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1 Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756-0001, USA2 Departments of Neurology and, Cellular & Molecular Physiology, Yale University, New Haven, CT, USA3 Advanced Targeting Systems, Inc, 11175-A Flintkote Ave, San Diego, CA, USA
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(Received 5 December 2003;
accepted after revision 7 January 2004;
first published online 14 January 2004)
Corresponding author E. E. Nattie: Department of Physiology, Dartmouth Medical School, Borwell Bldg, Lebanon, NH 03756-0001, USA. Email: eugene.nattie{at}dartmouth.edu
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Introduction |
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To study the function of single chemoreceptor sites in an unanaesthetized in vivo model we have applied a dual strategy. We examine the effects of (1) focal acidosis on breathing, or (2) focal cell specific lesions on the response to systemically applied CO2, the CO2 response. Focal CO2 stimulation at the retrotrapezoid nucleus (RTN) region stimulated breathing in wakefulness (Li et al. 1999), at the caudal NTS it stimulated breathing in both wakefulness and NREM sleep (Nattie & Li, 2002a), and at the medullary raphé it stimulated breathing only in NREM sleep (Nattie & Li, 2001). The state of arousal affects responses to focal stimulation (Nattie, 1998, 1999, 2000, 2001; Feldman et al. 2003). Non-specific chemoreceptor disruption at the ventral medullary surface and other sites by means of cooling, lesions produced by excitatory amino acid neurotoxins, and inhibition produced by muscimol dialysis decreased the response to systemic hypercapnia (Loeschcke, 1982; Berger & Cooney, 1982; Budzinska et al. 1985; Akilesh et al. 1997; Forster et al. 1997; Nattie & Li, 2000; Messier et al. 2002). But many types of neurones were affected in these studies.
As a first attempt to examine in vivo the role of a specific type of neurone in central chemoreception we used (Nattie & Li, 2002b) a conjugate of the ribosomal toxin saporin (SAP) and substance P (SP) the natural ligand for the NK1R (Wiley & Lappi, 1997). SP-SAP has been used successfully to specifically kill NK1R-expressing neurones involved in spinal cord pain responses (Mantyh et al. 1997) and in the generation of the normal respiratory rhythm in rats (Gray et al. 1999, 2001; Wang et al. 2002). We hypothesized that NK1R-expressing neurones are involved in chemoreception based on the similar distributions in the rat brainstem of NK1R immunoreactivity (Nakaya et al. 1994) and of central chemoreceptor sites (Nattie, 2000, 2001). SP-SAP injections bilaterally into one chemoreceptor site, the RTN and adjacent parapyramidal (Ppy) regions, destroyed 40–47% of NK1R-ir neurones and processes and produced both hypoventilation and a decrease in the CO2 response in both sleep and wakefulness. Unilateral destruction of 47% of NK1R-ir also decreased the CO2 response in both sleep and wakefulness. We concluded that NK1R-ir neurones or processes in the RTN–Ppy region are involved in central chemoreception and provide a tonic drive to breathe.
In this study we focus on a second chemoreceptor site, the region of the medulla containing serotonergic neurones. This site is of particular interest for a number of reasons. It contains both NK1R-expressing neurones and serotonergic neurones, which are likely to be separate populations (Léger et al. 2002). Serotonergic neurones are chemosensitive in vitro (Wang et al. 1998; Richerson et al. 2001; H. Wang et al. 2001) and are closely apposed to ventral medullary arteries (Bradley et al. 2002), a location that would be an advantage for neurones whose role is to sense blood CO2 levels. Focal CO2 stimulation of the rostral aspect of the medullary serotonergic cell group increases breathing in sleep (Nattie & Li, 2001). Large non-specific lesions of much of the region of the medullary serotonergic neurone distribution (Dreshaj et al. 1998) and muscimol dialysis focally in the rostral aspect of this distribution in newborn piglets (Messier et al. 2002) decrease the CO2 response. Chemical destruction of serotonergic neurones by systemic administration of 5,7-dihydroxytryptamine (DHT) in newborn rats induces, in the adult, hypoventilation (Olson et al. 1979; Mueller et al. 1984) and a decreased CO2 response (Mueller et al. 1984). Medullary serotonergic neurones may be involved in the pathogenesis of the sudden infant death syndrome (SIDS). There are serotonergic binding abnormalities in many SIDS cases (Panigrahy et al. 2000; Kinney et al. 2001) and there is an association of SIDS with a polymorphism in a promotor for the serotonin transport protein (SERT) gene (Weese-Mayer et al. 2003).
Here we use SP-SAP together with a novel conjugate, anti-SERT-SAP (anti-SERT antibody with saporin), to specifically kill (a) NK1R-ir neurones, (b) serotonergic neurones, or (c) both NK1R-ir and serotonergic neurones together. We find that these are separate neuronal populations and that destruction of a similar amount of either cell type alone or both together alters the breathing response to elevated levels of CO2 by a similar amount in adult rats in vivo during wakefulness and sleep.
