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1Department of Physiology, Medical College of Wisconsin, 2Zablocki Veterans Affairs Medical Center, and 3Department of Physical Therapy, Marquette University, Milwaukee, Wisconsin 53226
Submitted 10 June 2004 ; accepted in final form 17 August 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSREFERENCES |
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central chemoreception; control of breathing
An additional characteristic of raphe neurons is that they are chemosensitive (32, 37–39), which, as demonstrated by microdialysis-induced focal acidification, is capable of increasing breathing by 12–30% during wakefulness and by 20% during non-rapid eye movement (NREM) sleep in goats and rats, respectively (13, 26). Additionally, selectively lesioning serotonergic neurons in neonatal rats results in hypoventilation and reduced sensitivity to hypercapnia in adult life (21). Finally, injections of an antibody against the serotonin transporter (SERT) conjugated to saporin (anti-SERT-SAP) into the raphe decreased the CO2 response by 16% during wakefulness in rats (28). However, serotonergic neurons compose only 15–50% of the total neuronal population in the medullary raphe nuclei (16, 18). The sum of the evidence leads to two questions: 1) Are there cell types in the raphe other than serotonergic neurons that are involved in CO2/H+ "chemoreception?" and 2) Is the modulatory effect on breathing exclusively through chemosensitive serotonergic neurons? It is conceivable that raphe NK1R-expressing neurons in the raphe may also be a determinant of chemoreceptor responses. Indeed, Nattie et al. recently reported that rats exhibited a 21% reduction in the response to 7% inspired CO2 during wakefulness after injections of SP-SAP in the raphe (28), similar to the effects of unilateral and bilateral SP-SAP-induced lesions in the parapyramidal/retrotrapedoid (RTN/Ppy) region (27). However, a combination injection of two neurotoxins (SP-SAP and SERT-SAP) did not have an additive effect on the CO2 response, despite the evidence that serotonergic and NK1R-expressing neurons are separate cell populations. These findings led to the conclusion that "separate populations of serotonergic and NK1R-expressing neurons in the medulla are both involved in chemoreception" (28).
Glutamate receptor-expressing neurons are also important in the raphe influence on the control of breathing (3, 4, 8). For example, a decrease in phrenic and hypoglossal activities during progressive hyperoxic hypercapnia was reported after a glutamate receptor-specific neurotoxin ibotenic acid (IA) was injected into the raphe of decorticate, paralyzed, artificially ventilated piglets (8).
It is unclear from lesion studies whether reductions in CO2 sensitivity or any other effects are due to attenuation of central chemoreception or whether these effects are simply due to loss of non-chemoreceptor-related neuromodulatory inputs from the raphe. One potential means of distinguishing between these possibilities is to determine whether the effect of lesions is specific to CO2 sensitivity or whether it is a general effect on breathing at rest (awake and asleep) or during exercise. Accordingly, one objective of the present study was to test the hypothesis that raphe lesioning with sequential injections of SP-SAP and IA will decrease CO2 sensitivity, but not cause hypoventilation at rest (awake or asleep), or during exercise, nor induce transient changes in respiratory rhythm. The choice and order of neurotoxins was based on the knowledge that SP-SAP is neurotoxic to a subset of raphe neurons, whereas IA is a more general neurotoxin. A second objective was to test the hypothesis that the combination of SP-SAP and IA neurotoxic lesions will have a greater effect than the individual effects of each neurotoxin. This hypothesis is based on the finding that injections of SP-SAP in the pre-Bötzinger complex of goats resulted in transient changes in respiratory rhythm and pattern and that subsequent injections of IA caused terminal apnea (40, 41). A third objective was to determine whether there is plasticity (recovery) after a lesion-induced change in ventilatory control.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSREFERENCES |
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Experimental Design
The experimental animals (n = 13) underwent a series of two surgeries: an initial surgery for subcutaneous elevation of the carotid arteries and for placement of electrodes into respiratory muscles and a second surgery 3 wk later for implantation of microtubules into the medullary raphe (Fig. 1). Beginning 2 wk after microtubule implantation, physiological measurements were obtained at rest, during exercise, and while inhaling elevated inspired CO2 nearly daily throughout the duration of the protocol. Also, beginning 2 wk after the second surgery, we assessed the effects of microdialysis-induced focal acidosis in the raphe (13). Subsequently, we tested the acute and chronic effects of neurotoxic lesions with a sequence of three injections, made in the awake state on separate days: 1) a control injection of either 50 pM saporin (SAP) or mock cerebral spinal fluid (mCSF), 2) 50 pM SP-SAP, and 3) 50 mM IA. Breathing [arterial PCO2 (PaCO2)] during sleep was also assessed at four time points: before and 
5 days after SP-SAP injection, and 12 h and 5 days post-IA injection. Animals were then euthanized 8–13 days post-IA, the head was perfused and fixed, and the medulla was harvested for processing after completion of all protocols. Similarly, four medullas from unoperated goats were collected to obtain control values for raphe tissues in goats.
