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J Appl Physiol 90: 269-279, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 1, 269-279, January 2001

Nucleus raphé obscurus modulates hypoglossal output of neonatal rat in vitro transverse brain stem slices

John H. Peever, Aleksandar Necakov, and James Duffin

Respiratory Neuroscience Laboratory, Departments of Physiology and Anaesthesia, University of Toronto, Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nucleus raphé obscurus (NRo) modulates hypoglossal (XII) nerve motor output in the in vitro transverse brain stem slice of neonatal rats (1-5 days old); chemical ablation of NRo and its focal CO2 acidification modulated the bursting rhythm of XII nerves. We microinjected a 4.5 mM solution of kainic acid into the NRo to disrupt cellular activity and observed that XII nerve activity was temporarily abolished (n = 10). We also microinjected CO2-acidified (pH = 6.00 ± 0.01) artificial cerebrospinal fluid (aCSF) into the NRo (n = 6), the pre-Bötzinger complex (PBC) (n = 6), as well as a control region in the lateral tegmental field equidistant to NRo, PBC, and the XII motor nuclei (n = 12). CO2 acidification of the control region had no effect on XII motor output. CO2 acidification of the NRo significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 77%; integrated burst amplitude and burst duration increased by 64% and 52%, respectively. CO2 acidification of the PBC significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 65%, but neither integrated burst amplitude nor burst duration changed. These results demonstrate that chemical ablation of the NRo can abolish XII nerve bursting rhythm and that stimulation of the NRo with CO2-acidified aCSF can excite XII nerve bursting activity. From these observations, we conclude that, in transverse brain stem slices, the NRo contains pH/CO2-sensitive cells that modulate XII motor output.

respiratory neurons; brain stem; pH/carbon dioxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS HYPOTHESIZED THAT the site of respiratory rhythm generation resides in the pre-Bötzinger complex (PBC) located in the rostral ventrolateral brain stem (16, 29, 51). Transverse brain stem slice in vitro preparations of juvenile rodents contain the PBC, a functioning portion of the hypoglossal (XII) motor nuclei, and XII nerve rootlets. When placed in artificial cerebrospinal fluid (aCSF) maintained at appropriate temperature, pH, and K+ concentration, the XII nerve rootlets exhibit a spontaneously rhythmic discharge (see Fig. 1), termed respiratory rhythm by some investigators (16, 29, 51).


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  		Fig. 1.   Top: schematic depiction of a transverse brain stem slice showing the three injection sites [kainic acid 4.5 mM into nucleus raphé obscurus (NRo) and the control area, and CO2-acidified artificial cerebrospinal fluid (aCSF) into NRo, pre-Bötzinger complex (PBC), and the control area]. Bottom: typical example of hypoglossal (XII) nerve rootlet discharge illustrating how the measures of bursting frequency, burst duration, and integrated amplitude were obtained.

The brain stem slice preparation is therefore widely used to examine the neural mechanisms of mammalian respiratory rhythm generation, but with almost exclusive focus on the PBC and XII motor nuclei (16, 29, 48, 51, 55). However, although both are required to produce respiratory rhythmic discharge of XII nerves, other regions might modulate its expression. Although the nucleus raphé obscurus (NRo) is known to modulate respiratory rhythm in intact adult rats and cats (5, 6, 11, 17, 20, 30, 37), its involvement in respiratory rhythm generation in transverse brain stem slices is not well documented (2).

NRo neurons are located along the brain stem midline, dorsal to the pyramidal tracts and ventral to the XII motor nuclei (see Fig. 1). In adult rats and cats, the NRo contains neurons with respiratory-related discharge patterns (15, 23, 33, 34), which have axonal projections extending into brain stem regions corresponding to the PBC and XII motor nuclei (1, 9, 18, 21, 22). Neurons of the NRo contain serotonin, substance P, and thyrotropin-releasing hormone, all thought to excite XII motoneurons (3, 4, 13, 41, 49). In rhythmic transverse brain stem slices, microinjection of serotonin into the NRo increases the bursting frequency of XII nerve discharge (2). In intact adult rats, focal acidification of brain stem NRo increases respiratory motor output (6, 32), and, when transiently acidified, cultured NRo neurons increase their tonic discharge rate (56, 57).