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Methods |
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All procedures were within the guidelines of the National Institutes of Health for animal use and care and were approved by the Dartmouth College Institutional Animal Use and Care Committee. A total of 51 rats were used. Thirty male Sprague-Dawley rats (280–350g) were anaesthetized with ketamine (100mgkg–1I.M.) and xylazine (20mgkg–1, I.P.). Hindlimb withdrawal and corneal reflexes were tested to monitor the depth of anaesthesia. Responses resulted in administration of supplemental anaesthesia in the form of a quarter of the initial dose. The skull and a portion of the abdomen were shaved and the skin cleansed with betadine and alcohol. The head was placed into a Kopf stereotaxic holder and two EEG electrodes were screwed into the right side of the skull. One was over frontal cortex (2mm anterior to bregma and 2mm lateral to midline), another over parietal cortex (3.5mm posterior to bregma, 2mm lateral) and an earth lead placed between the two 3mm lateral to midline. For the EMG, a pair of wire electrodes was threaded through the nuchal muscles. A sterile telemetry temperature probe (TA-F20, Data Sciences, St Paul, MN, USA) was placed in the abdominal cavity. After recovery and control data collection, each rat was re-anaesthetized and received two microinjections into the midline of the rostral ventral medulla. The skin on the top of the skull was incised and one hole about 3mm long in the rostral–caudal dimension was drilled into the skull. The coordinates for the placement were 2.2–2.5mm and 3.3–3.6mm caudal from lambda in the midline with the bite bar at 9.5–10.1mm so that the top of the skull was level (Paxinos & Watson, 1982). The microinjections were made by using a 0.5µl Hamilton microsyringe with a 28 gauge needle inserted 10.3–10.6mm below the dorsal surface of the skull. Each microinjection lasted at least 5min and the needle remained in position for another 3–5min before removal. The wound was sutured.
Protocol
The rats were housed in a room with a light, rest period from midnight to noon and a dark, active period from noon to midnight. Food and water were available ad libitum. All the experiments were performed between 9a.m. and 4p.m. After 4–7 days recovery from the EEG/EMG implantation, baseline ventilation 
, tidal volume (VT), frequency (f), body temperature, and oxygen consumption were measured at least twice in each rat while breathing room air and 7% CO2 in air during sleep and wakefulness. Each rat then received two injections (100 nl each, 1µM in artificial cerebrospinal fluid) (Nattie & Li, 2002b) of one of the following agents, mouse IgG–saporin (IgG-SAP), substance P–SAP (SP-SAP), anti-SERT-SAP, or SP-SAP and anti-SERT-SAP combined (Advanced Targeting Systems, San Diego, CA, USA). The anti-SERT-SAP is a novel molecule. The antibody to the serotonin transporter was made to amino acids 376–388 of the rat sequence of the serotonin re-uptake transporter. This is a segment of the fourth extracellular loop. Immunization of mice, fusion, screening, isolation of monoclones and purification were as previously described (Harlow & Lane, 1988). Clones were also screened for ability to deliver saporin as described (Weltman et al. 1987; Kohls & Lappi, 2000). A single clone, 4A2.2, was used in these experiments for staining and immunotoxin synthesis. Immunotoxin was synthesized as described in Picklo et al. (1995). The anti-SERT-SAP conjugate had an ED50 of 543 pM against RBL-2H3 cells that express SERT but no effect at 10nM on PC12 cells, which do not (M. D. Kohls, unpublished observations). Ventilatory measurements were made on days 3, 6–8 and 13–15 after the injections. Sleep cycling was measured during 3 h of air breathing prior to and 8–13 days after the lesion.
Data analysis
We determined sleep and wakefulness using EEG and EMG electrode signals, the fast Fourier transform (FFT) of the EEG signal analysed in 3.6s epochs at delta (0.3–5Hz), theta (6–9Hz), and sigma (10–17Hz) frequency bands, and behavioural observations. We used criteria modified from those of Bennington et al. (1994) and Trachsel et al. (1988) as previously described (Nattie & Li, 2001, 2002a,b). In our case, we measured breathing only during quiet wakefulness. In active wakefulness the activity of the rat in the plethysmograph prevents reliable measurement of breathing. We analysed the amount of time the rat spent in NREM and REM sleep and in wakefulness during each experiment. Ventilation was measured using a flow-through whole body plethysmography as described by Pappenheimer (1977) and Jacky (1978). The volume of the plethysmograph was 7.6 l with a 3.5 l top to protect the head pedestal. The plethysmograph was connected by a high resistance leak to a similarly sized reference chamber. The inflow gas for the plethysmograph chamber was humidified and controlled by a flow meter at a minimum of 1.4 l min–1 to prevent rebreathing of exhaled gas. The outflow was matched to the inflow via a flow meter connected to a vacuum system. Approximately 100ml min–1 of outflow gas served O2 and CO2 analysers (Applied Electrochemistry). We measured chamber pressure by transducer and calibrated the plethysmograph with multiple 0.3ml injections. We measured chamber temperature by thermometer and rat body temperature by telemetry. For calculation of ventilatory data, we used the DataPac 2000 system to determine the pressure deflections and the respiratory cycle time for each of 100–300 breaths at defined sleep and wake periods breathing air or 7% CO2. Sighs, sniffing and recording artifacts were edited from analysis. These data were exported to Sigmaplot 4.0 (Jandel Scientific software) with f, VT and 
per 100 g of body weight calculated for each breath using plethysmograph and body temperatures for that time period. We obtained two to four measurement periods for NREM sleep and wakefulness as a baseline prior to 7% CO2 exposure and one or two periods during the 30min of CO2 exposure. In our four experimental groups, we compared 
, VT and f breathing air or 7% CO2 in NREM sleep or wakefulness on days 3, 6–8, and 13–15 to the baseline data for that treatment in each rat. We defined the ‘CO2 response’ as the 
breathing 7% CO2 minus that in air breathing. The change in this CO2 response was then expressed as a percentage of the baseline value for each rat in each treatment group on each measurement day. Oxygen consumption 
was calculated by the Fick Principle using the difference in O2 content between inspired and expired gas and the flow rate through the plethysmograph and normalized to ml (g body weight)–1 h–1. We monitored CO2 content of the outflow gas continuously to ensure that no build-up of CO2 took place within the chamber.
