ventromedial regions show immu-noreactive nuclei in both stimulated (left) and sham-operated controlcat (middle). Anesthesia alone resulted in a few Fos-like immunoreac-tive nuclei (FLI) in the PAG (right). aq, aqueduct; DR, dorsal raphe;EW, Edinger-Westphal nucleus; MLB, mediolateral bundle; III, oculo-motor nucleus; IV, Trochlear nucleus.

Fig. 4. Distribution of Fos-like immunoreactive nuclei (FLI) at thecaudal level (approximately at P 0.2 level in the Berman’s atlas) of theperiaqueductal gray (PAG) after internal branch of the superiorlaryngeal nerve (ISLN) stimulation (A, C, E), and after surgery withno stimulation (B,D). The lateral (l) and dorsolateral (dl) PAG showmany Fos-immunoreactive nuclei following stimulation (A and E).Sham surgery resulted in Fos-like immunoreactivity in the ventrolat-

eral region of the caudal PAG (F) and the ventromedial regionincluding the dorsal raphe (dr), with very little expression in thelateral or dorsolateral regions (B). Arrows in C and E indicate the Fosimmunoreactivity nuclei. aq, aqueduct; dl, dorsolateral; l, lateral.Scale bar 5 800 µm for A and B; 200 µm for C and D; 400 µm for Eand F.

Sham-operated control group. In contrast to theexperimental group, only a few scattered FLI were foundin the lateral regions of the PAG in the sham-operatedcontrol group (see examples in Figs. 3 and 4B,D). Similarnumbers of FLI were found in the juxta-aqueductal regionin the control group as in the experimental group espe-cially at the caudal levels. The ventrolateral and ventrome-dial regions (Fig. 3 and 4) of the PAG also expressed manyFLI in the control group to an equivalent level in theexperimental group. At rostral levels a few FLI werescattered in these regions.

Anesthetized controls. Anesthesia alone induced onlyoccasional FLI (2–5 per section), in the PAG (see exampleof one such animal in Fig. 3).

The numbers of FLI in the six different regions atdifferent rostrocaudal levels are given for the five experi-mental cats and five sham-operated controls in Figure 5.

Large differences were found in the numbers of FLIbetween different cats within the experimental group;some cats showed lower numbers in all areas than others.Examination of the muscle responses among the experi-mental cats indicated no major differences in the latenciesor amplitude of the muscle responses, which could accountfor differences in the overall numbers of FLI among theseanimals.

Statistical analyses

The total number of FLI was significantly higher in theexperimental cats than in the sham-operated controls (F 569.0292; P 5 0.00005). When group comparisons wereconducted for each of the individual regions, significantlygreater numbers of FLI were found in the lateral (P 50.002) and dorsolateral (P 5 0.001) regions of the PAG inthe stimulated group than the control group (Table 1).Although the numbers of FLI in the juxta-aqueductalregion were high, the counts were not significantly differ-ent (P # 0.00714) between the two groups. Similarly, thenumber of FLI in the ventrolateral, ventromedial, and

Fig. 5. The number of Fos-positive cells totaled from 3 sections(right and left sides) at each of the four rostrocaudal levels (caudal,caudal intermediate, rostral intermediate, and rostral) in individualcats (5 experimental and 5 sham-operated control cats) for thejuxta-aqueductal, dorsomedial, dorsolateral, lateral, and ventrome-

dial regions of periaqueductal gray (PAG). The ventrolateral columndoes not extend to the rostral and rostral intermediate levels and thelateral column does not extend to the rostral level. The ventromedialregion includes the dorsal raphe at the caudal and caudal intermedi-ate levels. FLI, Fos-like immunoreactive nuclei.

TABLE 1. The P Values for the Effects of Group, Level, and Group byLevel Interaction in Repeated Measures ANOVA

Region Group Level Group by level

Juxta-aqueductal 0.0195 0.000051 0.2842Dorsomedial 0.0085 0.121 0.121Dorsolateral 0.0011 0.096 0.126Lateral 0.0021 0.058 0.178Ventrolateral 0.028 0.00051 0.074Ventromedial 0.0525 0.0002 0.6813

1Significant at a 5 0.05 level after Bonferroni correction for the fact that seven analysisof variance (ANOVA)s were performed (a 5 0.05/7 5 0.00714).


dorsolateral regions did not differ significantly (P #0.00714) between the two groups.

