Ventilatory effects of muscimol microdialysis into the rostral medullary raphé region of conscious rats

Respiratory Physiology & Neurobiology 153 (2006) 203–216

Ventilatory effects of muscimol microdialysis into the rostral medullary raph´e region of conscious rats Natalie C. Taylor, Aihua Li, Eugene E. Nattie ∗ Department of Physiology, Dartmouth-Hitchcock Medical Center, Borwell Bldg., Lebanon, NH 03756-0001, USA Accepted 8 November 2005

Abstract We hypothesized that inhibition of the rostral medullary raphe region (MRR), a putative central chemoreceptor location, with the GABAA receptor agonist muscimol would decrease ventilatory responses to hypercapnia and hypoxia in conscious rats, and that its known effect at this site on body temperature might alter its effect upon these ventilatory responses. At ambient temperatures of 24.5–26.5 ◦ C (Cool), microdialysis of 1 mM muscimol into the MRR significantly decreased body temperature by approximately 0.5 ◦ C, increased the ventilatory response to 7% CO2 and decreased the response to 10% O2 . At ambient temperatures of 29.5–30.5 ◦ C (Warm), 1 mM muscimol microdialysis no longer decreased body temperature and increased the ventilatory response to hypercapnia and to hypoxia. Muscimol did not significantly affect the V˙ E /V˙ O2 ratio at either temperature. Muscimol significantly increased the hypercapnic ventilatory responses in Cool and Warm conditions and the hypoxic response in Warm conditions, which indicates the presence of an inhibitory effect of rostral MRR neurons sensitive to muscimol. In the Cool condition the ventilatory response to hypoxia is inhibited but appropriately so for the lower V˙ O2 . © 2005 Elsevier B.V. All rights reserved. Keywords: GABAA ; Central chemoreceptors; Thermoregulation; Caudal raphe

1. Introduction Neurons in the medullary raphe region (which we define as the raphe magnus, obscurus, and pallidus; as well as the lateral paragiganticellular nucleus, the parapyramidal region, and the ventral surface of the medulla, abbreviated MRR) project to several brain∗ Corresponding author. Tel.: +1 603 650 7726; fax: +1 603 650 6130. E-mail address: [email protected] (E.E. Nattie).

1569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2005.11.005

stem and spinal cord nuclei involved in respiratory function, including the dorsal and ventral respiratory groups, and the hypoglossal and phrenic motor nuclei (Holtman et al., 1984; Connelly et al., 1989; Smith et al., 1989; Manaker et al., 1992). The MRR is considered to be a site of central CO2 chemosensitivity, as reverse microdialysis there of artificial cerebrospinal fluid (aCSF) containing elevated PCO2 increases ventilation in conscious rats and goats (Nattie and Li, 2001; Hodges et al., 2004). Electrophysiological studies conducted in primary tissue culture have shown that there are two populations of MRR neurons in rats

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that specifically respond to PCO2 and/or decreased pH: one which increases firing rate with increased PCO2 /acidosis, and one which decreases firing rate in response to PCO2 /acidosis (Wang et al., 1998). The majority of MRR neurons that increase their firing rate with increased PCO2 in vitro were consequently found to be serotonergic (Wang et al., 2001), and selective lesioning or acute inhibition of MRR serotonergic neurons was shown to decrease the ventilatory response to hypercapnia in conscious rats and piglets (Messier et al., 2004; Nattie et al., 2004; Taylor et al., 2005). Muscimol dialysis into the MRR of newborn piglets also decreases the CO2 response (Messier et al., 2002) as it does in the rat at another near-by central chemoreceptor location, the retrotrapezoid nucleus (RTN) (Nattie and Li, 2000) where the chemosensitive neurons have been proposed to be glutamatergic (Mulkey et al., 2004; Guyenet et al., 2005) not serotonergic as in the MRR (Wang et al., 2001; Richerson, 2004). There is some evidence that neurons in the MRR are also involved in modulating the acute ventilatory response to hypoxia. Following acute hypoxic exposure or electrical stimulation of the carotid sinus nerve in rats, there is increased c-fos expression (indicative of neuronal activation) observed in the MRR, and 30% of fos-positive neurons counterstain for serotonin (Erickson and Millhorn, 1994). Also, electrical or chemical stimulation of the MRR in anesthetized rats abolishes the increased neuronal activity of the nucleus of the solitary tract (NTS) observed after acute peripheral O2 chemoreceptor activation (Perez and Ruiz, 1995). In conscious rats, lesions of the MRR with ibotenic acid result in an increased ventilatory response to 7% O2 breathing (Gargaglioni et al., 2003), although acute inhibition of serotonergic activity via microdialysis of the 5-HT1A agonist (R)-(+)-8-hydroxy-2(di-npropylamino)tetralin (DPAT) has no effect on the ventilatory response to hypoxia (Taylor et al., 2005). These results suggest that the MRR neurons can modulate the acute ventilatory response to hypoxia in rats. In addition to modulating ventilatory responses, neurons in the MRR mediate sympathetic thermoregulatory processes. In conscious rats, lidocaine or DPAT microinjection into the MRR eliminated the increases in oxygen consumption and electromyographic activity (i.e. shivering) observed following focal cooling of the pre-optic anterior hypothalamus (Berner et al., 1999). Morrison and colleagues have shown that microinjec-