In combination, these observations led us to hypothesize that, in transverse brain stem slices, NRo modulates XII motor output and may do so in a pH/CO2-sensitive manner. We conducted three series of experiments to test this hypothesis. First, we tested the postulate that elimination of the NRo would reduce XII motor output because its stimulation increases XII activity (2). To do this, we disrupted the cellular activity of the NRo by microinjecting the neurotoxin kainic acid into it. Then we postulated that brain stem slices contain the appropriate neural circuitry to detect and respond to pH/CO2 perturbations. We changed the pH/CO2 of the aCSF bathing medium and observed XII nerve discharge. Finally, we postulated that the NRo responds to focal pH/CO2 changes and tested this hypothesis by observing the effects of focal CO2 acidification of NRo, PBC, and a control area on XII nerve discharge. We microinjected CO2-acidified aCSF into the PBC because neurons in this region are supposedly pH/CO2-sensitive (27) and we wanted to compare the effects of acidifying the NRo and PBC. We demonstrate for the first time in rhythmically active transverse brain stem slices that chemical lesions of NRo disrupt the expression of XII motor output and that focal CO2 acidification of NRo transiently increases XII nerve discharge frequency, integrated amplitude, and duration. We conclude that NRo are involved in the expression of XII motor output and that, in transverse brain stem slices, cells in the NRo are capable of detecting and responding to pH/CO2 alterations.

This work was presented as an abstract (46).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The following procedures were approved by the University of Toronto animal care committee. Testing our hypotheses about NRo modulation of XII motor output in in vitro transverse brain stem slices involved a series of microinjections into specific regions of the slice while the discharge from XII nerves was recorded. We microinjected kainic acid into the NRo, as well as CO2-acidified aCSF into the NRo, PBC (as visually defined by Koshiya and Smith, Ref. 29), and a control region approximately equidistant from NRo, PBC, and XII motor nuclei (Fig. 1). We utilized fluorescence microscopy to visualize the location and extent of microinjections, saturating the injected aCSF with the fluorescent green dye fluorescein. XII motor nuclei were also made visible by retrogradely labeling XII motoneurons with the orange fluorescent dye dextran tetramethylrhodamine lysine (Fig. 2). In this way, we ensured that injections were confined to the specific regions tested and, moreover, did not spread into the XII motor nuclei (Fig. 2).


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  		Fig. 2.   Photomicrograph showing the fluorescence techniques used to make focal microinjections in the transverse slice preparation. XII motoneurons are labeled orange with dextran tetramethylrhodamine lysine. A microinjection of aCSF into the midline NRo is labeled green by saturating the injectate with fluorescein.

Labeling XII Motoneurons

We retrogradely labeled XII motoneurons 1-2 days before obtaining brain stem slices from 1- to 4-day-old Sprague-Dawley neonatal rat pups. The rat pups were lightly anesthetized in 1.5-2% halothane in O2. Then, by use of a 22-gauge Hamilton syringe, 5 µl of 10% solution (by molecular weight) of dextran tetramethylrhodamine lysine (10,000 molecular weight, Molecular Probes) in water were injected into the midline of the genioglossus tongue muscle. Pups were then returned to their mother and subsequently used to make transverse brain stem slices.

Respiratory Network Isolation

The transverse brain stem slices containing the respiratory network were isolated by the following procedures. Animals were deeply anesthetized in 3% halothane in O2 and decapitated after absence of response to foot pinch. The head was then immediately immersed in ice-cold aCSF, the composition of which (in mM) was 125 NaCl, 3 KCl, 1 KH2PO4, 2 CaCl2, 1 MgSO4, 25 NaHCO3, and 30 D-glucose. The aCSF was bubbled with 5% CO2 in O2 to produce a pH of ~7.4 (measured by using a pH microelectrode, Beetrode, World Precision Instruments). After the skin and muscle over the skull and cervical spinal column were cut away, the parietal and occipital bones were carefully removed, and a dorsal laminectomy was performed from the first to sixth cervical vertebrae. The olfactory, cranial, and spinal nerves were cut, and the whole brain stem-spinal cord (to the first cervical vertebrae) was then excised from the skull and vertebral column.