NADPH-diaphorase staining and itsrelationship to Fos-expressing neurons

NADPH-diaphorase-positive neuronal cell bodies, fi-bers, and dendrites were found mainly in the dorsolateralPAG along its rostrocaudal extent as an intensely labeledband (Fig. 2A–D), with the highest amount of staining inthe middle one-third of the PAG. At the level of theoculomotor nucleus, another smaller band of intenselystained fiber plexus and cell bodies were observed ventralto the aqueduct. Although fewer in number, clearly stainedneuronal cell bodies and processes were observed in thelateral region of the PAG (see Fig. 2E,G). The dorsomedialregion of the PAG did not show much staining for NADPH-diaphorase. The dorsal raphe and the dorsolateral tegmen-tal nuclei, which were embedded in the ventral part of thecaudal PAG, showed many densely stained neuronal cellbodies including their cytoplasmic processes.

Fos-immunoreactive neurons were codistributed withNADPH-diaphorase-positive neurons in the dorsolateral(Fig. 2D,F) and lateral (Fig. 2E,G) regions of the PAG.Sections were examined at a high magnification (3400) forcolocalization of Fos and NADPH-diaphorase in theseregions. No double-labeled neurons were observed eitherthe dorsolateral (Fig. 2F) or the lateral (Fig. 2G) regions.The NADPH-diaphorase-positive fibers and dendrites werein close vicinity to the FLI neurons (Fig. 2F,G).

DISCUSSION

Induction of Fos protein is indicative of neuronal activa-tion (Sagar et al., 1988). Using Fos as a marker forneuronal activity, the aim of the study was to determinewhether neurons in the PAG are activated followinglaryngeal afferent stimulation. The results clearly show anincrease in the number of FLI in the PAG following ISLNstimulation, indicating functional connections betweenthe larynx and the PAG. These FLI neurons did notcontain NADPH-diaphorase, indicating that these neu-rons do not produce NO as a neuronal messenger.

Potential drawbacks ofFos-immunocytochemistry

Fos immunocytochemistry is a powerful technique forthe study of functional pathways of different systems(Morgan et al., 1987; Sagar et al., 1988; Dragunow andFaull, 1989). Neurons in some regions of the brain andspinal cord, however, do not express Fos (Dragunow andFaull, 1989). Therefore, the absence of Fos in PAG neuronscannot be interpreted as lack of neuronal activation.Hyperpolarizing or inhibitory conditions have not beenreported to induce Fos. Thus, we do not know if some PAGneurons are inhibited by stimulation of the ISLN.

Significance of Fos activation in theperiaqueductal gray

The most striking result of our study was the Fosactivation in the lateral region of the PAG which has longbeen implicated in vocalization (Jurgens and Richter,1986; Carrive et al., 1987; Bandler and Carrive, 1988;Depaulis et al., 1992; Jurgens 1994; Jurgens and Lu, 1993;Lu and Jurgens, 1993; Davis et al., 1993b) and has

recently been shown to be crucial for the precise timing ofrespiratory, laryngeal, and oral muscle activity for vocaliza-tion (Zhang et al., 1994; Davis et al., 1993b,1996). Davisand her colleagues proposed that relevant afferent input tolateral PAG neurons can modulate vocal outputs (Davis etal., 1993b, 1996). Our study provides the first neuroana-tomical evidence that afferent information from the larynxreaches the lateral PAG and supports the view thatlaryngeal afferent feedback via the PAG may regulateintralaryngeal muscle activity during vocalization (Daviset al., 1993b; Shiba et al., 1995).

The activation of the dorsolateral PAG most likelyindicates a role for this region in the afferent control ofvocalization or related reflexes. Although direct stimula-tion of the dorsolateral PAG does not produce voicedsounds as occurs in the lateral PAG (Zhang et al., 1994),this region may be indirectly involved in vocal control.Larson and his colleagues (Larson and Kistler, 1984, 1986;Larson, 1991) recorded single unit activity from PAGneurons of monkeys during vocalization and the majorityof these were located in the dorsolateral region. Thepresent Fos results further suggest that this region of thePAG receives afferent information from the larynx. Theaxonal bundles arising from the neurons of the dorsolat-eral PAG column distribute fibers and terminals locallywithin the PAG (Cameron et al., 1995). These neuronstherefore may be modulating vocalization-related activityvia other PAG regions.

Fos immunoreactivity has been demonstrated in thelateral and dorsolateral PAG following increased arterialpressure (Bandler and Shipley, 1994; Murphy et al., 1995).Although cardiovascular changes have been reported fol-lowing respiratory afferent stimulation in dogs (Angell-James and de Burgh Daly, 1975; Kordy et al., 1975) andcats (Tomori and Widdicombe, 1969) at higher rates ofstimulation (10–20 Hz), no changes were observed at therate of stimulation used to the ISLN in our study (0.5 Hz).Further, in previous studies, either both the external andinternal branches of the SLN (Angell-James and de BurghDaly, 1975; Kordy et al., 1975), or the mucosa of therespiratory tract (Tomori and Widdicombe, 1969), werestimulated. In contrast, we stimulated only the internalbranch of the SLN. The respiratory rate was reduced by25% in only one of the three cats in group 4 as a result ofISLN stimulation in our study, whereas the other 2remained constant. Similarly, Ludlow and Luschei (1997)also observed an increase in respiratory cycle duration atthis stimulation rate in only one of the five cats theystudied. However, it cannot be ruled out that the cardiore-spiratory changes during ISLN stimulation are unlikely tohave contributed to the increase in FLI in the lateral anddorsolateral PAG in our study.