tion of DPAT or the GABAA agonist muscimol into the MRR of anesthetized rats specifically inhibits brown adipose tissue sympathetic activity and thermogenesis (Morrison, 2003, 2004a). Furthermore, MRR neurons mediate cutaneous sympathetic vasomotor responses in the rat tail and rabbit ear in response to thermal stress (Tanaka et al., 2002; Ootsuka et al., 2004). When studying ventilation in small mammals (such as rats), it is important to consider that thermoregulatory activity mediated by the MRR generally increases oxygen consumption and can affect the ventilatory responses to hypercapnia and especially hypoxia (Maskrey, 1990; Mortola and Maskrey, 1998; Mortola, 2001). In the current study, we hypothesized that inhibition of neurons in the MRR by dialysis of the GABAA receptor agonist muscimol would decrease the ventilatory responses to hypercapnia and hypoxia in conscious rats. Because muscimol microinjection into the MRR has also been shown to decrease body temperature in conscious rats studied at room temperature (Zaretsky et al., 2003), the second hypothesis of this study was that the potential effects of muscimol on ventilatory responses would be altered if its effects upon thermoregulation were minimized. Therefore, we observed hypercapnic and hypoxic ventilatory responses in both room temperature conditions (24.5–26.5 ◦ C) (Cool) and at a slightly warmer temperature of 29.5–30.5 ◦ C (Warm), in an attempt to eliminate the decrease in body temperature following MRR muscimol administration.

2. Methods 2.1. Animal instrumentation All animal experimentation and surgical protocols were approved by the Institutional Animal Care and Use Committee at the Dartmouth College Animal Resource Center. Twelve adult male Sprague–Dawley rats weighing 250–350 g were used in these experiments. Rats were individually housed in a light and temperature (70 ◦ F) controlled environment, with 12 h of light beginning at midnight and 12 h of darkness beginning at noon. Food and water were provided ad libitum. Prior to the ventilation experiments, animals were surgically instrumented with a telemetric temperature probe, a microdialysis probe cannula within the MRR, and electroencephalogram (EEG) and electromyogram

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(EMG) electodes. Briefly, animals were anaesthetized with an intramuscular injection of 100 mg/kg ketamine and an intraperitoneal injection of 20 mg/kg xylazine. Depth of anaesthesia was monitored throughout surgery by firm pinching of the hindpaw, and if necessary, additional quarter doses of ketamine and xylazine were administered. Hair on the neck, head and abdomen was thoroughly shaved, and the skin was sterilized with betadine solution and 70% ethanol. A sterilized telemetric temperature probe capsule (TA-F20, Data Sciences International, St. Paul, MN) was placed within the intraperitoneal space via a midline abdominal incision. Then, the animal’s head was centered on a Kopf stereotaxic frame, and a midline incision was made from the frontal bone to the base of the skull exposing the surface. Three EEG electrode screws were secured into the skull surface: one 2 mm rostral to bregma and 2 mm lateral to the midline, one 2 mm rostral to lambda and 2 mm lateral to the midline, and a ground was placed slightly lateral between the two. Two EMG electrodes were sutured within the skeletal muscle of the neck, and electrode wires were put into a six-prong plastic pedestal. A microdialysis probe cannula with dummy insert was stereotaxically inserted into the brainstem 10.3–11.6 mm caudal to bregma and 0 mm from midline, and 10.6–10.8 mm deep into the dorsal surface of the cerebellum. These coordinates assured placement into the MRR at the level of the facial motor nucleus according to the rat brain atlas of Paxinos and Watson (1986). The microdialysis cannula and six-prong pedestal were secured to the skull with cranioplastic cement and the wound was closed. All rats were allowed at least four days to recover, and any rats showing illness or infection were immediately euthanized with an overdose of sodium pentobarbital (intraperitoneal injection, >75 mg/kg) and not used in the experiments. 2.2. Reverse microdialysis drug administration The microdialysis probe used in this study had an 11 mm stainless steel shaft with a 1 mm cuprophane membrane tip (0.38 OD), allowing diffusion of molecules under 6000 Da (CMA 11, CMA Microdialysis, Solna, Sweden). Solutions were dialyzed through the probe at a constant rate of 0.05 mL/h maintained by a syringe pump (Model 100, KD Scientific, Holliston, MA). Artificial cerebrospinal fluid (aCSF) served