Anatomy
We performed anatomical analysis on the brains from the rats in these four groups but technical problems resulted in unreliable staining for TPOH-ir. We then repeated the injection portion of the protocol in 21 additional rats; five controls with no injection, four with IgG-SAP injections, four with SP-SAP, four with anti-SERT-SAP, and four with SP-SAP and anti-SERT-SAP. After 14 days, the rats were anaesthetized with ketamine and pentobarbital, then transcardially perfused with 200ml saline followed by 300–500ml of chilled 4% paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4). The brain was removed and postfixed overnight in 4% paraformaldehyde at 4°C, then cryoprotected for 48 h in 30% sucrose. The brains were sectioned at 30µm thickness on a Reichert-Jung cryostat. All immunohistochemical procedures were performed by using free-floating sections at room temperature. We used 0.1M phosphate buffer (PB) for NK1R-ir and 0.1M Tris buffer for TPOH-ir quenched by 3% H2O2 and then blocked with 5% normal goat serum (NGS). The sections were incubated with either a rabbit polyclonal antibody against the NK1R (1 : 10 000, Advanced Targeting Systems, San Diego, CA, USA) or a mouse monoclonal antibody against TPOH (1 : 10 000, Sigma) for 48h at 4°C followed by a biotinylated goat antirabbit or antimouse IgG overnight at 4°C (1 : 500, Vector Laboratories, Burlingame, CA, USA). An avidin–biotin–horseradish peroxidase procedure with diaminobenzidine was used to visualize NK1R and TPOH staining. These sections were used for all cell counts. Double staining was performed in three additional rats. The primary antibodies are the same as above, except with lower dilution (1: 2000). The following secondary antibodies diluted at 1 : 200 were used: goat antimouse IgG conjugated to Cy2 and goat antirabbit IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The two-step sequential methods was used, with first NK1 then TPOH. All the sections were mounted and dehydrated with graded alcohol (25–100% EtOH), cleared with xylene and cover-slipped.
To estimate the total number of TPOH-ir and NK1R-ir neurones in the medullary area of interest we averaged the counts of somatic profiles obtained from one to four 30µm sections within every 360µm segment for 11 such segments. Each rat contributed one value for cell (somatic profiles) counts for each of the 11 360-µm segments. These extended from the most rostral aspect of the medullary raphé caudally for some 3960µm (Fig. 5). In this manner we estimated the total number of TPOH-ir and NK1R-ir neurones in the medulla for each rat.
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The specificity of the anti-SERT antibody was determined using immunohistochemistry of tissue sections from the rat medulla. Rat brains were perfused using the methods described above. Transverse slices (40µm) were prepared on a vibratome, and then incubated as free floating sections in anti-SERT antibody (1 : 500 in 0.1M phosphate buffer) for 24–48 h at 4°C. They were then visualized on a confocal microscope (Zeiss LSM 410) using an antimouse IgG conjugated to fluorescein.
The anti-SERT-SAP dose that was used for in vivo experiments was based on results from cell culture experiments in which it was found that the anti-SERT-SAP conjugate selectively killed serotonergic neurones. Medullary raphé neurones were cultured using methods previously described (Wang et al. 1998). Briefly, the medullary raphé was microdissected from postnatal day zero to postnatal day 1 (P0–P1) rats. Neurones and glia were dissociated and plated onto glass coverslips and allowed to grow in vitro for at least 17days. Culture wells containing 1ml of culture medium were fed with 10µl of glial-conditioned culture medium containing anti-SERT-SAP to achieve a final concentration of between 2.7nM and 55nM (1 : 200–1 : 4000 dilutions of a 2.4 mg ml–1 stock solution). Control coverslips from sister culture dishes containing a similar density of neurones were fed with an equal volume of glial-conditioned culture medium without anti-SERT-SAP. After 1, 4, 7 or 10 days, cultures were fixed with 4% formalin for 1h. Plates were then processed for immunohistochemistry using the mouse monoclonal antibody against TPOH (1 : 2000) for 24–48h at 4°C. Serotonergic neurones were visualized with the Vectastain Elite ABC kit (Vector Laboratories) using the peroxidase method with diaminobenzidine as the chromogen. After staining, the total number of serotonergic neurones was counted on each plate by an individual unaware of the specific treatment. The effect on non-serotonergic neurones was determined by counting the number of such neurones per high power field (HPF; x 40 objective) within the portion of each plate containing the highest density of neurones (mean values for 4 HPFs per plate.)
Statistics
Counts of somatic cell profiles of TPOH-ir and NK1R-expressing neurones were compared by two-way ANOVA with 'treatment group' and 'distance along the medulla' as factors. Tukey's post hoc test was used for post hoc comparisons.
In analysis of ventilatory data we used three approaches. First, in unanaesthetized rats there is variability in 
, VT, and f during both air and 7% CO2 breathing. Our study design included a control group, which received two injections of IgG-SAP at the same volume and at the same site as in the treatment groups, with measurements made before injection, as in the treatment groups, and at 3, 7 and 14 days following the injections. We performed a one-way repeated-measures ANOVA on these data using Tukey or Dunnett's test for post hoc comparisons. If there is no effect of IgG-SAP we presume that effects observed with the one-way repeated-measures ANOVA following any treatment are the result of that treatment. This approach minimizes effects of interanimal variability and is a sensitive way to detect small treatment effects and show at which times they might occur. Second, we perform a two-way ANOVA on absolute ventilatory values with Treatment and Time as factors using a Tukey test for posthoc comparisons. This analysis directly compares absolute data values among the four groups and allows us to ask if the effects observed in any treatment group, or with time, reflect differences from those observed in the IgG-SAP control group. Third, we define a variable for ‘the CO2 response’ as the 
comparing 7% CO2 to air and express for each rat the percentage change in 
at each post injection time. For all four groups we apply to these data a two-way ANOVA with Treatment and Time as factors with Tukey test for posthoc analysis. This approach normalizes each rat's CO2 response and allows a comparison among all four treatment groups at all three post injection times while minimizing interanimal variability.