To isolate the brain stem, both the cerebrum and cerebellum were removed. The brain stem-spinal cord was then mounted on a block of agar by its dorsal surface (cyanomethacrylate, Sigma Chemical) and placed rostral end down. Thin slices were then cut by using a vibratome (752M Vibroslice, Campden Instruments) until the two most rostral XII nerve rootlets were seen. At this point, a 400- to 700-µm slice incorporating the nerve rootlets was made and constituted the transverse brain stem slice. The remaining dura mater covering the underlying tissue was removed, and the slice was transferred into a recording chamber perfused with aCSF fluid at 15-20 ml/min, where for 30 min it stabilized in aCSF at 20-25°C with temperature controlled by an in-line heater (TC-324B, Warner Instrument). During this time, KCl concentration was increased from 3 to 8 mM to establish and maintain a stable respiratory motor output (48, 51).

Recording

XII nerve discharge was recorded by use of glass-suction electrodes (Fig. 1). The signals were amplified (Axoprobe A1, Axon Instruments; Neurolog, NL106), band-pass filtered (0.1-50 kHz; Neurolog, NL104) with emphasis around 1 kHz using a 10-band stereo equalizer (Audio Reflex, EQ-1), and integrated (time constant = 50 ms; Neurolog, NL703).

Protocols

After aCSF temperature was increased to 26-28°C, XII motor output was recorded for 30 min to establish baseline discharge frequency, integrated burst amplitude, and duration. Then three specific protocols were followed, with the extent and location of the fluorescent green microinjections relative to fluorescent orange XII motoneurons visualized by using a fluorescent microscope (Olympus, BX50WI) with appropriate filters (Olympus, U-MWIG and U-MWIBA). XII motor output was continuously recorded during all procedures. A micromanipulator (MMN-333, Narishige) was used to stereotaxically position glass microelectrodes (tip diameter ~5 µm) for microinjections at depths (relative to the slice surface) that ranged from 200 to 300 µm.

Protocol 1. A microelectrode was placed into the midline NRo; aCSF (pH = 7.40 ± 0.02) saturated with fluorescein was microinjected under pressure (custom-built picospritzer, 20-25 psi; pulse duration 40-50 ms); and after 15-20 min, the control region was microinjected. NRo was defined visually under fluorescence microscopic examination as the midline region ventral to the labeled XII motoneurons and dorsal to the pyramidal tracts, and the control region was approximately equidistant to NRo, PBC, and XII motor nuclei (Fig. 1). If no XII nerve discharge changes resulted from these control injections, a 4.5 mM kainic acid (Sigma Chemical) solution dissolved in aCSF saturated with fluorescein (Molecular Probes) was microinjected into the control region. If no effect on XII discharge was observed, kainic acid was then microinjected into the NRo (Fig. 2), and XII motor output was monitored for 1 h.

Protocol 2. XII nerve discharge was monitored while the pH of the aCSF was maintained constant at 7.42 ± 0.03 and temperature at 27-29°C. After 15-20 min of stable recordings, the flow of CO2 was increased (in addition to the original 95% O2 and 5% CO2) until the pH of the aCSF decreased to 7.17 ± 0.05; XII nerve discharge was recorded after pH had remained constant for 15-20 min. The aCSF pH further decreased to 7.01 ± 0.03, and, after 15-20 min, XII nerve discharge was again recorded.

Protocol 3. Fifty milliliters of aCSF saturated with fluorescein were bubbled with 100% CO2 to produce a pH of 6.00 ± 0.01. Under fluorescence microscopic examination, CO2-acidified aCSF solution was then pressure-injected into one of three regions of the brain stem slice (NRo, PBC, or control region) while XII nerve activity was monitored, with at least a 20-25 min recovery period between injections.

Data Analysis

Ten consecutive bursts of XII nerve discharge were analyzed for each experimental condition, and their characteristics (burst frequency, burst duration, and integrated burst amplitude) were averaged. Once the effects of microinjections caused an obvious change in XII discharge, the time from injection to response characterized the latency (s). The frequency of bursting (bursts/min) was determined from the time required for the 10 bursts to occur. The burst duration (s) was measured directly, and integrated burst amplitude (mm) was determined by measuring the distance from baseline to peak of the integrated signal.

