A. Ruangkittisakul and L. Secchia contributed equally to this work.
Corresponding author K. Ballanyi: Department of Physiology and Perinatal Research Centre, 220 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. Email: email@example.com
The pre-Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstem–spinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K+] and [Ca2+] (both of which determine neuronal excitability) is not clear. We report here that inspiratory-related rhythms in newborn rat slices and brainstem–spinal cords with defined boundaries were basically similar in physiological Ca2+ (1.2 mm) and K+ (3 mm). Hypoglossal nerve rhythm was 1 : 1-coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC-related slice rhythms were inhibited in 1.5 mm Ca2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mm Ca2+. Ca2+ depression of slice rhythms was antagonized by raising superfusate K+ to 8–10 mm. This strong extracellular Ca2+/K+ antagonism of inspiratory (motor) rhythms was also revealed in brainstem–spinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K+, respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscles
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The neural control of breathing is studied in reduced preparations such as isolated newborn rat brainstem–spinal cords (Smith et al. 1990; Ballanyi et al. 1999). Microsectioning of this in vitro model showed that neurons of the pre-Bötzinger Complex (preBötC) inspiratory centre remain active in a transverse brainstem slice (Smith et al. 1991). In both, slice and en bloc models, rhythmogenic preBötC networks and preBötC-driven motor circuits are studied under quite divergent experimental conditions. preBötC slice thickness ranges from 200 to 1000 μm, and rostrocaudal boundaries of slices and brainstem–spinal cords vary notably (Rekling et al. 1996; Ballanyi et al. 1999; Koshiya & Smith, 1999; Ruangkittisakul et al. 2006). This may affect fictive inspiratory rhythms as the preBötC is neighboured by other ventral respiratory column structures such as the more rostral Bötzinger complex (BötC) and the parafacial respiratory group (pFRG), which is presumably closely associated with the retrotrapezoid nucleus (RTN) (Monnier et al. 2003; Feldman & Del Negro, 2006). While differences in the content of ventral respiratory column structures may modulate preBötC rhythms specifically, unspecific modulation may arise from interstitial accumulation or, conversely, washout of neuromodulator(s), which both depend on physical dimensions of isolated preparations (Ballanyi, 1999; Ruangkittisakul et al. 2006). preBötC activity may also be affected by superfusate constituents such as Ca2+ and K+ that modulate excitability in other neuronal systems (Hille, 2001; Somjen, 2002). The most common values of interstitial [Ca2+] and [K+] in diverse mammalian brain regions in vivo are 1.2 mm and 3 mm, respectively (Somjen, 2002). This contrasts with a wide range of superfusate Ca2+ (0.8–2.4 mm) and K+ (3–11 mm) in studies on isolated respiratory networks (Suzue, 1984; Smith et al. 1991; Johnson et al. 1996; Rekling et al. 1996; Ruangkittisakul et al. 2006).
Differences in dimensions of preparations and/or superfusate composition may be responsible for discrepant findings, e.g. regarding the capability of the isolated preBötC to generate rhythm. The above transection study (Smith et al. 1991) showed that the rate of inspiratory-related cervical nerve bursting is not affected by transection between preBötC and facial motonucleus (VII) in 1.5 mm Ca2+ solution. Other studies on this model showed that such transection depressed inspiratory burst rate in 2 mm Ca2+ (McLean & Remmers, 1994), whereas cervical rhythm was abolished following transection in ‘Suzue-type’ solution with 2.4 mm Ca2+ (Onimaru & Homma, 1987). We hypothesize that effects of brainstem transection on inspiratory-related rhythms depend critically on superfusate Ca2+.
The constancy of extensions of respiratory brainstem marker nuclei in early postnatal rats enables ‘online’ histology for generation of preBötC slices with defined boundaries that are capable of stable rhythm in 3 mm K+ (Ruangkittisakul et al. 2006). Our finding here that cranial nerves and blood vessels are ventral brainstem surface landmarks with a constant anatomical relation to the marker nuclei allowed generation of brainstem–spinal cords with defined rostral boundary. As one major aim of our study, we quantified in these en bloc preparations in physiological (1.2/3 mm) Ca2+/K+ the relation of their boundaries to the rate and duration of inspiratory cervical/hypoglossal nerve bursts and longevity of these rhythms, and compared the findings with those in the above slices. We then studied the Ca2+ dependence of preBötC (related) rhythms in both models. Finally, we elucidated the dependence on the rostral brainstem boundary of pre/postinspiratory lumbar nerve activity driving expiratory muscles (Janczewski et al. 2002; Janczewski & Feldman, 2006).