For analysis of effects on vigilance state, we applied a one-way ANOVA comparing the effects of treatment on percentage time awake, in NREM sleep or in REM sleep and a two-way ANOVA with Treatment and State as factors. For analysis of the effects of vigilance state on ventilation we performed a two-way ANOVA with State and Time as factors using the Tukey test for post hoc comparisons.
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Results |
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The initial and final mean (S.E.M.) body weights were: for the IgG-SAP group 320 ± 12 and 359 ± 11 g; for the SP-SAP group, 302 ± 8 and 332 ± 12 g; for the anti-SERT-SAP group, 282 ± 24 and 341 ± 12; and for the combined SP-SAP and anti-SERT-SAP group, 340 ± 7 and 376 ± 12. The increases in body weight were not significantly different among the groups. We normalized our ventilatory and oxygen consumption data using body weight values. Over the small increase in body weight observed in our 15 day protocol there was no detectable difference in results if ventilation was normalized by body weight or oxygen consumption with one exception in the IgG-SAP group as discussed below. Neither body temperature nor 
expressed as ml O2100 g–1 h–1 differed significantly among the four groups during this 15-day period.
In vitro experiments
The anti-SERT antibody specifically stained a subset of neurones (Fig. 1A) in regions of the medulla that contain serotonergic neurones. Outside of these regions, there was punctate staining consistent with nerve terminals, but no staining of somata (Fig. 1B). There was also staining of ependymal cells lining the fourth ventricle (Fig. 1B).
Exposure of cultures to anti-SERT-SAP led to killing of serotonergic neurones without an effect on other neurones on the same plates. The destruction of serotonergic neurones occurred in a dose-dependent manner with concentrations greater than 11nM (= 1 : 1000 dilution of 2.4mg ml–1 stock solution) leading to loss of 90–95% of TPOH-ir neurones within 4–7 days after exposure (Fig. 1C). Control coverslips (n= 24) contained a mean (±S.E.M.) of 50 ± 9 neurones. After 7–10 days of exposure to anti-SERT-SAP, the number of TPOH-ir neurones on treated plates was 0.5 ± 0.5 for 22–55nM (n= 4), 4 ± 1 for 11nM (n= 4), and 17 ± 5 for 2.7–5.5nM (n= 4). Cell killing by anti-SERT-SAP was also delayed as expected (Mantyh et al. 1997) with a significant effect present at 4 days after treatment, and maximum killing reached by 7–10 days (Fig. 1D). There was sparing of non-serotonergic neurones on these same plates; with 6.4 ± 3.9 neurones per high power filed (HPF) in control plates (n= 8), 7.4 ± 3.9 neurones per HPF in plates treated with 11nM anti-SERT-SAP (n= 4), and 9.0 ± 6.6 neurones per HPF in plates treated with 22–55nM anti-SERT-SAP (n= 4). The highest concentration of anti-SERT-SAP caused some alteration in the morphology of a subset of non-serotonergic neurones, and caused some glia to detach from the surface of the plates. For in vivo injections we used a 1µM concentration in a volume of 100 nl to obtain a wide killing field.
Anatomy
Medullary serotonergic neurones extend from a level about 800–900µm rostral to the rostral pole of the facial nucleus caudally for about 4–5mm ending at the beginning of the spinal cord. We include all TPOH-ir neurones seen in each cross section (Fig. 2A–D). In this paper we refer to the medullary serotonergic neurones as a single cell population and do not use the anatomical designations of raphé pallidus, obscurus, and magnus. We include extra-raphé TPOH-ir neurones, ones that are outside of classic midline raphé nuclei, e.g. in the nucleus paragigantocellularis lateralis or parapyramidal region.
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Given the intermingling of TPOH-ir and NK1R-ir neurones and the hypothesis that both might be involved in central chemoreception, we performed double labelling of the same neurones using goat antirabbit IgG conjugated to Cy3 as the secondary antibody for NK1R and goat antimouse IgG conjugated to Cy2 as the secondary antibody for TPOH. It was important to stain first for NK1R-ir then for TPOH-ir. The reverse order resulted in a non-specific cross-reaction. In three rats we counted 1188, 1211 and 1036 TPOH-ir neurones and 110, 107 and 148 NK1R-ir neurones, respectively, finding a total of three with double labelling. Figure 3(A–C) shows at a level 200µm caudal to the caudal pole of the facial nucleus an example of a single section showing TPOH-ir cells (A) NK1R-ir (B) cells, and both together (C). There was no double labelling. We conclude that TPOH-ir neurones in the medullary serotonergic group do not express NK1Rs, that they are separate cell populations.
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The mean ±S.E.M. of the total cell counts of NK1R-ir (top) and TPOH-ir (bottom) neurones for each of the five groups are shown on Fig. 4. The stars refer to significant differences determined by two-way ANOVA analysis of cell counts along the rostral to caudal length of the area of interest using Treatment and Distance as factors. Figure 5 displays these cell counts for each 360µm segment along the rostral to caudal length of the medulla. The top panel shows the NK1R-ir results with the pooled control data (No Injection controls, N= 5, plus IgG-SAP controls, N= 4) and the SP-SAP data (N= 4); the bottom panel shows the TPOH-ir results for the pooled controls and the anti-SERT-SAP group (N= 4). The figure also shows the rostral to caudal location of the facial nucleus as a convenient landmark and the locations of the injections in each of the 4 SP-SAP (top) and anti-SERT-SAP (bottom) injected rats. For NK1R-ir neurone counts, two-way ANOVA using Treatment and Distance as factors showed a significant effect for each (P < 0.001) but no significant interaction. Tukey post hoc analysis showed that there was no difference between the No Injection and IgG-SAP controls thus allowing them to be pooled. The number of NK1R-ir cells was significantly less in the SP-SAP and combined anti-SERT-SAP plus SP-SAP groups compared to either the No Injection or the IgG-SAP control groups (P < 0.05, Tukey multiple comparisons). For the TPOH-ir neurone counts, two-way ANOVA using Treatment and Distance as factors showed a significant effect for each (P < 0.001) but no significant interaction. Tukey post hoc analysis showed that there was no difference between the No Injection and IgG-SAP controls thus allowing them to be pooled. The number of TPOH-ir cells was significantly less in the anti-SERT-SAP and combined anti-SERT-SAP plus SP-SAP groups compared to either the No Injection or the IgG-SAP control groups (P < 0.05, Tukey multiple comparisons).