Statistical Analysis

Data are expressed as means ± SE. Statistical inferences were made using one-way ANOVA. Significant differences between means were assessed by Student-Newman-Keuls test, with the level of significance considered at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kainic Acid Injection into NRo

In 10 slices, injection of fluorescein-labeled aCSF (pH = 7.42 ± 0.02) into NRo (Fig. 2) had no effect on XII nerve discharge nor did injection of kainic acid into the control region (Fig. 3C). The general response to kainic acid injected into the NRo was an initial excitation of XII nerve discharge (Fig. 3, A and B) followed by three types of responses. In five slices, we observed a statistically significant increase in XII nerve bursting frequency after 10-25 s and then a tonic discharge followed by a 25- to 30-min period of silence, after which bursting reappeared at preinjection levels (Fig. 3A). In three slices, the response was similar, but after the rhythmic discharge stopped it did not return after 1 h. In two slices, the response was a transient increase in the frequency of XII nerve bursting lasting only ~20 s, a transient period of XII inactivity (~20 s), and then return of the frequency to baseline (Fig. 3B).


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  		Fig. 3.   Typical responses of XII motor output (upper traces) and its integration (lower traces) to microinjections of kainic acid (4.5 mM) into the NRo (A and B) and into the control region in the lateral tegmental field approximately equidistant to NRo, PBC, and XII motor nuclei (C). All traces start at the time when kainic acid was microinjected, as indicated by solid arrows.

Whole Brain Stem Slice CO2 Acidification

In six slices, the XII nerve bursting frequency, burst duration, and integrated burst amplitude were 7.0 ± 1.4 bursts/min, 0.90 ± 0.03 s, and 1.7 ± 0.3 mm, respectively, at 26-27°C and pH = 7.42 ± 0.03 (Fig. 4). After aCSF, pH decreased to 7.17 ± 0.05 and XII nerve bursting frequency, burst duration, and integrated burst amplitude were 9.0 ± 2.1 bursts/min, 0.90 ± 0.04 s, and 1.6 ± 0.4 mm (Fig. 4), respectively, with none of these values changing significantly from those previously recorded at pH = 7.42. The pH of the aCSF was then decreased to 7.01 ± 0.03, and XII nerve bursting frequency significantly increased to 11.0 ± 2.4 bursts/min (56% increase from pH = 7.42) whereas neither burst duration nor integrated burst amplitude changed from previous values in pH = 7.42 or 7.17 (Fig. 4).


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  		Fig. 4.   Effects (means ± SE) of CO2 acidification of aCSF bathing transverse brain stem slices on XII nerve bursting frequency, integrated burst amplitude, and burst duration. Arrows and stars show significant differences (P < 0.05).

Focal CO2 Acidification of NRo, PBC, and Control Region

In six rhythmically active slices, NRo, PBC, and the control region (see Fig. 1) were individually CO2 acidified by microinjecting an aCSF solution of pH = 6.00 ± 0.01 into each region. CO2 acidification of the control region had no effect on XII motor output (Figs. 5C and 7C). However, CO2 acidification of the NRo and PBC had significant and differential effects on XII motor output (Figs. 5, 6, and 7).


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  		Fig. 5.   Typical responses of XII motor output (upper traces) and its integration (lower traces) to microinjections of CO2-acidified aCSF into the NRo (A and B) and into the control region in the lateral tegmental field approximately equidistant to NRo, PBC, and XII motor nuclei (C). All traces start at the time when CO2-acidified aCSF was microinjected, as indicated by solid arrows.



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  		Fig. 6.   Effects (means ± SE) of focal CO2 acidification of NRo, PBC, and control on XII nerve bursting frequency, integrated burst amplitude, and burst duration. Arrows and stars show significant differences (P < 0.05).



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  		Fig. 7.   Typical responses of XII motor output (upper traces) and its integration (lower traces) to microinjections of CO2-acidified aCSF into the PBC (A) and into the control region in the lateral tegmental field approximately equidistant to NRo, PBC, and XII motor nuclei (B). All traces start at the time when CO2-acidified aCSF was microinjected, as indicated by solid arrows.