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For all surgeries, the goats were anesthetized with a cocktail of ketamine and xylazine (24:1 vol/vol), intubated, and mechanically ventilated, and anesthesia was maintained with 1–1.5% halothane in oxygen. In the initial surgery (under sterile conditions) the carotid arteries were isolated from the vagi and elevated superficial to the muscle, and the skin was sutured. Electromyographic (EMG) electrodes were placed in the diaphragm in all animals. In some animals, upper airway electrodes were implanted through an anterior midline incision in the neck into laryngeal and pharyngeal muscles and into expiratory abdominal muscles. Three electroencephalographic (EEG) electrodes were implanted along the midline of the cranium evenly spaced at 2 cm centered at the level of bregma. Electrooculographic (EOG) electrodes were implanted 8–10 mm dorsal to the orbital ridge bilaterally. After surgery, the goats received ceftifur sodium (2 mg/kg) daily as an antibiotic for 1 wk.
Three weeks later, a second surgery was performed to chronically implant one, two, or three microtubules into the medullary raphe nuclei. Microtubules (cannulas) were implanted through an occipital craniotomy, using the dorsal surface of the medulla, obex, and the midline as reference points (42). A single 70-mm "double-barrel" polyethylene [(PE) PE-50 and/or PE-90 inside a PE-205 tube] microtubule (n = 8), or one (n = 3), two (n = 1) or three (n = 1) 70-mm stainless steel (18.5 gauge) microtubules were chronically implanted at the midline into either the caudal (raphe obscurus) and/or rostral (raphe pallidus) raphe nuclei. The double-barrel PE microtubules were implanted for additional purposes unrelated to the present studies (13). After placement, the microtubules were secured with screws in the bone and dental acrylic.
Laboratory personnel closely monitored the goats continuously for a minimum of 24 h after the brain implantation surgery. Typically, the goats were unable to stand without assistance for 1.5–6 h postsurgery, and one animal was unable to stand for 2 days after the implant. Food and water intake were monitored closely in all goats daily after the implantation surgery. Brain edema was minimized with dexamethasone injections (0.4 mg·kg–1·day–1 iv for 2 days, then decreasing by 0.05 mg·kg–1·day–1) three times a day for 1 wk. Infection was minimized with chloramphenicol injections (20 mg/kg iv) for 3 days and with daily injections thereafter of ceftifur sodium (2 mg/kg) and gentamyacin (3 mg/kg). Buprenorphine was administered 3–12 h after implantation to minimize pain.
Physiological Measurements
For all studies except sleep and exercise, a fitted mask was taped firmly to the snout, and a two-way breathing valve was attached to the mask. The inspired port of the valve was connected to a pneumotachograph and computerized data acquisition system to measure inspiratory flow (
I). The expired port was connected to a spirometer (Tissot) for collection of expired air [expired minute ventilation (E)] and analysis of O2 [O2 uptake (O2)] and CO2 concentrations. Diaphragm, upper airway, and abdominal EMG activities were continuously measured. A chronically placed catheter in the elevated carotid artery was used to monitor arterial blood pressure (BP) and heart rate (HR) and for arterial blood sampling to obtain pH, arterial PO2 (PaO2), and PaCO2 values (model 278, Ciba-Corning). Rectal temperature (Tre) of the animal was measured at regular intervals.
Assessment of CO2 sensitivity.