In certain regions of the PAG, the number of FLI did notdiffer between the experimental and sham-operated con-trols. In particular, the juxta-aqueductal, ventromedial,and ventrolateral regions expressed many FLI in bothgroups. The ventrolateral region of the PAG is thought tobe involved in mediating hypotension, quiescence, andimmobility (Carrive, 1993). It receives inputs from theupper cervical spinal cord in the cat (Keay and Bandler,1992), and the cervical (Blomquist and Craig, 1991) andlumbar spinal cord in the cat and monkey (Yeziserki, 1988;Blomquist and Craig, 1991; Vanderhorst et al., 1996). Theventrolateral and juxta-aqueductal regions of the PAG alsoreceive second-order visceral afferent input from the me-


dial and commisural subnuclei of the NTS in the rat(Herbert and Saper, 1992) where most of the cardiovascu-lar afferent fibers are found. These inputs from the NTSare most dense at the caudal levels of the PAG (Herbertand Saper, 1992), the levels in which many FLI were foundin our study. In our experiments, surgery to expose theISLN involved handling of the neck regions, and arterialand urinary catheterization were also performed in bothgroups of animals. Although it is difficult to distinguish thespecific stimuli responsible for the expression of FLI in theventrolateral and juxta-aqueductal regions, expression inboth groups of animals may indicate the effect of surgeryand/or cardiovascular and respiratory effects due to han-dling.

Potential afferent pathways inducing Fos inthe periaqueductal gray

Electrical stimulation of the ISLN activates afferentneurons which terminate in the NTS (Tanaka et al., 1996).At the same time, the adductor reflex produces bilateralTA muscle contractions, which in turn will induce stimula-tion of the tendon afferents and laryngeal mechanorecep-tors. Some or all of these laryngeal responses, as well asthe initial afferent stimulation, may be involved in theelicitation of PAG neuronal activity.

A strong mono- or polysynaptic activation is effective toinduce Fos in neurons (Morgan and Curran, 1986; Sagar etal., 1988). As there are no reports of direct termination oflaryngeal afferents in the PAG, our results suggest thatsecond-order neurons of the NTS conveyed the afferentinformation to the PAG neurons. For example, neurons inthe NTS, particularly the interstitial subnucleus, receivelaryngeal afferent terminals in the cat (Kalia and Mesu-lam, 1980; Nomura and Mizuno, 1983) and the rat (Patrick-son et al., 1991; Mrini and Jean, 1995), and show FLIfollowing ISLN stimulation in the cat (Tanaka et al., 1995,1996). As previous tracing studies have demonstratedconnections between the NTS and the PAG (Mantyh, 1983;Yoshida et al., 1985; Bandler and Tork, 1987; Herbert andSaper, 1992; Cameron et al., 1995), the laryngeal afferentinformation most likely was conveyed to the PAG via theNTS, through second- (or third-) order afferents. Thelateral PAG region, which is highly implicated in vocaliza-tion, receives projections from the ipsilateral medial andbilateral ventrolateral NTS in the cat (Bandler and Tork,1987). In the rat, it receives bilateral projections from themedial and caudal commissural subnuclei of the NTS(Herbert and Saper, 1992), whereas in the rabbit, projec-tions are from the contralateral NTS to the ventrolateral,lateral, and dorsal regions of the PAG (Mellor and Dennis,1986). Interestingly, the subnuclei of the NTS that connectto the PAG are not the major laryngeal afferent termina-tion zones. Therefore, it is possible that some other brainregions are also involved in this pathway.

Following ISLN stimulation, increases in FLI were alsoobserved in the parabrachial nucleus with ipsilateralpredominance (Ambalavanar, R., Tanaka, Y., and LudlowC.L, unpublished observations). The lateral parabrachialregion has reciprocal connections with the PAG (Saper andLoewy, 1980; Beitz, 1982; Fulwiler and Saper, 1984) andwith the NTS (Norgren, 1978; Herbert et al., 1990). Thus,the afferent information from the NTS may have reachedthe PAG via the parabrachial region.