CO2 acidification of the midline NRo resulted in significant increases in XII nerve bursting frequency from 4.8 ± 1.3 to 8.5 ± 0.8 bursts/min (77%), integrated burst amplitude from 1.4 ± 0.1 to 2.3 ± 0.3 mm (64%), and burst duration from 0.71 ± 0.05 to 1.07 ± 0.12 s (52%) (Figs. 5 and 6). The latency of response ranged from 25 to 60 s, and tonic discharge also increased after CO2 acidification. In two preparations, this tonic discharge was pronounced, resembling that observed for kainic acid injection (Fig. 5B), whereas in the others it was similar to that shown in Fig. 5A.

CO2 acidification of the PBC caused a significant increase in XII nerve bursting frequency from 5.4 ± 1.4 to 8.9 ± 2.1 bursts/min (65%), with the response time ranging from 10 to 30 s (Fig. 7A). However, burst duration and integrated burst amplitude were unchanged. Although there was a tendency for integrated burst amplitude to decrease, it was not statistically significant (Figs. 6 and 7A).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We found that chemical ablation of NRo temporarily abolished the expression of XII motor output and that focal CO2 acidification of NRo significantly increased the frequency, amplitude, and duration of XII nerve rhythmic bursting, whereas CO2 acidification of the PBC increased only the bursting frequency of XII nerve discharge. We conclude that NRo modulates XII motor output, that neurons in the NRo are pH/CO2 sensitive (as they are in the PBC), and that NRo and PBC respond differently to focal CO2 acidification. We suggest the NRo is part of the neuronal circuitry required for the expression of respiratory motor output in transverse brain stem slices for the reasons discussed in the following paragraphs.

Critique of Methods

We used the in vitro transverse brain stem slice as a model of the central respiratory control system. Although some investigators accept it as a model of the intact central respiratory rhythm generator (51, 52, 55), others do not (14, 53). Certainly the slice tissue is relatively hypoxic and at a lower temperature (26-28°C) than tissue from intact neonates. It represents a very reduced preparation with no afferents and only a fraction of the brain stem respiratory neurons present in the intact animal. Perhaps the reduction in the number of active synaptic inputs to the respiratory neurons retained in the slice accounts for the requirement of an increase in potassium ion concentration to produce XII nerve bursting (51).

Despite such differences between intact and in vitro brain stem slice preparations, the slices contain sufficient neural circuitry to spontaneously generate periodic bursts of neural activity on XII nerve rootlets (29, 48, 51). In addition, other investigators have observed respiratory neurons with firing patterns similar to those of the ventral respiratory group (analogous to the PBC in this preparation) in adult rats, including preinspiratory, inspiratory, expiratory, and phase-spanning neurons (43, 44, 48, 52). Furthermore, when the temperature of brain stem slice preparations is increased to within physiological limits (35°C), the frequency and pattern of XII motor output approaches that of intact age-matched neonatal rats (47). We therefore suggest that in vitro transverse brain stem slices contain the appropriate neural circuitry to generate and transmit respiratory rhythm to XII motoneurons. It offers superior control of the extracellular environment, making it well suited for these experiments.

Our methods relied on restricting microinjections of kainic acid and CO2-acidified aCSF into the NRo, PBC, and a control region of transverse slices. We used anatomic methods rather than electrophysiological techniques to identify the injection sites. To this end, we labeled the injected aCSF with a fluorescent green dye, fluorescein, and the XII motoneurons with a fluorescent orange dye, dextran tetramethylrhodamine lysine. The extent and location of the microinjections were therefore clearly discerned relative to fluorescent XII motoneurons, which provided a directly visible anatomic landmark. The extent of the spread of injectate, as well as being visualized in this way, was also limited in both space and time by the washout of the injectate by the aCSF flowing at a high rate (15-20 ml/min). Although over time the fluorescence of fluorescein decreases due to photodamage (specifications, Molecular Probes), we observed no such decrease because of the brief time of microinjection. Our confidence in the limited radius of effectiveness of our microinjections was supported by our observations. First, there was a lack of response to injections of kainic acid and CO2-acidified aCSF in the control region, approximately equidistant to NRo, PBC, and XII motor nuclei. Second, the fact that NRo and PBC responded to focal CO2 acidification differently implies that they were specifically and independently activated. We are therefore confident that our microinjections were confined to their target areas and did not spread into XII motor nuclei or adjacent regions.