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Physiology
We measured the amount of time spent in wakefulness, NREM and REM sleep during a 3 h continuous air breathing period from 9 AM to noon (the last three h of the light, resting period in our midnight to noon imposed diurnal cycle) during the final few days post injection. The IgG-SAP group (N= 5) spent 38 ± 3 (mean ±S.E.M.)% of this time awake, 54 ± 2% in NREM sleep, and 7 ± 1% in REM sleep. The anti-SERT-SAP group (N= 6) spent 46 ± 4% awake, 46 ± 4% in NREM sleep, and 7 ± 1% in REM sleep. The SP-SAP group (N= 5) spent 42 ± 3% awake, 54 ± 4% in NREM sleep, and 3 ± 1% in REM. The combined SP-SAP and anti-SERT-SAP group (N= 5) spent 48 ± 5% awake, 49 ± 4% in NREM sleep, and 3 ± 1% in REM sleep. There is a tendency for all three treatment groups to be awake more but this is not statistically significant (one-way ANOVA comparing the effects of treatment on percentage time awake, in NREM sleep or in REM sleep; two-way ANOVA with Treatment and State as factors).
IgG-SAP injection effects on ventilation
In the control group, which received two injections of IgG-SAP, there was no substantial effect on breathing or on the CO2 response in wakefulness or sleep (Fig. 6). 
in wakefulness was significantly decreased (P < 0.01, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test at 14 days versus baseline) but the magnitude of this effect was small. Examination of 
showed that it too was decreased at this 14 day time point such that the 
/
ratio was unchanged. Breathing in air and in 7% CO2 was less in NREM sleep than in wakefulness (P < 0.02, two-way ANOVA with state and time as factors, P < 0.05 Tukey post hoc for state).
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The SP-SAP injections had no effect on air breathing in wakefulness or sleep (Fig. 7). During 7% CO2,
was significantly decreased in wakefulness (P<0.03, one-way repeated-measures ANOVA; P<0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline). The effect was entirely due to a decrease in VT (P<0.02, one-way repeated-measures ANOVA; P<0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline). During 7% CO2 in NREM sleep there was an overall significant decrease in 
(P= 0.05, one-way repeated-measures ANOVA). Posthoc analysis using a Bonferroni modification showed significance at 14 days compared to baseline (P<0.05). Breathing in air and in 7% CO2 was less in NREM sleep than in wakefulness (P<0.05, two-way ANOVA with state and time as factors, P<0.05 Tukey post hoc test for state).
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The anti-SERT-SAP injections had no effect on air breathing in wakefulness or sleep (Fig. 8). During 7% CO2, 
was significantly decreased in wakefulness (P < 0.02, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline) and sleep (P < 0.001, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline). In wakefulness, the effect was due to a decrease in VT and f (both P < 0.01, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 7 and 14 day values versus baseline). In NREM sleep the effect was due to a decrease in VT (P < 0.01, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 7 and 14 day values versus baseline). Breathing in air and in 7% CO2 was less in NREM sleep than in wakefulness (P < 0.01, two-way ANOVA with state and time as factors, P < 0.05 Tukey's post hoc test for state).
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The combined SP-SAP and anti-SERT-SAP injections had no effect on air breathing in wakefulness (Fig. 9). In sleep, 
was significantly decreased (P<0.001, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test for day 7 and 14 values versus baseline). During 7% CO2, 
was significantly decreased in wakefulness and sleep (both P < 0.001, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline). In wakefulness, the effect was due to a decrease in VT (P < 0.05, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 3, 7 and 14 day values versus baseline). In NREM sleep the effect was due to a decrease in VT (P < 0.005, one-way repeated-measures ANOVA; P < 0.05, Dunnett's post hoc test of 7 and 14 day values versus baseline). Breathing in air and in 7% CO2 was less in NREM sleep than in wakefulness (P < 0.002, two-way ANOVA with state and time as factors, P < 0.05 Tukey's post hoc test for state).
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We also applied a two-way ANOVA to examine among the four groups the effects of treatment and time on the absolute 
values (a) while breathing room air and (b) while breathing 7% CO2. This approach does not minimize interanimal variability but allows a comparison of responses among the different treatment groups to those in the IgG-SAP control group. During air breathing in wakefulness there is a significant effect of treatment (P<0.05) and of time (P<0.05) but no post hoc significance. During air breathing in NREM sleep there was no significant treatment or time effect. During 7% CO2 breathing awake there were significant treatment (P < 0.001) and time effects (P < 0.001) but no significant interaction. Tukey's posthoc test showed the SP-SAP, anti-SERT-SAP, and the combined SP-SAP and anti-SERT-SAP treatment groups being different from the IgG-SAP control (P<0.05) and times 3, 7, and 14 days as different from the preinjection control day (P < 0.05). This analysis supports that of the one-way repeated-measures ANOVA. During 7% CO2 breathing in NREM sleep there was no significant treatment effect but there was a time effect (P<0.001). Tukey's post hoc test showed times 7 and 14 days as different from the preinjection control day (P < 0.05). There was not a significant interaction. Here the between-group analysis does not support the results of the one-way repeated-measures ANOVA. By inspection, the reason for this is that the IgG-SAP control group had, before any treatment, a larger NREM sleep effect on the 
while breathing 7% CO2 than did any of the other three groups before treatment.