Breathing, BP, HR, and Tre were measured for 30 min before and during exposure to three levels of elevated inspired CO2 (2.5, 5.0, and 7.5% CO2 in RA). Arterial blood samples were drawn during the control period and during the fourth and fifth minute of each CO2 exposure level. The relationship between E and PaCO2 (CO2 slope) was used as an index of CO2 sensitivity.
Exercise protocol. The goats stood on a treadmill during a 15-min control period before walking for 4 min at 1.8 miles/h at both 5 and 15% grades. Arterial blood samples were drawn during the last 2 min of the control period and during the final 2 min of each exercise level. BP was measured continuously, except for periods of blood sampling, and Tre was monitored throughout the study.
Sleep protocol.
The goats were trained to sleep in a stanchion for 4–6 days before study. Goats were studied for 6 h (9 PM to 3 AM) during the natural dark cycle in the environmental chamber isolated from the equipment and investigators. Diaphragm and upper airway EMG, EEG, and EOG and arterial BP were continuously monitored (Grass recorder and computer acquisition system) in all studies, and in some cases I and abdominal EMG were also recorded. Arterial blood samples were drawn at regular intervals, or with changes in state [awake, NREM sleep, or rapid eye movement (REM) sleep], and Tre was recorded after each blood withdrawal.
Injections of mCSF, SAP, SP-SAP, and IA.
Breathing, BP, HR, and O2 were measured for a 30-min control period, during and for 5 h after the injection of 1 or 10 µl mCSF, SAP, SP-SAP, and IA. Arterial blood was drawn during the control period and at 30-min intervals after each of the injections. Tre was continuously measured throughout the studies.
Histological Studies
Medullary tissues from experimental goats were harvested 8–13 days post-IA injection (10.0 ± 0.8 days), placed in a 4% paraformaldehyde solution for 24–48 h, and then placed in a 30% sucrose solution for an additional 48 h. The medullas were then frozen and serial sectioned (20–25 µm) in a transverse plane, and the sections adhered to chrom alum-coated slides. The tissue from experimental (n = 13) and unoperated control (n = 4) goats was then stained with hematoxylin and eosin and a tryptophan hydroxylase (TPOH) or a NK1R immunostain, and coverslipped for microscopic examination.
Hematoxalin and eosin stain. Serial sections were thawed and dried for 24 h before hematoxylin and eosin staining. The tissue was sequentially bathed in 100 and 95% ethanol solutions before a tap water rinse for >5 min. The tissue was exposed to filtered hematoxylin (Harris formula, Surgipath) for 3 min, rinsed, dipped in 0.5% acid alcohol, exposed to Scott's tap water (Surgipath) for 30 s, and rinsed again. Then the tissue was bathed in eosin for 30 s, and sequentially bathed in two 95% and three 100% ethanol solutions before bathing in xylenes for >1 min, and coverslipped.
TPOH immunostaining.
After the tissue sections were thawed and dried for 1 h, the samples were washed twice with 10 mM PBS for 10 min. The tissue was then treated for 15 min with 0.25% trypsin for antigen retrieval and washed in PBS for 10 min (3 times). H2O2 (0.5%) in PBS was applied to the sections for 30 min and washed with PBS (3 times). A 5% normal horse serum (0.2% Tris-buffered saline) blocking solution was applied for 1 h before washing in PBS. Tissue was then incubated with the primary antibody (anti-TPOH, 1:250 in serum) overnight (
12 h), washed in PBS, and incubated with the secondary antibody (biotinylated anti-mouse in serum-blocking solution, 1:60 vol/vol) for 1–3 h. ABC solution was then applied for 50 min (Vector ABC Elite), and the tissue was washed, exposed to diaminobenzidine for 5 min before a final wash, and coverslipped.
NK1R immunostaining.