It is possible that the fluorescein-labeled aCSF itself was responsible for the observed responses. However, microinjections into NRo and the control region did not change XII motor output; therefore, we eliminated this possibility.

Finally, we realize that the aCSF used to locally CO2 acidify discrete regions of tissue had a low pH (6.00 ± 0.01), beyond the physiological range. Nevertheless, we believe that the actual pH/CO2 stimulus achieved was not this extreme. The effectiveness of the microinjections was limited not only by the high flow rates through the tissue chamber (15-20 ml/min) but also by the higher pH (~7.4) of the bathing aCSF. The injected CO2-acidified aCSF was therefore quickly washed out of the tissue by both rapid flow and diffusion. This contention is further strengthened by the estimations of Lui et al. (35), who found that drug concentrations around cells 100-200 µm below the surface of transverse brain stem slices were roughly 10% of the microinjected concentration. On the basis of this estimation, we predict that the pH around the tissue microinjected (200-300 µm below slice surface) with aCSF of pH = 6.0 would be ~7.0, a stimulus level comparable to that used to CO2 acidify the entire slice in these experiments and those by Johnson et al. (27).

Injection of either kainic acid or acidic aCSF into the NRo and PBC altered XII motor output at latencies ranging from 10 to 60 s. Why motor responses occur at such long latencies is unclear; however, comparable latencies were observed when serotonin was microinjected into the NRo of rhythmically active transverse brain stem slices from neonatal rats (2).

NRo and Respiratory Rhythm

Brain stem raphé nuclei are known to alter the expression of respiratory rhythm both in vivo and in vitro, yet assessments of their function have been contradictory. Stimulation of raphé nuclei in intact adult cats and rats and in in vitro brain stem-spinal cord and transverse brain stem slice preparations from neonatal rats elicits either an inhibition or an excitation of respiratory motor output (2, 11, 12, 30, 31, 37, 38). Circumstantial evidence of their crucial role in the expression of respiratory rhythm in neonatal rats comes from lesioning experiments. Destruction of brain stem midline structures by scalpel lesioning results in the complete cessation of respiratory motor output in both brain stem slice and brain stem-spinal cord in vitro preparations, as well as in intact neonatal rats (26, 36, 45). These findings imply that the functional integrity of midline raphé nuclei may be required for the expression of respiratory motor output in neonatal rats. In the current experiments, we specifically targeted the NRo and found that chemical ablation of this region abolished the expression of respiratory rhythm.

For the chemical lesioning, we used microinjections of kainic acid with concentrations similar to those used by others to excite cells to inactivity and thereby remove their function (40). In all preparations tested, injection of kainic acid into the NRo caused the frequency and tonic discharge of XII motor output to initially increase; in all preparations, respiratory activity was abolished either temporarily (n = 7) or permanently (n = 3). We interpret these findings as showing that kainic acid initially acts to depolarize NRo neurons. Depolarization causes a release of endogenous thyrotropin-releasing hormone, serotonin, and substance P; and both serotonin and substance P are known to excite XII motoneurons (3, 4, 13, 41), as well as neurons within the PBC (2, 16). Serotonin release within the NRo causes an increase in XII tonic discharge (2), whereas substance P injected into the PBC causes an increase in XII burst frequency (16). Because kainic acid overexcites NRo neurons into quiescence, the excitatory effects on PBC and XII motoneurons are withdrawn, and XII motor output is no longer expressed.

Two factors, effectiveness and localization, explain the variability of effects of kainic acid injection on XII motor output. First, kainic acid may not have completely disrupted all cellular activity (10), so that in some cases cells within the NRo recovered their function and XII nerve rhythmic discharge returned; or kainic acid may not have affected all the cells of the raphé nuclei because of the extensive dorsoventral distribution of the nucleus and the restricted area of microinjection. Second, some regions of the NRo may have fewer projections to XII motoneurons and the PBC; disruption of their activity would therefore be less effective in altering XII nerve motor output. Regardless, these results demonstrate that the NRo is involved in the expression of respiratory rhythm in transverse brain stem slices.