Normalized effects on the CO2 response in wakefulness and sleep
To normalize the treatment effects on the CO2 response and minimize any initial interanimal variability and to allow an intergroup comparison we first defined this response as 
breathing 7% CO2versus breathing air for each measurement day for each rat. We then compared 
at days 3, 7 and 14 after treatment to that of the pretreatment baseline period, expressing it as the percentage change in the CO2 response for each rat in each of the four groups. The mean results (+S.E.M.) for days 7 and 14 after injection are shown in Fig. 10. In wakefulness and in sleep there was a significant decrease in the CO2 response in the SP-SAP, anti-SERT-SAP, and combined SP-SAP and anti-SERT-SAP groups compared to the IgG-SAP control group (P < 0.001, one-way ANOVA; P < 0.05, Tukey post hocversus IgG-SAP control). There was no significant effect of time after lesion nor was there a significant interaction. There was no significant difference between SP-SAP and anti-SERT-SAP or between anti-SERT-SAP and combined SP-SAP and anti-SERT-SAP.
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We found that medullary serotonergic and adjacent NK1R-expressing neurones are separate cell populations. Specific killing of medullary serotonergic neurones, or of adjacent NK1R-expressing neurones, reduced the ventilatory response to systemic CO2 in wakefulness and in NREM sleep. Specific killing of both cell types simultaneously did not result in a greater effect.
Analysis
We used three analytic approaches for the ventilatory data; (1) a one-way repeated-measures ANOVA applied to absolute ventilatory values for each group separately, (2) a two-way ANOVA comparing the overall response in terms of absolute ventilatory values among groups, and (3) a two-way ANOVA using values of 
, the ‘CO2 response’, to compare normalized percentage changes among all groups. In wakefulness, all three indicated that these treatments did not affect air breathing but did decrease the CO2 response. In NREM sleep, there was a small but significant decrease in 
during air breathing at 7 and 14 days after combined SP-SAP and anti-SERT-SAP treatment, uncovered only by the repeated-measures one-way ANOVA. Further experiments will have to examine whether or not this is a real treatment effect. With respect to the CO2 response in sleep, both the one-way repeated-measures ANOVA and the two-way group comparison of normalized 
indicated that all three treatments decreased the CO2 response. The two-way ANOVA of absolute 
values at 7% CO2 did not show a treatment effect. By inspection, the reason for this is that the IgG-SAP control group had, before any treatment, a larger NREM sleep effect on the 
while breathing 7% CO2 than did any of the other three groups before treatment. We conclude, based on the two approaches that normalize initial value variability, that the three treatments decreased the CO2 response in NREM sleep.
Anatomy of medullary serotonergic and associated NK1R-expressing neurones
The serotonergic neurones in the rat medulla are distributed in long columns beginning rostral to the facial nucleus and extending caudally to the very end of the medulla. They are located in the raphé nuclei, i.e. raphé magnus, obscurus and pallidus, and in extra-raphé sites like the parapyramidal region and the nucleus paragigantocellularis lateralis. We evaluate our lesions within the entire medullary serotonergic system.
NK1R-expressing neurones are intermingled among these serotonergic neurones and, in addition, are distributed widely within the brainstem (Nakaya et al. 1994). They are large with prominent and extensive processes (Figs 2E and F and 3B). In some locations, e.g. the RTN–Ppy region, there is an extensive reticular-like network of NK1R-ir processes (Nattie & Li, 2002b); in others, e.g. the pre-Bötzinger region, there is a cluster of NK1R-ir soma (Gray et al. 2001; W. Wang et al. 2001). Here we focus on NK1R-expressing neuronal soma present within the medullary serotonergic neurone region of interest.
In this region of interest, the TPOH-ir neurones do not express NK1 receptors. We found essentially no double labelling of NK1R-ir and TPOH-ir neurones in an extensive examination of the entire medullary serotonergic cell group, which verifies an earlier report by Léger et al. (2002). While NK1 receptors are not present on serotonergic neurones, they are present on many other brainstem neurones. These include: noradrenergic neurones of the locus coeruleus (Chen et al. 2000), GABA-ergic neurones in periaqueductal grey, dorsal raphé (Ma & Bleasdale, 2002), and ventrolateral medulla (W. Wang et al. 2001), glutamatergic neurones in the pre-Bötzinger complex (Guyenet et al. 2002) and dorsal raphe (Liu et al. 2002), and undefined neurones in the NTS (Ljungdahl et al. 1978). A small number of C1 cells also express NK1-receptors (Makeham et al. 2001). NK1R-ir is prominent in central chemoreceptor regions, which include the locus ceruleus, RTN, parapyramidal region, medullary raphé, caudal NTS, caudal ventral medulla, pre-Bötzinger complex, and rostral nucleus ambiguus (Nakaya et al. 1994; Nattie & Li, 2002b).
Cell specific lesions and chemoreception
SP-SAP has been previously used to specifically kill NK1R-expressing neurones or processes that mark a functionally important anatomical site. For example, in the pre-Bötzinger complex, which contains a dense NK1R-ir cell group (Gray et al. 1999, 2001; Wang et al. 2002), selective and substantial bilateral lesions (loss of > 75% NK1R-ir neurones) produced a dramatic respiratory phenotype with irregular breathing, hypoventilation, and a reduced response to increased CO2.