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as the drug vehicle and for the control, and was comprised of (in mM) 152.0 sodium, 3.0 potassium, 2.1 magnesium, 2.2 calcium, 131.0 chloride, and 26.0 bicarbonate in sterile deionized water. Directly before the ventilation experiments and prior to adding the calcium, aCSF was equilibrated with 5% CO2 . 1 mM muscimol HBr (Sigma-Aldrich, St. Louis, MO) was prepared in aCSF. This concentration was chosen based upon previous microdialysis studies in conscious rats (Nattie and Li, 2000). 2.3. Data acquisition To measure ventilation in conscious rats, we used the whole body plethysmograph technique as previously described (Nattie and Li, 2001, 2002a,b; Nattie et al., 2004), based upon the original setup of Bartlett and Tenney (1970). The volume of the plethysmograph was 7.6 L with a 3.5 L top to protect the head pedestal. Analog output from the pressure transducer was sampled at 150 Hz and was converted into a digital signal by a computer using the DATAPAC 2000 system (RUN Technologies, Laguna Hills, CA). The rates of inflow and outflow to the plethysmograph were balanced to one another with a flow meter such that flow was maintained at ≥1.4 L/min (flow meter model 601E, Matheson Tri-gas, Montgomeryville, PA), and the plethysmograph remained at atmospheric pressure. CO2 and O2 fractions were sampled from the outflow line at ∼100 mL/min by a combination CO2 and O2 gas analyzer (Gemini Respiratory Gas Analyzer, CWE Inc., Ardmore, PA). To calibrate the plethysmograph before each experiment, we obtained pressure data from five 0.3 mL air injections made with a 1 cm3 syringe. Raw EEG and EMG outputs from the skull and skeletal muscle electrodes were sampled at 150 Hz, filtered at 0.3–50 and 0.1–100 Hz, respectively, and were recorded continuously using the DATAPAC 2000 system. Oxygen consumption (V˙ O2 ) was calculated by application of the Fick principle, using the difference in O2 content between inflow air and outflow air: [V˙ O2 = (V˙ in × FIO2 ) − (V˙ out × FOO2 )] where V˙ in and V˙ out are inflow and outflow rates, and FIO2 and FOO2 are the fraction of inflow and outflow O2 . In the plethysmograph chamber used in these experiments, any gas mixture (i.e. 7% CO2 or 10% O2 ), when added to the inflow (at a flow rate of 1.4 L min−1 ),

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took approximately 10–15 min to reach appropriate concentrations in the outflow. V˙ O2 was thereby calculated from outflow oxygen content data after 10-min intervals during the ventilation experiments, and analyzed as an average value during room air, 7% CO2 , or 10% O2 conditions. Core body temperature was measured every 10 min also, using the signal from the telemetric temperature probe within the abdomen, and animal chamber temperature was measured using a thermometer within the plethysmograph. 2.4. Experimental protocol The rats were broken up into two separate groups, six were run at temperatures between 24.5 and 26.5 ◦ C (the room temperature or “Cool” group) and the other six were run at temperatures between 29.5 and 30.5 ◦ C (the “Warm” group). In the Warm group, the temperature of the plethysmograph chamber was maintained at 29.5–30.5 ◦ C throughout all of the ventilation experiments with a heating lamp and thermometer connected to a thermostat (model 73A, Yellow Springs Instrument Co, Yellow Springs, OH). Ventilation experiments began following recovery from surgery, and animals gained weight similarly over the course of the experiments in the Cool and Warm groups. After the EEG/EMG pedestal was connected and the microdialysis probe inserted, animals were placed into the plethysmograph and allowed to acclimatize for at least 30 min. Once the animals appeared calm and ceased exploring, aCSF or 1 mM muscimol HBr in microdialysis was started. Rats were microdialyzed with aCSF or 1 mM muscimol for 30 min prior to starting the room air ventilation recording, and microdialysis was continued throughout the entire ventilation experiment. Thirty minute of room air ventilation was recorded, following which the gas concentration entering the plethysmograph was changed to either 7% CO2 balanced with air, or 10% O2 balanced with nitrogen. Once the gas analyzer connected to the plethysmograph outflow read 7% CO2 or 10% O2 , 30 more minutes of ventilation data were recorded. In each rat in both the Cool and Warm groups, four ventilation experiments were performed: two aCSF microdialysis experiments with a 7% CO2 and 10% O2 challenge, and two 1 mM muscimol microdialysis experiments with a 7% CO2 and 10% O2 challenge. Designing the experiment this way allowed each rat to be used as its own aCSF con-

trol in both the hypercapnic and hypoxic experiments in a paired fashion. 2.5. Data analysis and anatomy For behavioral state analysis, a fast Fourier transform was performed on the EEG–EMG signal at 3.6 s long epochs, in addition to studying the raw EEG– EMG signals. We designated frequency bands delta, theta, and sigma as 0.3–5, 6–9, and 10–17 Hz, respectively, based upon algorithms for rat sleep scoring by Heller (Bennington et al., 1994). Experiments were performed either between 9 AM to noon (right before the light period ended) or noon to 3 PM (directly after the dark period began) such that both wakefulness and sleep states were observed. Sleep states were categorized in the following way as previously described (Li et al., 1999; Nattie and Li, 2001, 2002a): (1) wakefulness—raw EEG low, EMG present, delta power low; (2) non-rapid eye movement sleep—low, delta power high, the product of sigma and theta power high; (3) REM sleep—raw EEG low, EMG absent, delta power low, theta to delta power ratio high. REM periods were short and were not present in every experiment, and on some instances sleep state could not be determined from the EEG–EMG record. Because of this, ventilation events that occurred during REM or when sleep state was indeterminate were excluded from our analysis. Using the DATAPAC 2000 software, breath events were individually selected. Breaths occurring during rat movement (sniffing, grooming, etc.) were excluded. Breath events were grouped into bins of 50–500 events depending upon arousal state. The minute ventilation (V˙ E ) and tidal volume (VT ) of each breath were calculated using rat body temperature, plethysmograph chamber temperature, and the barometric pressure of the day the experiment was performed, and normalized against rat body weight (Bartlett and Tenney, 1970). Ventilatory values are expressed as means for quiet wakefulness and NREM sleep during room air, 7% CO2 , or 10% O2 conditions. We analyzed data using a two-way repeated measures ANOVA with aCSF and muscimol as repeated measures and Warm–Cool and room air-hypercapnia (or hypoxia) as categorical variables. Post hoc evaluation was via modified (Bonferroni) t-test or Dunnett’s test (SYSTAT Software Inc, Point Richmond, CA). Body temperature was analyzed