After the tissue sections were thawed and dried for 1 h, the samples were washed twice with 10 mM PBS for 10 min. The tissue was then treated for 20 min with heat antigen retrieval (DAKO), and washed in PBS for 10 min (3 times). H2O2 (0.5%) in PBS was applied to the sections for 30 min, and washed with PBS (3 times). A 5% normal horse serum (0.2% Tris-buffered saline) blocking solution was applied for 1 h before washing in PBS. Tissue was then incubated with the primary antibody (anti-NK1R, 1:10,000 in serum) over two nights (48 h), washed in PBS, and incubated with the secondary antibody (biotinylated anti-mouse in serum-blocking solution, 1:60) for 1–3 h. ABC solution was then applied for 50 min (Vector ABC Elite), and the tissue washed, exposed to diaminobenzidine for 5 min before a final wash, and coverslipped.
Location of the microtubules. The site of implantation was identifiable by visualization of an area of absent or disrupted tissue, which extended over a finite rostrocaudal distance and was related to the size of the implanted microtubule (0.9–2.3 mm). The implantation site was defined as being at the tip (ventral-most aspect) and middle of the microtubule-induced tissue disruption.
Neuronal count regions.
The medullary raphe area count region was in an area that included (but was not restricted to) the traditional location(s) of serotonergic neurons, beginning ventral to the dorsal motor nucleus of the vagus and 1.0 mm lateral to the midline bilaterally and extending to the ventral surface of the medulla. We defined the count region to include an area (volume) of tissue slightly greater than that used in previous investigations due to the presence of both TPOH-expressing and NKIR-expressing neurons at a distance 
1.0 mm lateral to the midline in the goat. Neurons in the inferior olivary nuclei were excluded from all midline neuron counts.
A second region near the ventral lateral medullary surface (VLM) was counted bilaterally in TPOH- and NK1R-stained tissue to serve as a control, nonlesion count region. This region begins 3 mm lateral to the midline 0–1 mm from the ventral surface, ventromedial to nucleus ambiguus and the facial nucleus, and extends from the obex to the level of the caudal pole of the facial nucleus. The described region closely resembles the Ppy/RTN region described for the rat (27).
Lesion quantification. The lesion was quantified two ways: 1) neuron counts from the raphe area and VLM region and 2) volumetrically. Midline raphe area and VLM neuron counts were made every 100–250 µm and averaged every 500 µm from –5.0 mm caudal to and +10.5 mm rostral to obex in unoperated control tissues (n = 4), and greater than –1.5 mm caudal to and +1.5 mm rostral to the effected tissue in experimental tissues (n = 13). Total living, dead, TPOH-expressing, and NK1R-expressing neurons were counted in the raphe area, and TPOH-expressing and NK1R-expressing neurons counted in the VLM.
With the use of hematoxylin and eosin staining, living neurons stain purple, whereas dead neurons stain pink and are circular in shape (Fig. 2, A and B). Initially, we intended to quantify the extent of the on the basis of the reduction in total living neurons remaining at the lesion site compared with the expected number of living neurons from unoperated control animals. However, we found considerable variation in number of living neurons in the control goats; thus, in some lesion goats, living neurons counts were equal to or greater than average control levels. Accordingly we determined that the reduction in living neurons in the lesion goats was not a suitable measure to quantify the lesion.
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The lesion regions within the raphe area describing the effects on TPOH-expressing and NK1R-expressing neurons were defined on the basis of the location of effected tissue volumes (described above), such as 1) the injection site, which includes all TPOH-expressing and NK1R-expressing neurons within the raphe area bounded by the overall effected tissue volume, and 2) region of raphe area tissue caudal or rostral to the injection site. Direct comparisons of raphe area and VLM counts between unoperated control and experimental goats were used to determine the lesion effect in TPOH-expressing and NK1R-expressing stained tissues (examples are shown in Fig. 2, C and D), with VLM counts serving as an internal control site and the neuron counts from unoperated control goats serving as the raphe area standard.
Data and Statistical Analyses
Anatomy. Total TPOH-expressing and NK1R-expressing neurons at the midline raphe and VLM sites were compared with counts from unoperated control goats, and they were tested with a one-way ANOVA with repeated measures and a Bonferroni post hoc test and with a t-test to determine significant changes from control. Because of the few numbers of animals that received no neurotoxin, only SP-SAP or only IA, we did not perform a statistical analysis on volumetric and/or total dead neuron counts, but we include these data to quantitatively compare lesion size among these groups.