We found that preBötC (related) rhythms in 1.2 mm Ca2+ and 3 mm K+ were basically similar in slices and en bloc preparations. Hypoglossal rhythm was stable and 1 : 1-coupled to preBötC population activity in slices and to cervical bursting in brainstems without VII. In contrast, hypoglossal rhythm was depressed in en bloc preparations with more rostral boundaries, while pre/postinspiratory lumbar nerve bursting was only present in brainstems with a major portion of VII. Most importantly, preBötC bursting and associated motor rhythms in slices and en bloc medullas without VII were greatly depressed upon Ca2+ elevation by only 0.5 and 0.6 mm, respectively, and were reactivated by 8–10 mm K+. Conversely, in 1 mm instead of 1.2 mm Ca2+ solution, slice rhythms had a substantially greater longevity (> 3.5 h vs.∼1.5 h) and higher long-term burst rate. After spontaneous arrest of rhythm in 3 mm K+ and 1–1.2 mm Ca2+ in (slice) preparations with exposed preBötC, similar rhythm was reevoked by 6–7 mm K+ and cAMP elevation by rolipram.
We discuss whether lack of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory (pFRG) interneurons to abdominal expiratory muscles. We hypothesize that depression of hypoglossal rhythm and attenuated Ca2+ sensitivity of preBötC rhythm in less-reduced en bloc medullas is probably not related to increased content of rostral respiratory structures, but rather due to larger dimensions creating interstitial gradients for inhibitory neuromodulator(s) and K+. The strong dependence of inspiratory rhythms on an extracellular Ca2+/K+ antagonism suggests use of 1 mm Ca2+ for analysing isolated preBötC functions in 3 mm K+.
The University of Alberta Animal Care and Ethics Committee approved all procedures and provided governance for the animal care.
Preparations and solutions
Brainstem–spinal cord preparations were generated from 57 Sprague–Dawley (SD) and 54 Wistar (W) rats between postnatal day 0 (P0) and P4. Animals were anaesthetized with 2–3% isoflurane and rapidly decerebrated after the paw withdrawal reflex disappeared. The neuraxis was isolated at 19–22°C in superfusate (for composition, see below) (Brockhaus & Ballanyi, 1998, 2000). The brainstem was transected rostral to the trigeminal nerve (Fig. 1), and the spinal cord cut at the cervical level (C6−8), except for one series of experiments in which the entire spinal cord was isolated for recording from ventral lumbar (L1−2) nerve roots. To obtain preparations with a defined boundary, rostral brainstem tissue including the pons was manually sectioned with a razor blade based on the constancy of ventral surface anatomical landmarks that we have revealed here (Fig. 1; see also online supplemental material Fig. S1). The desired rostrocaudal sectioning level was histologically verified after the electrophysiological experiments (supplemental Figs S1–S3). Preparations were fixed with the ventral side up at the spinal cord to the bottom silicone layer of the superfusion chamber with insect pins (volume 1.5 ml). Further stabilization was achieved by suction electrodes used to record nerve activities (see below). Superfusate was administered at a flow rate of 5 ml min−1 via a peristaltic pump (Watson-Marlow Alitea-AB, Sin-Can, Calgary, Alberta, Canada). Temperature in the recording chamber was kept at 25–27°C (TC-324B, Harvard Apparatus, Saint-Laurent, Quebec, Canada).
preBötC-related rhythms in brainstem–spinal cords in standard solution were compared with those in solution of elevated Ca2+, K+ and Mg2+ (2.4, 6.2 and 1.3 mm, respectively), very similar to that in the first study (Suzue, 1984) and numerous follow-up reports on this and other isolated respiratory networks (e.g. Oshima et al. 2000; Herlenius et al. 2002; Iizuka, 2004; St. John et al. 2005; Kuwana et al. 2006; Onimaru et al. 2006). This ‘Suzue-type’ solution contained (mm): 120 NaCl, 6.2 KCl, 2.4 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 30 d-glucose (pH adjusted to 7.4 by gassing with 95% O2, 5% CO2). In a related series of experiments, Ca2+ and K+ in the standard solution were varied between 1.2 and 6 mm, and 3 and 10 mm, respectively.