In this study, the SP-SAP injections focally killed 31% of the NK1R-expressing neurones in the region of the medullary serotonergic neurone distribution. Two lines of evidence support the conclusion that the lesions are focal. First, caudal to the preponderance of injection sites (Fig. 5) the numbers of NK1R-ir neurones were similar in control and treated rats. Second, SP-SAP injections did not decrease the numbers of NK1R-ir neurones in a specifically defined region of the rostral ventrolateral medulla (W. Wang et al. 2001). The 31% loss of NK1R-ir neurones reduced the ventilatory response to systemic CO2, averaged over the postinjection period, by 21% in wakefulness and 16% in NREM sleep. In prior experiments, slightly greater destruction of NK1R-ir neurones and processes in the RTN/Ppy region (Nattie & Li, 2002b) reduced the CO2 response in wakefulness and sleep by 17–22% after bilateral lesions and by 28–30% after unilateral lesions. We conclude that approximately similar amounts of destruction of NK1R immunoreactivity in the RTN/Ppy region or in the medullary serotonergic system produce similar effects on chemoreception. NK1R-expressing neurones at both sites are involved in central chemoreception. Bilateral destruction in the RTN/Ppy region also resulted in hypoventilation during air breathing indicating the presence of a tonic drive for breathing from NK1R-expressing neurones in this region. While we did not observe hypoventilation following SP-SAP injection in the medullary serotonergic system, it is possible that greater loss of NK1R-expressing neurones would uncover a tonic drive emanating from the region of the medullary serotonergic system as well.
We used a novel approach to selectively kill serotonergic neurones. Lappi and colleagues conjugated an antibody to the external portion of the serotonin transport protein with the cell toxin, saporin, in order to produce selective destruction of serotonergic neurones. We tested this conjugate using serotonergic cells in culture and found specific killing of TPOH-ir neurones within days by exposure to an 11nM concentration with more efficacious killing at 2 x and 5 x greater doses. From these data we chose a dose of 1µM for in vivo injection. This proved useful in that two 100 nl injections of this concentration killed 28% of medullary serotonergic neurones. That our injections had no effect on TPOH-ir neurones located in the caudal aspect of the dorsal raphe argues against any distant effects of our focal injections. But the presence of SERT protein on axons and terminals indicates that some of the medullary neurones may have been killed by anti-SERT-SAP access via these sites.
The 28% loss of medullary serotonergic neurones by our anti-SERT-SAP injections significantly reduced the ventilatory response to systemic CO2, averaged over the postinjection period, by 15% in wakefulness and 18% in NREM sleep. There was no effect on air breathing. Other studies have examined the role of the medullary raphé in chemoreception. Extensive non-specific cell killing in the ventral midline medulla of decerebrate piglets substantially decreased the responses to CO2 of both phrenic and hypoglossal nerve activities (Dreshaj et al. 1998). Focal non-specific neuronal inhibition in the rostral aspect of the medullary serotonergic distribution of unanaesthetized newborn piglets by dialysis of muscimol, a GABA-A receptor agonist, decreased the breathing response to CO2 by 17% in wakefulness (this treatment also disrupted sleep cycling) (Messier et al. 2002). Both studies suggest that neurones, of unknown phenotype, in the region of the medullary serotonergic system are involved in central chemoreception.
More specific inhibition of medullary serotonergic neurones was produced by focal microdialysis of the serotonin 1A receptor agonist, 8-OH DPAT, in unanaesthetized newborn piglets. This treatment, designed to inhibit serotonergic neurones that express the serotonin 1 A auto-receptor, decreased the CO2 response in wakefulness (sleep cycling was disrupted) in an age-dependent manner. Linear regression analysis showed a significant negative correlation (P<0.001) between the percent change in the CO2 response with piglet age over a 3–16 days of age range (Messier et al. 2004). In a previous experiment, Mueller et al. (1984) injected the serotonin chemical toxin 5,7 DHT in 3-day-old rats and measured chemoreception in adult rats under anaesthesia. An approximately 90% reduction in tissue serotonin levels was associated with a reduction in the response to CO2 by approximately 50%. Our anti-SERT-SAP data provide specific evidence for involvement of serotonergic neurones in the medullary serotonergic system of the adult rat in chemoreception in both sleep and wakefulness. The data from these 8 OH-DPAT and 5, 7 DHT experiments provide further support for a role for medullary serotonergic neurones in chemoreception. Based on all of these data we suggest that the serotonergic neurones may account for from 20 to 50% of the total ventilatory response to CO2.
Other ventral chemoreceptor sites can contribute importantly to the ventilatory response to CO2. These include the RTN/Ppy region, caudal ventrolateral medulla, caudal NTS, LC, and rostral VRG and they do not involve serotonergic neurones (Forster et al. 1997; Nattie, 1998, 1999, 2000, 2001; Okada et al. 2002; Feldman et al. 2003; Ribas-Salgueiro et al. 2003). The relative and specific roles of these different sites in normal chemoreceptor physiology remain to be fully defined. Responses to moderate intensity focal stimulation by CO2 dialysis at RTN/Ppy, medullary raphé, and caudal NTS varied in sleep and wakefulness suggesting a state dependence in the function of some central chemoreceptor locations (Nattie, 1998, 1999, 2000, 2001; Feldman et al. 2003). In addition, the peripheral chemoreceptors at the carotid bodies are intact in all of these experiments and contribute significantly to the ventilatory responses to systemic hypercapnia. It is possible that some of the reduction in the CO2 response observed after these lesions could be attributed to loss of modulation of this peripheral reflex by serotonergic or NK1R-expressing neurones.
How might serotonergic and NK1R-expressing neurones interact in chemoreception?