Acute effects of neurotoxins. For each goat, physiological variables were averaged into 5-min mean values for each injection study and statistically analyzed by using a one- or two-way ANOVA with repeated measures and a Bonferroni post hoc test.
Chronic effects of the neurotoxin injections. For each physiological variable, a one-way ANOVA with repeated measures and Bonferroni post hoc test were used to determine treatment effects. Sleep state was determined by standard EEG-EOG and behavioral criteria (33). Effects on breathing during wakefulness, NREM sleep, and REM sleep were assessed by a one-way ANOVA with repeated measures.
Analysis of altered breathing periods. Analyses of augmented breaths and irregular breathing (altered breathing periods) was performed in all experimental animals. The analysis included quantification of the frequency of fractionated inspiration (with and without associated prolonged TE), frequency of augmented breaths per hour and associated postsigh apnea (PSA; in s), and the frequency and duration (relative to control TE) of central apneas (prolonged TE events). Treatment effects on the duration and frequency of central apneas, fractionated breaths, a combination of all altered breathing periods, and augmented breaths were assessed by one-way ANOVA with repeated measures and Bonferroni post hoc analysis (P < 0.05).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSREFERENCES |
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Histological analysis. Serotonergic neurons in unoperated control goats (n = 4) were present in the raphe area and VLM sites (Fig. 3, 1–4). NK1R-expressing neurons (n = 4) were located in medullary regions similar to those previously described (22), including the hypoglossal motor nucleus, nucleus ambiguus, pre-Bötzinger complex, spinal trigeminal tract, the VLM and midline raphe area. In general, the total living, TPOH-expressing, and NK1R-expressing raphe area neuron counts increase (P < 0.001) from the caudal to rostral regions of the medulla, ranging from 50.2 ± 3.2 to 960 ± 82 (total neurons), from 19.9 ± 1.6 to 84.3 ± 14.3 (TPOH-expressing), and from 7.8 ± 0.8 to 19.5 ± 0.6 (NK1R-expressing) neurons per tissue section. Additionally, TPOH-expressing and NK1R-expressing neuron counts from the VLM area also increase (P = 0.001) from the caudal to rostral medulla, reaching a peak [86.3 ± 8.1 (TPOH-expressing) and 20.0 ± 2.9 (NK1R-expressing) neurons per tissue section] +6.0 mm rostral to obex.
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The total volume of affected tissue within the raphe area was greater in goats that received SP-SAP, IA, or both SP-SAP and IA compared with the animal that only had microtubules implanted and no neurotoxins (Fig. 5A). Among the subvolumes in the raphe area (attributed to the volume occupied by the microtubule, tissue volume devoid of neurons, and tissue volume containing dead neurons), the tissue volume containing dead neurons represented the greatest volume. Also, both the volume containing dead neurons and volume devoid of neurons were consistently larger in goats that received neurotoxins. In contrast, there was no appreciable difference between the goat that did not receive neurotoxins and all other lesion goats in the volume occupied by the microtubule.
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Physiology
Acute responses of mCSF or SAP, SP-SAP, or IA injections.
Injections of either mCSF (10 µl, n = 7) or SAP (1 or 10 µl, n = 2 or 1) had no acute effect on I, BP, HR, or O2 throughout the 5 h after the injections (P 0.289). Similarly, there were no acute effects on I, BP, HR, or O2 after injections (1 or 10 µl) of SP-SAP (P = 0.131; n = 8) or IA (P = 0.794; n = 7) in the caudal raphe. However, IA injections in more rostral regions of the medullary raphe nuclei had a substantial effect on I (n = 3), increasing to 163% of control. Additionally, mean BP and HR were elevated by 33.6 and 30.1%, respectively, after IA injection in this rostral group.
Chronic effects of mCSF, SAP, SP-SAP, or IA injections.