preBötC-related rhythms were also studied in 600 μm thick ‘m-preBötC slices’ with the preBötC in the middle (Ruangkittisakul et al. 2006). After removal of the cerebellum and sectioning close to the caudal cerebellar artery (De Araujo & Campos, 2005) (Fig. 1), the brainstem of P0–4 SD (n= 8) or W rats (n= 28) was glued rostral side down to a metal plate and transferred to a vibrating microtome (Leica VT1000S, Leica Microsystems, Richmond Hill, Ontario, Canada). Serial transverse sectioning (100 μm) was stopped, and one ‘rhythmic’ slice was cut, when specific landmarks were reached, in particular subregions of the inferior olive (Fig. 1 and supplemental Fig. S1). Slices were fixed caudal side up with insect pins in the recording chamber and superfused with standard saline in which Ca2+ and K+ were varied between 1 and 1.8 mm and 3 and 10 mm, respectively, at either 1 or 2 mm Mg2+ (Ruangkittisakul et al. 2006).
Discharge of preBötC-driven motoneurons providing inspiratory motor output in brainstem–spinal cord preparations (Smith et al. 1990; Ballanyi et al. 1999) was recorded from ventral spinal (C3–4) and cranial (hypoglossal) nerve roots with suction electrodes (outer diameter 80–250 μm) filled with standard superfusate. In brainstem preparations with a complete spinal cord, activity was recorded simultaneously from C3–4 and ventral L1−2 roots, the latter containing axons of pre/postinspiratory active motoneurons that appear to be driven by the pFRG and innervate expiratory abdominal muscles (Janczewski et al. 2002; Iizuka, 2004). Neuronal population activity was recorded with a differential amplifier (×10 000, DAM 50, World Precision Instruments, Sarasota, FL, USA), bandpass-filtered (0.3–3 kHz) and integrated. In the slices, suction electrode recording was done at the caudal surface from the ventral respiratory column containing the preBötC (Alheid et al. 2004), in most cases combined with recording from one hypoglossal nerve root. Signals were fed into a computer (sampling rate 1 kHz) via a digital recording system (Powerlab/8SP, ADInstruments, Colorado Springs, CO, USA).
Respiratory-related activity of brainstem–spinal cord preparations and slices was continuously recorded and quantified by measuring every 20 min over 2 min time periods the frequency of rhythm and single burst duration. Longevity of rhythms was defined as the time from start of recording until the time when the interval between consecutive bursts exceeded 1 min. Burst duration was defined, using Clampfit software (Molecular Devices, Union City, CA, USA), as the time interval from when the signal increased above and decreased below a threshold set at 10% of the peak amplitude value for that burst. Rhythms modified by changing superfusate cation composition or adding rolipram were analysed over a time period of 2 min at steady-state. Values are reported as means ±s.e.m. except for histological analyses where means ±s.d. were determined. Significance of values was determined by Student's one-sample t test using SigmaPlot (Systat software, Point Richmond, CA, USA). Significance was defined as *P < 0.05 and **P < 0.01.
For histological analysis after the experiments, both aspects of transected brainstem–spinal cords were fixed in phosphate buffer (1 : 2 mixture of 0.1 m NaH2PO4+ 0.1 m Na2HPO4 in H2O, pH 7.2) containing 4% paraformaldehyde (all agents from Sigma). For staining after > 15 min of fixation, preparations were incubated in phosphate buffer for at least 2 min and subsequently immersed 90 s for en bloc brainstems and slices and 45 s for 200–250 μm thick sagittal brainstem–pons slices in a solution consisting of 1% thionin acetate (Sigma) in a mixture of 0.1 m sodium acetate trihydrate and 0.1 m acetic acid (Fisher). After staining, preparations were first ‘washed’ with phosphate buffer (2 min) and then with 50% ethanol (4 min) before returning to phosphate buffer for 2 min. Subsequently, preparations were transferred to a Petri dish and photographed (PL-A686 6.6 megapixel camera, Capture-SE software, PixeLINK, Ottawa, Ontario, Canada) in phosphate buffer under a stereo microscope (Zeiss-SR15, magnification ×32; Carl Zeiss, Jena, Germany).