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using a two-way repeated measures ANOVA with Treatment (aCSF or muscimol) and Time (repeated measure) as factors and Dunnett’s test used for post hoc comparisons. At the conclusion of the experimental protocol, rats were euthanized with an overdose of sodium pentobarbital injected intraperitoneally (>75 mg/kg). The brainstem of the animals was quickly dissected and frozen rapidly on dry ice, and stored at −15 ◦ C until being sliced crosswise on a Leica crystat into consecutive 50 ␮m sections. After being mounted upon gelatincoated slides, sections were fixed in 4% paraformaldehyde and stained with cresyl violet. The distance between bregma and the probe tip (rostral–caudal level) was calculated by measuring the distance between the center of the probe and the most caudal section showing the entire facial nerve (−10.04 relative to bregma in the rat brain atlas of Paxinos and Watson (1986)). Animals that had probes falling well outside of the MRR were not included in the data analysis. 3. Results 3.1. Microdialysis probe locations The locations of the microdialysis probe tips for the six animals in the room temperature group and

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the six animals in the warmed group are illustrated schematically in Fig. 1A and B, respectively. In the room temperature (Cool) group, the average distance from bregma was −11.5 ± 0.3 (S.E.M.) mm, ranging from −10.3 to −12.3 mm. The facial nucleus ranges from −10.3 to −11.6 mm from bregma and all but one probe center was located within that range. Rats in the Warm group had an average distance from bregma of −11.4 ± 0.1 mm, ranging from −10.3 to −11.8 mm from bregma with one probe lying just outside the limits of the facial nucleus. Repeated placement of the microdialysis probe resulted in some tissue damage and gliosis surrounding the cannula, but never amounted to more than approximately a 0.4 mm radius from the center of the probe tip. 3.2. Effects on body temperature Muscimol dialysis into the MRR produced a significant and continual fall in body temperature during the length of the CO2 and hypoxic experiments in the Cool temperature conditions (Figs. 2A and 3A). Twoway repeated measures ANOVA showed an effect of Treatment (P < 0.005), Time (P < 0.001); and a significant interaction between them (P < 0.001). Muscimol treatment had no such effect when ambient temperature was in the thermoneutral zone (Warm

Fig. 1. Panel (A) shows a schematic diagram of probe locations within the rat brainstem (black circles) in the Cool group (n = 6), panel (B) shows a schematic of probe locations (black squares) in the Warm group (n = 6). Top schematics represent −11.0 mm, and bottom, −11.6 mm from bregma (Paxinos and Watson, 1986). In the Cool group probe centers were located between −10.3 and −12.3 mm from bregma; in the Warm group, between −11.0 and −11.8 mm from bregma. VII, facial nerve genu; VII nuc, facial nucleus; pyr, medullary pyramid; Rpa, raphe pallidus; Rmg, raphe magnus; and Rob, raphe obscurus.

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Fig. 2. Mean ± S.E.M. absolute body temperature over time is shown for the 7% CO2 ventilation experiments in (A) the Cool group (n = 6) and (B) the Warm group (n = 6). In (A), 1 mM muscimol dialysis decreased body temperature compared to aCSF microdialysis but in (B) it had no effect. In (A) and (B), there was a decrease in body temperature during 7% CO2 breathing compared to that at t = 0 (# P < 0.05 effect of time, * P < 0.05 effect of treatment, two-way RM ANOVA with treatment and time as variables, Bonferroni post hoc pair wise comparison to t = 0).

Fig. 3. Mean ± S.E.M. absolute body temperature over time is shown for the 10% O2 ventilation experiments in (A) the Cool group (n = 6) and (B) the Warm group (n = 6). In (A) and (B), 1 mM muscimol dialysis decreased body temperature compared to aCSF microdialysis but in (B) it had no effect. In (B), there was a decrease in body temperature during 10% O2 breathing compared to that at t = 0 (# P < 0.05 effect of time, * P < 0.05 effect of treatment, two-way RM ANOVA with treatment and time as variables, Bonferroni post hoc pair wise comparison to t = 0).

group) (Figs. 2B and 3B). Two-way repeated measures ANOVA revealed no Treatment effect, but there was a significant Time effect (P < 0.001) without any significant interaction. In the aCSF control experiments, 7% CO2 and 10% O2 breathing alone also produced a significant decrease in body temperature in both the Cool and Warm groups (P < 0.05, two-way repeated measures ANOVA, Dunnett’s post hoc comparison to t = 0). Exposure to either 7% CO2 or 10% O2 decreased body temperature in both Cool and Warm

conditions. Muscimol dialysis decreased body temperature in the Cool conditions but maintaining the animals in the Warm conditions prevented this muscimol effect. 3.3. Muscimol effects on the responses to 7% CO2 : warm and cool temperature groups Fig. 4 summarizes the effects of muscimol dialysis on oxygen consumption (A), ventilation (B) and the

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ratio of V˙ E /V˙ O2 (C) in the hypercapnia experiments. Muscimol did not have any significant effect on V˙ O2 during room air or 7% CO2 breathing in Warm or Cool conditions compared to aCSF. Similarly 7% CO2 did not significantly decrease V˙ O2 values compared to room air during either aCSF or muscimol treatment.