Injections of mCSF (n = 7) or SAP (n = 3) had no effect on resting variables (P > 0.05) or the ventilatory response to CO2 (98.4 ± 2.0% of control; P > 0.05) 1–9 days after injection. Compared with preneurotoxin injection values, CO2 sensitivity decreased (23.8%) 3–7 days after SP-SAP injections (P = 0.48, n = 10; Fig. 6). However, CO2 sensitivity returned to control values >7 days post-SP-SAP injections (P = 0.197). Similarly, CO2 sensitivity decreased (26.8%, P < 0.001) 3–7 days after subsequent IA injections but again returned to control levels >7 days post-IA injection (P = 0.709). However, SP-SAP or IA had no chronic effect on room air breathing (E, breathing frequency, tidal volume, O2, PaCO2, PaO2, and pH) during these time periods, with the exception of a transient reduction in breathing frequency at rest 7 days post-SP-SAP injections (Table 1; P > 0.05).
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65% of the total study time awake, 32% in NREM sleep, and 4% in REM sleep. This sleep pattern was unaltered >5 days after SP-SAP, <12 h after, or >5 days after IA injections (n = 7; P > 0.17). PaCO2 during nighttime wakefulness, NREM sleep, and REM sleep was also unaffected by neurotoxic lesions during these time periods (n = 7; P > 0.05). The ventilatory response (change in PaCO2) to two levels (mild and moderate) of steady-state exercise was variable among the goats tested (n = 4). The change in PaCO2 with mild exercise was –2.6 ± 1.1 Torr, and it was unaffected after SP-SAP (–3.2 ± 2.5 Torr) or IA (–3.3 ± 1.1 Torr) injections (P > 0.05). Similarly, the change in PaCO2 with moderate exercise preneurotoxin was –4.1 ± 1.2 Torr, which also was unaffected after SP-SAP (–4.6 ± 2.2 Torr) or IA (–4.1 ± 1.2 Torr) injections (P > 0.05).
Altered breathing periods.
After microtubule implantation (before and after neurotoxin injections) in all animals studied we observed a number of spontaneous, irregular breathing events that we collectively termed altered breathing periods, including 1) fractionated breaths (Fig. 7, goat L) similar to those previously reported with raphe microtubule implantation (10), 2) altered fractionated breaths with prolonged TE before and/or after (Fig. 7, goat K) the fractionation, and 3) central (Fig. 7, goats A and B) or obstructive (Fig. 7, goat L) apneic events. There was no specific microtubule implantation site that correlated with any of the types of altered breathing periods. The apneic events were predominantly central in nature (9 of 13 goats), where the prolonged TE was observed in both the airflow and diaphragm signals. Before neurotoxin injection, the average ratio of the prolonged TE to normal TE was 2.53 ± 0.15, occurring 8.2 ± 1.4 times/h (n = 13). Associated with the prolonged TE were elevated activity of the pharyngeal constrictor (thyropharyngeus) EMG and a cessation of expiratory abdominal EMG activity, similar to that reported with hyperventilation-induced central apnea (10). In five of these goats, single or multiple short breaths interrupted the apnea. Overall, SP-SAP and subsequent IA injections had no effect on the ratio of prolonged TE to normal TE (3.6 ± 0.9, 3.4 ± 0.7, respectively) or the frequency of the apneas during daytime (11.7 ± 2.7, 9.6 ± 2.0, respectively) or nighttime wakefulness, or NREM sleep (P 0.141).
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7 mm rostral to obex. The obstructive apneas began during NREM sleep, but the goats were aroused midway during the apneas (Fig. 7, goat L). When apneas were >10 s in duration, a brief hyperpnea followed both apnea types.
We observed fractionated breathing in 7 of 13 goats studied, two of which exhibited fractionation with an associated prolonged TE (2 times greater than eupneic TE). The frequency of fractionation postmicrotubule implantation was 15.0 ± 3.62/h (n = 5). In goats studied after SP-SAP, the frequency tended to increase (+11 ± 6.0/h, n = 2), and one goat only exhibited fractionated breathing after IA injection in the caudal raphe. Five goats exhibited fractionated breathing at night during wakefulness, and only two had fractionated breathing during NREM sleep.