Potential efferent modulation

We do not know whether the FLI are the same neuronsthat produce vocalization when stimulated chemically. Ifthey are, then it would imply that the vocalization-responsive neurons are under direct afferent feedbackcontrol. On the other hand, the FLI neurons are possibleinterneurons that modulate the PAG neurons that producevocalization upon chemical stimulation.

Descending projections of the PAG have been describedbased on neuroanatomical studies in the monkey (Jurgensand Ploog, 1970; Jurgens and Pratt, 1979b; Mantyh, 1983),the rat (Yajima and Hayashi, 1983; Cameron et al., 1995;Chen and Aston-Jones, 1996; Ennis et al., 1997; Holstegeat al., 1997), and the cat (Holstege, 1989; Vanderhorst andHolstege,1996). The nucleus retroambiguus (NRA) is apremotor region in the caudal part of the medulla, thoughtto play a critical role in the mediation of vocalization(Zhang et al., 1992, 1995). Holstege and his colleagues(Holstege, 1989; Holstege and Ehling, 1996; Vanderhorstand Holstege,1996) provided evidence, based on theirneuroanatomical studies, of a final common pathway, fromthe lateral PAG via the NRA to the motor neurons involvedin vocalization. Neurons in the lateral PAG column projectto the NRA in the caudal part of the medulla, which in turnprojects to vocalization-related motor neurons in the cat(Holstege, 1989; Chen and Aston-Jones, 1996; Holstegeand Ehling, 1996; Vanderhorst and Holstege, 1996). Ourfinding of FLI in the lateral PAG following laryngealafferent stimulation may indicate afferent influences onNRA projecting PAG neurons that may modulate laryn-geal muscle activity during vocalization. Although directprojections to the nucleus ambiguus region have beenreported in the monkey (Jurgens and Pratt, 1979b; Man-tyh, 1983) and the rat (Cameron et al., 1995; Ennis et al.,1997), it is unlikely that these are specific to the laryngealmotor neurons. According to Zhang et al. (1995), the PAGmediates the laryngeal muscle control mainly via aninitial synapse in the NRA. Sessle et al. (1981) demon-strated that stimulation of the PAG, as well as swallowingand coughing, can suppress the activity of interneuronsinvolved in this reflex in the NTS. Thus, descending reflexmodulation may also occur at the level of the NTS via thedescending projections from the PAG to the NTS (Yoshidaet al., 1985; Bandler and Tork, 1987).

Laryngeal afferent stimulation induced Fosexpression and NADPH-diaphorase

The neuronal NADPH-diaphorase is colocalized withNOS, the enzyme that synthesizes the neuronal messen-ger NO (Bredt et al., 1991). Immunocytochemical studiesby Onstott et al. (1993) demonstrated the presence of NOSin the dorsolateral region of the PAG. Therefore, thedistribution of NADPH-diaphorase staining in our studymost likely indicates the presence of NOS in the PAGneurons in agreement with the previous reports of thedistribution of NADPH-diaphorase-containing cell bodiesand fibers in the dorsolateral region (Herbert and Saper,1992; Onstott et al., 1993; Carrive and Paxinos, 1994) andthe supraoculomotor cap. We also observed many NADPH-diaphorase-stained neurons in the lateral PAG. The FLIneurons in our study did not show NADPH-diaphorasestaining, but were codistributed closely with NADPH-diaphorase-positive neurons and fibers. Thus, NO gener-ated by the NOS-positive cells in the PAG may influence


the activity of the non-NOS-containing FLI neurons thatare embedded in the matrix of NOS-positive cell bodiesand processes. The NO may be an inhibitory neuronalmessenger for PAG neurons (Lovick and Key, 1996) andtherefore the NADPH-diaphorase-positive neurons mayexert an inhibitory control on some of the PAG neurons.

CONCLUSIONS

The present Fos study demonstrates that laryngealafferent stimulation activates neurons in the lateral regionof the PAG that has previously been shown to producevocalization upon stimulation. This provides an anatomi-cal basis for previous demonstrations of afferent influenceson PAG-evoked vocalization. Although stimulation of thedorsolateral region of the PAG has not been shown toproduce voiced sounds, the present Fos results indicatesome neurons in these regions are responsive to laryngealafferent inputs. The neuronal excitation in the PAG mayindicate a role for these neurons in the reflex control of thelarynx during vocalization. The results of the doublelabeling studies indicate that those neurons activated bylaryngeal afferent stimulation do not contain NOS butmay be modulated by other NOS-containing PAG neurons.

ACKNOWLEDGMENTS

The authors thank Dr. Paul Smith for his assistancewith statistical analyses, Dr. Robert Strominger for hisconstructive comments on the manuscript, and Dr. FrankEvans for his assistance with data acquisition.

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Laryngeal afferent stimulation enhances fos immunoreactivity in periaqueductal gray in the cat

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