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V˙ O2 was significantly lower in the Cool as compared to the Warm conditions (P < 0.01, categorical effect of Temperature). Muscimol had no significant effect on V˙ E during air breathing in wakefulness (Fig. 4B) in either Warm or Cool conditions. In contrast, muscimol significantly increased V˙ E during 7% CO2 breathing in both the Warm and Cool conditions (P < 0.025 interaction of Treatment and CO2 with P < 0.01 post hoc t-test modified as per Bonferroni). If we defined the ventilatory response to 7% CO2 as V˙ E during room air breathing subtracted from V˙ E during 7% CO2 there was also a significant effect of muscimol when compared to aCSF microdialysis (P < 0.01) but there was no significant interaction. While this effect was significant, it was small. In the Warm conditions absolute VE increased by 15%; in the Cool conditions, 9%. Hypercapnia caused a large significant increase in the V˙ E /V˙ O2 ratio during both aCSF and muscimol microdialysis (P < 0.001) (Fig. 4C). The hypercapniainduced increase in the V˙ E /V˙ O2 ratio was significantly lower in the warm temperature group (P < 0.03). Muscimol increased this response in each temperature group but this was not significant. Analysis of the CO2 response as the change in the V˙ E /V˙ O2 ratio also did not show significance. We conclude that muscimol dialysis in the MRR did not decrease the CO2 response as expected but actually increased the VE response without a significant effect on the V˙ E /V˙ O2 response. This pattern of responses was independent of Warm and Cool conditions, it was not affected by the different body temperatures in the two conditions.

Fig. 4. The effects of muscimol treatment (open symbols) as compared to control (solid symbols) on oxygen consumption (A), ventilation (B), and the ratio of V˙ E /V˙ O2 (C) in air and 7% CO2 breathing in the animals exposed to Cool and to Warm ambient temperature. Note that body temperature in the Warm group remains relatively constant while muscimol treatment decreases body temperature on the Cool group (see Fig. 2). These results were analyzed using a two-way repeated measures ANOVA with control-treatment as the repeated measure and air–CO2 and Warm–Cool as categorical variables. Post hoc analysis was with a t-test modified as per Bonferroni (* P < 0.01). For oxygen consumption, the values in Cool are significantly lower than in Warm. For ventilation, the values at 7% CO2 are significant greater after muscimol treatment. For V˙ E /V˙ O2 , the values at 7% CO2 in Cool are significantly greater than in Warm but there was no effect of muscimol.

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3.4. Muscimol effects on the responses to 10% O2 : warm and cool temperature groups Fig. 5 summarizes the effects of muscimol dialysis on oxygen consumption (A), ventilation (B) and the ratio of V˙ E /V˙ O2 (C) in the hypoxia experiments. Muscimol did not have any significant effect on V˙ O2 during room air or 10% CO2 breathing in Warm or Cool conditions compared to aCSF. Overall there was no Treatment effect on V˙ O2 but there was a significant interaction between the repeated measures Treatment effect and Temperature (P < 0.002) but no post hoc comparisons reached significance. There were significant effects of the categorical variables Hypoxia (P < 0.001) and Temperature (P < 0.05) and a significant interaction of them with the repeated measures variable Treatment (P < 0.05) without significant post hoc effects. Hypoxia significantly decreased V˙ O2 values compared to air breathing during both aCSF or muscimol treatment as did Cool Temperature. Muscimol had no significant effect on V˙ O2 . Muscimol had no significant effect on V˙ E during air breathing in wakefulness (Fig. 5B) in either Warm or Cool conditions but V˙ E was less in the Cool conditions (P < 0.01, interaction of categorical variable Temperature with the repeated measures variable Treatment; post hoc P < 0.05). Muscimol significantly increased V˙ E during 10% O2 in the Warm condition and decreased it in the Cool condition (P < 0.001, interaction of categorical variables Temperature and Hypoxia with the repeated measures variable Treatment; post hoc P < 0.05). If we defined the ventilatory response to 10% O2 as V˙ E during room air breathing subtracted from V˙ E during 10% O2 , muscimol significantly increased the

Fig. 5. The effects of muscimol treatment (open symbols) as compared to control (solid symbols) on oxygen consumption (A), ventilation (B), and the ratio of V˙ E /V˙ O2 (C) in air and 10% O2 breathing in the animals exposed to Cool and Warm ambient temperature. Note that body temperature in the Warm group remains relatively constant while muscimol treatment decreases body temperature on the Cool group (see Fig. 3). These results were analyzed using a two-way repeated measures ANOVA with control-treatment as the repeated measure and air–CO2 and Warm–Cool as categorical variables. Post hoc analysis was with a t-test modified as per Bonferroni (* P < 0.01; + P < 0.05). For oxygen consumption, the values in Cool are significantly lower than in Warm. For ventilation, the values at 10% O2 are significant greater after muscimol treatment in Warm and significantly less in Cool. For V˙ E /V˙ O2 , there are no significant effects.

response in the Warm condition and decreased it in the Cool condition when compared to aCSF microdialysis (P < 0.001 interactive term between the categorical variable Temperature and the effect on Treatment on the change in V˙ E , post hoc Dunnett’s test, P < 0.01).