Augmented breath frequency (4.2 ± 0.6/h) and the PSA (4.1 ± 1.1 s) were variable between goats after microtubule implantation (n = 12). SP-SAP had no effects on augmented breath frequency (4.5 ± 0.9/h) or the PSA (6.4 ± 1.3 s) at any time after the injection (P > 0.05; n = 7). Similarly, IA had no effects on either augmented breath frequency (5.1 ± 0.9/h) or the PSA (5.8 ± 1.4 s) at any time after injection (P > 0.05; n = 8). We also noted that, when augmented breaths occurred at night, they were during wakefulness or associated with arousal and that they rarely occurred during NREM sleep.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSREFERENCES |
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Lesion Quantification and Estimation
Historically, there has been a great deal of difficulty in quantifying lesion size and severity in acute and chronic studies, leading to the construction and application of several approaches (1, 23–25). One useful approach has been the identification of dead neurons within or around the injection site (42; see also Fig. 1). Application of the hematoxylin and eosin stain to unoperated control tissues showed that dead raphe area neurons were rare (only 7 in 86,345 neurons). However, in goats with implanted microtubules, dead neurons were found over a 1- to 3-mm range around the microtubule. Virtually no dead neurons were found outside the midline raphe area. Our laboratory previously found that dead neurons are greatest in number at the injection site but that there was a 10–15% reduction of total neurons (living plus dead) compared with the contralateral side 2–3 wk after injection. This deficit is presumably due to absorption of dead neurons (42). Therefore, the dead neuron counts in the present study may underestimate the lesion size and severity, and the deficit of neurons may be closer to 25–30%.
As a result, we used two additional measures to estimate lesion size: 1) volumes (containing dead neurons, devoid of neurons, and occupied by the microtubule) and 2) loss of (TPOH-expressing and NK1R-expressing) neurons. The greatest volume in goats that received neurotoxin injections was the raphe area volume containing dead neurons, followed by the volume devoid of neurons. Both observations indicate that these aspects of the lesion were quantitatively larger than the tissue lesion associated with the implanted microtubule. Additionally, there was a 36 and 35% reduction in midline raphe TPOH-expressing and NK1R-expressing neurons, respectively, with VLM neuron counts unaltered. These lines of evidence lead us to believe that, although dead neurons were found at raphe sites more caudal and rostral to the injection site, the lesion was specific to the midline raphe and more severe at the neurotoxin injection site.
Clearly, implantation of the microtubules through the dorsal surface and into the medullary raphe, as well as initial microdialysis and pH studies, caused a degree of tissue disruption-induced neuronal death (13). Subsequently, the neurotoxins also created additional lesions in the raphe, indicated by the histological analysis and observed physiological effects on CO2 sensitivity days after injection. It is noteworthy that two of the three largest lesions (assessed by the total number of dead neurons) were in the goats that exhibited obstructive apnea during NREM sleep (goats L and M). Additionally, the goat that had the largest lesion (in terms of overall affected tissue volume) had the most frequent central apneas. However, we found no further correlation between other types of altered breathing periods (central apneas and fractionated breaths) and location or severity of the raphe lesions. It is therefore unclear whether loss of a specific raphe cell population is linked to the observed altered breathing periods.
Serotonergic and NK1R-Expressing Neurons of the Midline Raphe
The medullary raphe nuclei are classically defined as serotonergic regions distributed in a vertical column along the midline throughout the medulla, where the serotonergic neurons represent 15–50% of the total population in cats and rats (16, 18, 36). Serotonergic neurons are similarly distributed in the goat, but they tend to be found at slightly greater distances from the midline than previously reported. Additionally, recent reports in rats and piglets point to a second area with a high degree of TPOH-expressing and/or NK1R-expressing neurons termed the nucleus paragigantocellularis lateralis (PGCL) or Ppy/RTN (27–29). We also found (in goats) serotonergic neurons in the VLM region, with many in this region intersecting the PGCL and near the caudal RTN. Thus it appears that the serotonergic neurons of the goat medulla are similar to other species in spatial distribution.
NK1R-expressing neurons were found at medullary sites similar to those previously described (22) and relative to TPOH-expressing neurons are fewer in number. This finding is also similar to previous reports that indicate TPOH-expressing neurons are greater in number in the raphe than NK1R-expressing neurons (28). Additionally, there were a greater number of NK1R-expressing neurons bilaterally in the VLM than found in the midline raphe. We did not observe NK1R-expressing neuronal loss at the VLM sites, indicating that our neurotoxic lesions did not affect this area of cells and that the NK1R-expressing loss was specific to the midline raphe area.