On the basis of these observations, we hypothesize that NRo facilitation of XII motoneurons is withdrawn after kainic acid injection; although the rhythm generated by the PBC may still be transmitted to XII motoneurons, it is no longer expressed. This hypothesis also agrees with our postulate that NRo not only provides a tonic excitatory drive to XII motoneurons, which would account for their effects on burst amplitude, but also affects the PBC such that bursting frequency is altered. Neurons in the NRo discharge at a constant frequency (57) so that their influence is confined to a tonic excitation. Projections to XII motoneurons therefore would be expected to alter burst amplitude, and complete withdrawal could reduce the XII motor output to undetectable levels, as we observed. Tonic excitation of neurons in the PBC would be expected to alter the generation of the bursting rhythm. Indeed, Gray et al. (16) found that application of a substance P agonist increased the burst frequency of neurons within the PBC. Because the receptor for this transmitter is unique to PBC neurons (16), and NRo neurons not only contain substance P but also project to the PBC region (1, 18, 21, 22), we suggest that it is this pathway by which NRo stimulation increases XII nerve bursting frequency. Further support for this interpretation comes from the observation that microinjection of glutamate agonist, DL-alpha -amino-3-hydroxy-5-methylisoxazole-propionic acid, and serotonin into the NRo increases the frequency of respiratory bursts in transverse brain stem slices (2). The mathematical models developed by Butera et al. (7) also support this hypothesis because they demonstrate that increased tonic excitation of PBC neurons increases their oscillatory rate and presumably respiratory frequency.

In these studies, we demonstrate that the activity of XII motoneurons is affected by manipulating the activity of the NRo. This effect is likely due to the withdrawal of excitatory inputs from neurons within the NRo, which are active before their removal. In intact cats in vivo, the activity of raphé neurons is profoundly affected by sleep-wake states, discharging maximally during wakefulness and active during slow-wave sleep but quiescent during rapid-eye-movement sleep (25). We therefore suggest the interesting possibility that alteration of NRo activity in the slice might mimic the effects of sleep-wake state on XII motoneurons.

The pH/CO2 Response of Transverse Brain Stem Slices

Preliminary studies report that transverse brain stem slices from neonatal rats responded to decreased pH by increasing the bursting frequency of XII nerve discharge, and this response is mediated by pH-sensitive pacemaker cells within the PBC (27). However, Peever et al. (47) reported that XII motor output was not affected by altered pH levels. The difference between studies was likely due to either the extent of the pH change (7.4 to 7.0-6.5 vs. 7.4 to 7.1) or the thickness of the slices (300-400 µm vs. 400-700 µm). Presently, we report that slices made in our laboratory do respond to decreased pH by increasing the XII nerve bursting frequency, but only at pH = 7.0, thereby confirming both previously published results. We conclude that, although transverse brain stem slice preparations contain sufficient and appropriate neural circuitry to detect and respond to changes in tissue pH, there is a threshold for this effect just above pH = 7.0.

Transverse brain stem slices respond differently to decreases in pH than do in vivo adult and neonatal rats exposed to hypercapnia. In decerebrate, unanesthetized, ventilated, and vagotomized rats 0-10 days old, inhalation of 3 and 5% CO2 in O2 caused an increase in the amplitude of phrenic nerve discharge, with no changes in bursting frequency or duration (58). These different responses could result not only from different nerves being recorded (phrenic vs. XII nerves), but also from the obvious differences between preparations, such as the reduction in the brain stem regions preserved in the slice and the elimination of afferent inputs as discussed previously.