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The effect though significant was small. In Warm conditions V˙ E was increased by 19%; in Cool conditions V˙ E decreased by 23%. Hypoxia caused a large significant increase in the V˙ E /V˙ O2 ratio during both aCSF and muscimol microdialysis (P < 0.001) (Fig. 5C). There was a significant interaction between the repeated measures Treatment effect and Temperature (P < 0.05) but no post hoc significance. There was no significant effect of muscimol treatment. In respect to V˙ E alone, muscimol increased the V˙ E hypoxic response in the Warm condition and decreased it in the Cool condition without a significant effect on the V˙ E /V˙ O2 response. 3.5. Effect of muscimol microdialysis on arousal state Table 1 portrays the percent experimental time spent in wakefulness and NREM sleep states during the 7% CO2 and 10% O2 experiments in both the Cool and Warm groups. The percent time spent in REM sleep or when sleep state was indeterminate never amounted to more than 3% of the total experimental time, so these data are not included in the analysis. Muscimol microdialysis had no significant effect on arousal state during the ventilation experiments in either rat group. Ten percent O2 breathing during both aCSF and muscimol microdialysis in the Cool group resulted in an increased time spent in wakefulness (P < 0.05, paired t-tests). Hypoxia caused an increase in the time spent in wakefulness in the Warm group during aCSF or 1 mM muscimol microdialysis, but this increase was not significant, and 4/6 rats in this group slept as compared to 2/6 rats in the room temperature group. Because we could not obtain V˙ E data in sleep during hypoxia we have only presented data for quiet wakefulness.

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4. Discussion 4.1. Main results 1. The known effect of muscimol application in the MRR to decrease rat body temperature requires ambient temperature to be below the thermoneutral zone if studied in conscious rats. This temperature lowering effect of muscimol likely reflects inhibition of cool-induced tail vasoconstriction as muscimol had no significant effect on V˙ O2 . 2. Muscimol did not decrease the V˙ E response to CO2 as expected; instead it increased the response. 3. The normal response to hypoxia in the rat is an increase in V˙ E along with a decrease in V˙ O2 , the proportions of these two effects varying depending on ambient temperature. Muscimol in the MRR did not affect the overall V˙ E /V˙ O2 ratio response but increased the V˙ E response in Warm conditions while decreasing it in Cool conditions. 4.2. Body temperature responses Before discussing the results we point out that insertion of the microdialysis probes into the brainstem does produce tissue damage. This is shown in various figures in our previous papers (Nattie and Li, 2000, 2001, 2002a; Taylor et al., 2005). This damage is unavoidable. Control rats receive that exact same probe insertion and the constancy of their responses provide reasonable assurance that the treatment effects are specific. The room temperature (Cool) experiments took place at ambient temperatures between 24.5 and 26.5 ◦ C, which represents a mild cold stress for the rat, as the rat thermoneutral zone is closer to 30 ◦ C

Table 1 Percent time spent in wakefulness and NREM sleep in the room temperature (RT) group (n = 6) and the warmed group (n = 6) Room air

7% CO2

Room air

10% O2

aCSF

Muscimol

aCSF

Muscimol

aCSF

Muscimol

aCSF

Muscimol

RT group

Wake NREM

59.9 ± 9.5 37.2 ± 9.2

59.3 ± 8.8 39.1 ± 9.0

66.4 ± 9.4 32.0 ± 8.5

77.1 ± 7.0 22.0 ± 6.9

60.7 ± 3.3 36.6 ± 3.4

71.6 ± 6.3 28.2 ± 6.3

92.7 ± 7.3 ± 5.7

96.0 ± 3.3# 4.0 ± 3.3

Warm group

Wake NREM

56.7 ± 7.7 40.9 ± 7.3

51.0 ± 9.4 47.0 ± 9.0

61.1 ± 11.6 33.0 ± 9.7

69.0 ± 12.7 30.0 ± 12.4

45.2 ± 11.4 52.2 ± 10.8

64.8 ± 5.2 33.2 ± 5.3

60.2 ± 9.6 28.6 ± 10.1

90.6 ± 5.1 9.4 ± 5.1

5.7#

Percents are given ±S.E. In the room temperature group, 10% O2 breathing led to a significant increase in the percent experimental time spent in wakefulness. # P < 0.05, paired t-tests.

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(Gordon, 1990; Gautier, 2000). When aCSF was microdialyzed, hypercapnia itself led to a small but significant decrease in body temperature, which most likely can be attributed to increased respiratory heat loss (Gautier et al., 1993; Mortola and Maskrey, 1998). Hypoxia leads to a larger well-documented decrease in body temperature in small mammals and newborns, due to a decrease in the thermoregulatory set point by the hypothalamus, and withdrawal of thermoregulatory effector responses such as BAT thermogenesis and shivering (Gautier, 2000; Mortola, 2001; Steiner and Branco, 2002). Therefore, the drop in body temperature we observed during hypoxia is a standard response. Muscimol microdialysis into the MRR significantly decreased core body temperature during the hypercapnia and hypoxia experiments in Cool conditions. This decrease was present at the start of the ventilatory experiments, which occurred following 30 min of initial microdialysis (unfortunately, body temperature was not recorded during the initial period). The body temperature effect of MRR muscimol microdialysis in conscious rats is not surprising, as the MRR is the site of sympathetic input to both the subscapular BAT pads (Morrison, 2004b) and the cutaneous vasomotor nerves in the tail arteries (a major point of heat loss in the rat) (Smith et al., 1998; Tanaka et al., 2002; Ootsuka et al., 2004). Additionally, focal muscimol administration into the MRR has been shown to significantly inhibit BAT sympathetic nerve activity and lower temperature in anesthetized rats (Cao et al., 2004), and is capable of decreasing the core body temperature of conscious rats studied between 24 and 25 ◦ C (Zaretsky et al., 2003). In the Warm temperature group, muscimol had little effect on body temperature during either the hypercapnia or hypoxia experiments. This indicates that the fall in body temperature observed in the Cool conditions reflects an interaction between mild cold stress and muscimol. Since we did not observe a significant muscimol effect on V˙ O2 even as body temperature decreased continually in the Cool condition we can attribute the muscimol effect in these conditions to an inhibition of heat conservation mechanisms, which in the rat likely involved inhibition of tail vasoconstriction (Smith et al., 1998; Tanaka et al., 2002; Ootsuka et al., 2004).