In the experiment with combined injections of both anti-SERT-SAP and SP-SAP we expected to observe an additive effect, that the CO2 response would be reduced by the sum of the two individual lesions. The amount of cell killing in the combined toxin experiments was comparable to that in the individual toxin experiments. Yet the ventilatory response to systemic CO2 was reduced by similar amounts. We conclude that both types of neurones are required for a normal response.
While there are no in vitro data to support a role of NK1R-expressing neurones in chemoreception per sé there are ample such data for serotonergic neurones. Richerson and colleagues have shown that serotonergic neurones in brain slices and in tissue culture are highly sensitive to focally applied CO2 at physiological levels (Richerson et al. 2001; W. Wang et al. 2001) and that 73% of medullary TPOH-ir neurones are chemosensitive in culture (W. Wang et al. 2001). Many serotonergic neurones are located in close association with medullary arteries, which would aid in faithfully sensing arterial CO2 (Bradley et al. 2002).
Based on the observation that serotonergic neurones are themselves chemosensitive, we put forth two explanations for our observation that simultaneous killing of both NK1R-expressing and TPOH-ir neurones has no greater effect on the CO2 response than does killing of either population alone.
The medullary raphé, sleep and body temperature regulation
Disruption of the medullary raphé or rostral ventrolateral medulla seems to promote wakefulness. Studies with SP-SAP induced lesions of NK1R-ir neurones and processes in the RTN–Ppy region of adult rat (Nattie & Li, 2002b), muscimol inhibition of RTN (Curran et al. 2001; Darnall et al. 2001) and medullary raphe neurones (Messier et al. 2002) of newborn piglet, and 8-OH-DPAT inhibition of medullary serotonergic neurones of newborn piglets (Messier, A. Li & E. E. Nattie, unpublished observations) have all shown a significant tendency for sleep cycle disruption with less NREM sleep and more wakefulness. However, in this experiment, we did not observe a statistically significant effect on the amount of time spent in NREM or REM sleep or wakefulness among the four treatment groups, although there was a tendency for sleep disruption. It may be that lesions that develop over days as compared to acute inhibition allow a return to a more normal sleep cycle.
Medullary serotonergic neurones have been implicated in body temperature regulation via effects on brown fat metabolism and cutaneous blood flow (Cano et al. 2003). We observed no significant difference in body temperature or resting oxygen consumption in our lesioned animals. This is surprising in that room temperature is in the 22–24°C range, which is below the thermoneutral zone for the rat and we might have expected to see some body temperature effect. Perhaps our lesions did not kill a sufficient number of temperature-related neurones.
Summary and significance
NK1R-expressing neurones are involved in sensory processing (Mantyh et al. 1997) and in the regulation of blood pressure and breathing (Li & Guyenet, 1997; Chen et al. 1988, 1990; Urbanski et al. 1989; Seagard et al. 2000; Riley et al. 2002; Gray et al. 2001). As shown in this and in our prior study (Nattie & Li, 2002b), NK1R-expressing neurones are also important in central chemoreception.
In contrast, the activity of medullary serotonergic neurones, studied in vivo by Jacobs and colleagues (Jacobs et al. 2002), seems most reliably correlated with motor acts, especially repetitive motor acts, and with arousal state, their firing being highest in wakefulness. Azmitia (1999) suggested that serotonergic neurones are important in brain tissue homeostasis while Lovick (1997) and Jacobs et al. (2002) have suggested that serotonergic neurones help to coordinate autonomic activity with motor output. Serotonergic neurones are known to be involved with pain processing (Mason, 2001), the regulation of breathing (Lalley, 1986; Holtman et al. 1987), upper airway tone (Haxhui et al. 2001; Horner, 2001), body temperature regulation (Cano et al. 2003), and possible glucose sensing (Maekawa et al. 2000).
Richerson et al. (2001) has proposed from in vitro studies of slices and single neurones in culture that medullary serotonergic neurones are primary CO2 chemosensors. The results presented here support the hypothesis that medullary serotonergic neurones are involved in the ventilatory response to an increase in CO2. The overall importance of the serotonergic chemoreceptors in vivo is unclear, given the presence of other central and peripheral chemoreceptor sites, but the data suggest that they contribute substantially. Our results also point to an adjacent non-serotonergic population of NK1R-expressing neurones that are important for full expression of the chemoreception response in vivo. We suggest that these two different cell types interact in some as yet undetermined manner to affect chemoreception in vivo.
These serotonergic neurones may represent the neurobiological substrate for a subset of cases of the Sudden Infant Death Syndrome (SIDS) that express abnormalities in serotonergic receptor binding (Panigrahy et al. 2000; Kinney et al. 2001). We do not know how developmental defects in these neurones might lead to SIDS. One hypothesis is that these neurones participate in a number of homeostatic processes, including chemoreception, that when disrupted could in the appropriate circumstance contribute to sudden death.
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Azmitia EC (1999). Serotonin neurons, neuroplasticity, and homeostasis of neural tissue. Neuropsychopharm 21, 33s–45s.[CrossRef]
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; N= 4). The line within the axes at bottom shows the rostral to caudal location of the facial nucleus. The lines at the bottom outside the axes show the rostral-to-caudal locations of sections with noticeable tissue disruption from probe placement in the SP-SAP group, i.e. the injection sites. Two-way ANOVA showed a significant effect of treatment (P < 0.001) and of distance (rostral–caudal location) (P < 0.001) but no significant interaction. Tukey's post hoc test showed that the 'no injection' and IgG-SAP control data did not differ, and hence we pooled them. SP-SAP and the 'BOTH' (not shown) groups differed significant from either the 'no injection' or the IgG-SAP control groups (P < 0.05) but not from each other. Bottom, mean ±S.E.M. TPOH-ir cell counts for each 360µm segment for Control (•; N= 9; 5 'no injection' and 4 IgG-SAP) and for anti-SERT-SAP (