The pH/CO2 Sensitivity of NRo and PBC

Although the transverse brain stem slice responds to pH changes, the precise cellular mechanisms and location of pH detection are unresolved. In rats, both in vitro and in vivo experiments demonstrate that different brain stem regions respond to local changes in pH. These include the ventral respiratory group, a region approximately analogous to the PBC in the transverse brain stem slice (24, 27, 39, 50), and the brain stem raphé nuclei (6, 11, 57). Here, we demonstrate that both the NRo and PBC in transverse brain stem slices respond to focal CO2 acidification by augmenting XII motor output but in different ways: bursting frequency alone for the PBC, and bursting frequency, duration, and amplitude for the NRo. These different responses might be expected because of the projections of the NRo to both XII motoneurons and PBC neurons (1, 9, 18, 21, 22), so that NRo activation should affect bursting amplitude and duration via excitation of XII motoneurons and bursting frequency via excitation of PBC neurons.

Indeed, focal CO2 acidification of the NRo significantly increased both bursting frequency and integrated burst amplitude. However, CO2 acidification of the whole slice failed to cause a significant effect on integrated burst amplitude, despite increasing bursting frequency as expected. Perhaps the effects of focal CO2 acidification of the PBC, which tended to decrease integrated burst amplitude, nullified the effects of NRo on integrated burst amplitude.

Previous work has shown that, in both in vitro neonatal rat brain stem-spinal cord and transverse brain stem slice preparations, as well as in cell cultures, neurons of the PBC region and NRo respond to changes in pH in the bathing medium by altering their discharge frequency (27, 28, 42, 50, 56). In the latter case, however, Wang and Richerson (57) concluded that raphé neurons do not become pH/CO2 sensitive until ~12 days of age. Because our slices were obtained from younger neonates, this finding contradicts ours. Although individual NRo neurons undoubtedly alter discharge rates in response to minor pH fluctuations (7.4 to 7.2) (57), we demonstrate that such changes do not affect XII discharge until pH <=  7.0. Nevertheless, we conclude that cells within the NRo and PBC are pH/CO2 sensitive in 1- to 4-day-old brain stem slice preparations.

With respect to other regions of the brain stem thought to be pH/CO2 sensitive, we found that the focal CO2 acidification of the control region did not alter XII motor output. In adult rats, this region in the lateral tegmental field approximates the dorsal respiratory group (19). Because the dorsal respiratory group in adult rats contains respiratory neurons that when acidified provoke an increase in phrenic nerve activity (8), we might expect it to do so in our preparation. However, these neurons may not be present in the neonate; there is an absence of respiratory-related neuronal activity in this region in neonatal rats (19). Furthermore, the pathways mediating such a response may be disrupted in our slice preparation, or else the target motor outputs may not include the XII motoneurons. The latter suggestion is supported by the observation that electrical stimulation of pontine raphé nuclei in brain stem-spinal cord in vitro preparations augments the respiratory motor output of phrenic nerves but suppresses it in XII nerves (38).

Conclusions

We demonstrate for the first time that the NRo is required for expression of respiratory rhythm by XII motoneurons in transverse brain stem slice in vitro preparations. On the basis of this finding, we deduce that the NRo is part of the neural circuitry required for the expression of respiratory rhythm, because it provides an essential tonic excitatory drive to XII motoneurons.

In addition, we demonstrate not only that transverse brain stem slices respond to global tissue pH/CO2 changes, but also that the NRo and PBC contain pH/CO2-sensitive cells, which when stimulated modify respiratory rhythm but in different ways. Furthermore, we show that respiratory rhythm is not altered in response to focal pH/CO2 changes applied to a region in the lateral tegmental field, approximately equidistant to the PBC, NRo, and XII motor nuclei. Thus CO2 acidification of different regions produces different effects on XII discharge. We conclude that this preparation represents a model system for studying the fundamental mechanisms by which the central respiratory network detects and responds to alterations in pH/CO2.


    ACKNOWLEDGEMENTS

We thank the members of the Respiratory Research Group in the Department of Physiology at the University of Toronto for helpful comments.


    FOOTNOTES

We are grateful for the funding provided by the Medical Research Council of Canada and for the salary support provided to J. H. Peever.

Address for reprint requests and other correspondence: J. Duffin, Respiratory Neuroscience Laboratory, Depts. of Physiology and Anaesthesia, Univ. of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8 (E-mail: j.duffin{at}utoronto.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 June 2000; accepted in final form 10 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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