4.3. Ventilatory responses to hypercapnia in the room and warm temperature groups CO2 is detected by the carotid body and by central chemoreceptors, which are located at many sites including the MRR (Feldman et al., 2004). Serotonergic neurons are sensitive to CO2 in culture (Wang et al., 2001) while focal acidosis in the MRR increases V˙ E (Nattie and Li, 2001; Hodges et al., 2004) and focal inhibition or lesion of MRR serotonergic neurons decreases the V˙ E response to CO2 (Nattie et al., 2004; Taylor et al., 2005). Chronic focal dialysis of fluoxetine into the MRR increased the response to CO2 (Taylor et al., 2004). CO2 induced excitement of MRR serotonergic neurons stimulates respiratory premotor neurons and increases V˙ E (Richerson, 2004). Muscimol dialysis in the near-by RTN, another central chemoreceptor site, decreases ventilation in air breathing and decreases the CO2 response (Nattie and Li, 2000) with the putative chemosensitive neurons in the RTN being glutamatergic (Mulkey et al., 2004; Guyenet et al., 2005). Further, Messier et al. (2002) reported that 10 mM muscimol microdialyzed into the MRR of unanaesthetized newborn piglets decreased the absolute V˙ E during 5% CO2 without effects on metabolic rate or core body temperature. To our surprise, muscimol dialysis into the MRR in the present study did not decrease the CO2 response; it increased it. We suggest that the difference in the response to MRR muscimol dialysis in adult rats and newborn piglets reflects the developmental stage of the animal studied. In the MRR muscimol microdialysis study performed by Messier et al. (2002) piglets were approximately 4–12 days old, and the decrease in the ventilatory response to CO2 observed following muscimol dialysis was 18%. This group of researchers later performed a similar MRR microdialysis study in piglets targeting serotonergic neurons with the specific 5-HT1A agonist DPAT, and found that 30 mM DPAT significantly decreased the ventilatory response to hypercapnia in piglets 10–16 days old, but significantly increased the response in younger animals (Messier et al., 2004). In adult rats, 1, 10, and 30 mM DPAT significantly decreased the ventilatory response to CO2 by 22, 21, and 40%, respectively (Taylor et al., 2005). The results of these three previous studies, when considered together with our present hypercapnic

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ventilatory response data, suggest that the function of MRR neurons in chemoreception and the CO2 response may change during post-natal development in mammals. Muscimol dialysis into the MRR decreases the CO2 response in young but not older animals while inhibition of serotonergic neurons by DPAT decreases the CO2 response in adult but not newborn animals. There are examples of age dependent effects of muscimol on GABA-ergic neurons in the hippocampus. Because immature neurons have high intracellular concentrations of Cl− , GABA actually acts as an excitatory neurotransmitter until Cl− levels fall and the K+ /Cl− cotransporter KCC2 is fully expressed (for review see Herlenius and Lagercrantz, 2004). In the rat, this “switching over” takes approximately 1–2 weeks to occur after birth (Rivera et al., 1999), however it is unknown when it occurs in the piglet, or in the human (Herlenius and Lagercrantz, 2004). Thereby, it is likely that developmental differences contribute to the differences in the ventilatory effect of muscimol microdialysis into the MRR in the adult rat compared to the newborn piglet. The difference in the effects of muscimol on the CO2 response in the MRR versus the RTN point to differences in GABA-ergic function in these two central chemoreceptor sites. In the Cool and Warm temperature groups, MRR muscimol microdialysis increased the ventilatory response to CO2 , and did not alter metabolic rate significantly, as found by others (Gautier et al., 1993). Thereby the V˙ E /V˙ O2 ratio was also increased during hypercapnia as well. Muscimol had no significant effect on the V˙ E /V˙ O2 ratio although it was increased on average by 25% in Cool and 26% in Warm conditions. 4.4. Ventilatory responses to hypoxia in the room and warm temperature groups V˙ O2 , ambient and body temperature, and the ventilatory responses to hypoxia are all tightly interrelated (Gautier, 1996). Specifically, when small animals (including rats) are exposed to hypoxia, there is a decrease in oxygen consumption (hypometabolism), and a concurrent decrease in body temperature due to inhibition of heat producing metabolism (Frappell et al., 1992). The decrease in V˙ E /V˙ O2 can be large in ambient temperatures well below thermoneutrality (Frappell et al., 1992; Matsuoka et al., 1994; Saiki et al.,