Disruption of Respiratory Rhythm: Interpretation of Altered Breathing Periods
Although still debated, there are data from in vitro and in vivo preparations indicating that the pre-Bötzinger complex is critical for eupneic respiratory rhythm generation (11, 40, 41). Among others, the pre-Bötzinger complex contains overlapping subpopulations of neurons expressing serotonin and neurokinin-1 receptors (11, 17). Chemical and/or electrical stimulation of the raphe, or endogenous and/or exogenous increases in serotonin at the pre-Bötzinger complex, increases respiratory frequency (2, 7, 34). On the other hand, serotonin antagonists block these effects and can elicit respiratory arrests and rhythm irregularity (2, 7, 31). Moreover, exogenous SP strongly modulates, and destruction of NK1R-expressing neurons in the pre-Bötzinger complex disrupts regular respiratory rhythm (11, 12, 35, 40). Collectively, these data implicate modulatory roles for serotonin and SP from the raphe to the respiratory rhythm generating mechanism at the pre-Bötzinger complex, which potentially contribute to the various altered breathing periods.
We found that fractionated breathing and other forms of disrupted eupneic respiratory rhythm were present after dorsal-to-ventral microtubule placement in the midline raphe area. These altered breathing periods were not all identical to those described in our laboratory's two previous reports; they were not associated with a discoordination of inspiratory and expiratory pump muscles (40), nor were they associated with swallows (10). Particularly different from past studies were the often-observed central apneas. We believe that it is conceivable that all of the altered breathing periods presently observed were due to imbalances of excitation and/or inhibition of respiratory rhythm-generating neurons as a result of 1) a destruction of raphe neuromodulatory neurons and/or 2) physical destruction of fibers of passage from the raphe to other respiratory nuclei such as the pre-Bötzinger complex. Alternatively, the microtubule may have destroyed fibers of passage between the bilaterally located pre-Bötzinger complex neurons. This alternative is suggested by findings that perturbations in respiratory rhythm occur after midline transection of medullary slices in solution (6). Transection along the midline severs axonal connections between the bilateral pre-Bötzinger complex nuclei that lie dorsal to the midline raphe nuclei. Therefore, fibers of passage between pre-Bötzinger complex nuclei may play a role in coordinating rhythmic output (6). However, this alternative is not supported by our findings that transient, strong stimulation of raphe neurons by IA eliminated the altered breathing periods (Fig. 8), which seems to support the concept that it was a deficit in raphe-associated neuromodulation that caused the altered breathing periods.
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Similar to previous reports, we observed attenuation of the ventilatory response to elevated inspired CO2 after SP-SAP- and IA-induced raphe lesions (8, 28). However, the attenuation of CO2 sensitivity was transient, lasting 3–7 days postinjection. This plasticity differs from earlier studies that report attenuation of the CO2 response up to 14 days post-SP-SAP injections in rats (28). Although we have no data to speak to the mechanism of plasticity observed, it is conceivable that other raphe or other chemoreceptor sites (peripheral and/or central) can compensate for the neuronal loss.
The SP-SAP- and IA-induced attenuation of CO2 sensitivity could be due to either 1) a deficit of CO2/H+ chemoreceptors or 2) a non-chemoreceptor-related loss of neuromodulation from the raphe neurons. As stated earlier, one potential means of distinguishing between these alternatives is to determine whether the effect is specific to CO2 sensitivity or whether it is a general effect on breathing. Our findings support the former alternative because the neurotoxic lesions did not cause hypoventilation at rest during wakefulness, sleep, or exercise. Furthermore, the neurotoxic lesions did not affect the frequency and characteristics of altered breathing periods, which were induced by the microtubule implantation and for which there was no recovery over time after the implant. In other words, there was a clear dissociation between regulation of CO2 sensitivity and the regulation of other aspects of the ventilatory control system consistent with the concept that the lesions induced a deficit in CO2/H+ chemoreception.
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Present address of M. Hodges: Dept. of Neurology, Yale University, 333 Cedar St., New Haven, CT 06520 (E-mail: matthew.hodges{at}yale.edu).
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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