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1994; Gautier, 1996; Mortola, 2001). Therefore, during hypoxia, the V˙ E /V˙ O2 ratio is affected by both a drop in metabolism and an increase in ventilation (Gautier et al., 1993). Hypoxia is detected primarily by peripherally located chemoreceptors in the carotid and aortic bodies. Information from peripheral chemoreceptors is carried via the carotid sinus nerve and is integrated by the NTS (Housley and Sinclair, 1988) and ventilation increases accordingly. Neurons in the MRR have been suggested to have an inhibitory influence on NTS activity, and therefore, the hypoxic ventilatory response (Feldman, 1994; Perez and Ruiz, 1995; Gargaglioni et al., 2003). The MRR sends serotonergic and nonserotonergic (including GABA-ergic) afferents to the NTS (Thor and Helke, 1987; Schaffar et al., 1988; Sim and Joseph, 1992; Beart et al., 1994; Zec and Kinney, 2003), the primary termination point of peripheral O2 chemoreceptor afferents (Housley and Sinclair, 1988). In anesthetized rats, electrical stimulation or l-glutamate microinjection into the MRR prevents the increase in NTS activity as a result of peripheral chemoreceptor activation (Perez and Ruiz, 1995). Gargaglioni et al. also found that non-selective lesioning of MRR neurons with microinjection of ibotenic acid results in an increase in the ventilatory response to hypoxia in unanaesthetized rats (Gargaglioni et al., 2003). It is not clear whether this inhibitory modulation of the hypoxic ventilatory response at the NTS involves serotonergic or non-serotonergic neurons. In the Cool group, muscimol decreased the hypoxic ventilatory response (Fig. 5), but the V˙ E /V˙ O2 ratio in hypoxia was not significantly affected. In the Warm group, prevention of the muscimol-induced fall in body temperature led to an increased hypoxic ventilatory response compared to aCSF but again the V˙ E /V˙ O2 ratio was not significantly affected. The rats produced an appropriate overall response in either temperature despite muscimol inhibition of rostral medullary raphe neurons. The inhibitory effect on NTS neurons and hypoxic ventilatory responses that arises from the MRR appears to be dependent on body temperature. Prevention of the decrease in body temperature by Warm conditions allows muscimol to disinhibit the MRR effect at the NTS so V˙ E increases. In Cool conditions with a lower body temperature muscimol results in a decreased V˙ E .

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Maskrey reported that decreasing core body temperature in conscious rats significantly decreased the ventilatory response to hypoxia but had a lesser inhibitory effect on the ventilatory response to hypercapnia (Maskrey, 1990). We speculate that the decrease in body temperature that is present as a result of MRR muscimol microdialysis in the Cool group contributes to the decreased ventilatory response to hypoxia. When body temperature remained unchanged in slightly higher ambient temperatures, muscimol increased the ventilatory responses to hypoxia via the suggested disinhibition.

GABA-ergic MRR neurons function oppositely of acidosis-stimulated serotonergic neurons, that there is normally a balance of inputs from neurons that are excited or inhibited by CO2 . The muscimol microdialysis in the current study could be affecting this balance perhaps by inhibition of GABA-ergic neurons. The MRR neurons that modulate NTS function and the ventilatory response to hypoxia are of unknown phenotype so it is possible that the muscimol effect in this case could be via serotonergic or non-serotonergic neurons.

4.5. MRR neurons affected by muscimol microdialysis

4.6. Conclusions

Muscimol is a specific GABAA agonist. In the MRR, GABA was originally believed to co-localize with serotonin (Belin et al., 1983; Millhorn et al., 1988; Kachidian et al., 1991). Closer analysis achieved by fluorescence microscopy and cell counting revealed that only 3.6% of serotonergic cell bodies stain positive for glutamic acid decarboxylase (GAD, the rate-limiting enzyme in GABA synthesis) in the raphe magnus, 1.5% of serotonergic cell bodies co-localize with GAD in the raphe obscurus, and no double labeled cells are observed in the extra-raphe regions (Stamp and Semba, 1995). The GABAA receptor, which could be expected to inhibit MRR serotonergic neurons, is only present on a modest percentage of serotonergic cells here as well, about 20% of the total serotonergic population (Hama et al., 1997). Considering that serotonin neurons are only one third of the total MRR cell population, and neurons expressing GABAA receptor are much more widespread throughout the MRR, we assume that our muscimol microdialysis affects both serotonergic and non-serotonergic neurons. Given that serotonergic neurons stimulate ventilation when excited by CO2 we interpret the enhanced ventilatory response to CO2 after muscimol to be the result of disinhibition, that muscimol inhibits neurons that are inhibitory to chemoreception. For example, electrophysiological studies of MRR neurons in primary tissue culture show the presence of neurons inhibited by CO2 the majority of which are GABA-ergic (stain positive for GAD) (Hodges and Richerson, personal communication). Consequently, it is possible that acidosis-inhibited

Muscimol dialysis into the MRR did not have the expected effects; inhibition of the CO2 and hypoxic ventilatory responses. For CO2 , our results suggest that the neuronal organization of the MRR as a central chemoreceptor location includes neurons that are normally inhibitory to chemoreception, which respond to muscimol inhibition. Small changes in body temperature do not affect this response. For hypoxia, the changes in body temperature qualitatively altered the muscimol effect on V˙ E but the V˙ E /V˙ O2 ratio remained unchanged. We have no insight into the neuronal phenotype involved in the effect on V˙ E but conclude that the ability to maintain the V˙ E /V˙ O2 ratio at an appropriate value in hypoxia does not requires MRR neurons sensitive to muscimol.

Acknowledgements This work was supported by National Institutes of Health HL 28066 and the Albert J. Ryan Foundation. Drs. Hannah Kinney, George Richerson and James C. Leiter helped with data interpretation and experimental design.

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