Defined sectioning of brainstem–spinal cords
Brainstem–spinal cords with defined rostral boundary were generated using ventral brainstem surface landmarks (Fig. 1 and supplemental Fig. S1). Specifically, we found that the vagal nerve is located 0.06 ± 0.05 mm (n= 5) caudal to the caudal end of VII (VIIc) used as reference, whereas the location of the caudal cerebellar artery matches well with the rostral border of VII, i.e. 0.74 ± 0.16 mm (n= 14) vs. 0.76 ± 0.07 mm (n= 16) rostral to VIIc, respectively. The area covered by the most rostral hypoglossal nerve root spans the proposed (Smith et al. 1991; Ruangkittisakul et al. 2006) extension of the preBötC between 0.4 and 0.6 mm caudal to VIIc, with its rostral end located 0.41 ± 0.13 mm (n= 5) caudal to VIIc (Fig. 1 and supplemental Fig. S1).
preBötC slice boundaries can be determined ‘online’ by comparing respiratory marker nuclei in (pre)rhythmic slices with those in a reference brainstem atlas (Ruangkittisakul et al. 2006). The rostrocaudal locations of the marker structures, and thus slice boundaries, are referred to their distance from VIIc. Here, we used the atlas to identify the rostral boundary of transected brainstem–spinal cords (Fig. 1 and supplemental Figs S1–S3). This method was not suited to determine the boundary of preparations transected between VIIc and the inferior olive due to lack of marker structures. In these cases, the boundary was determined by measuring the distance in sagittal slices between VIIc and the caudal surface of the rostral aspects of the sectioned preparations (supplemental Figs S2 and S3). Analysis of the sagittal sections revealed a distance between inferior olive and VIIc of 0.16 ± 0.03 mm (n= 5), similar to our previous value (0.13 ± 0.03 mm, n= 26) based on 50 μm transverse serial sections (Ruangkittisakul et al. 2006).
Ventral respiratory column structures in newborn rat brainstem–spinal cords
As one major aim, we investigated the dependence of fictive respiratory rhythms in brainstem–spinal cords on their rostral boundary. We restricted our analysis to preparations without pons as influences of this structure on respiratory activity in this model were previously studied (Monteau et al. 1989; Errchidi et al. 1991; see also Alheid et al. 2004).
First, we studied inspiratory cervical nerve rhythms in three groups of brainstem–spinal cords with the boundary (i) slightly rostral to VII (thus including the BötC and RTN/pFRG), (ii) between VII and preBötC, and (iii) at or into the rostral preBötC border (Fig. 1).
Inspiratory cervical rhythm in transected brainstem–spinal cords
The mean boundary of six preparations with VII was 0.83 ± 0.08 mm rostral to VIIc (Fig. 2). In standard 1.2 mm Ca2+, 3 mm K+ solution, these brainstems generated cervical rhythm with an initial frequency of 6.9 ± 0.8 bursts min−1 and a burst duration of 0.60 ± 0.05 s (Fig. 2). After 3 h of recording, burst rate did not change (7.1 ± 0.35 bursts min−1), whereas burst duration decreased to 0.40 ± 0.02 s. Raising superfusate K+ from 3 to 6.2 mm (as in ‘Suzue-type’ solution) after the 5 h recording period reactivated rhythm, which stopped after 298 min in one preparation lacking ∼10% of VII, and increased mean burst rate above control (8.7 ± 1.2 bursts min−1, n= 6) with no stimulating effect on burst duration (Fig. 2).
The mean boundary of seven medullas cut between VII and preBötC was 0.25 ± 0.04 mm caudal to VIIc (Fig. 3). The initial burst rate of 9.4 ± 0.7 bursts min−1 decreased to 8.1 ± 0.6 bursts min−1 within 20 min and remained rather stable for > 2 h in five cases. Burst duration decreased from initially 0.47 ± 0.02 s to 0.24 ± 0.06 s within 2 h, but remained stable thereafter (Fig. 3). In three preparations, rhythm stopped after 2–3 h, whereas four preparations showed rhythm for > 4 h (mean longevity 236 ± 26 min, n= 7). 6.2 mm K+ and the cAMP-elevating agent rolipram (1 μm) reactivated rhythms with rates (6.7 ± 0.6 vs. 6.9 ± 0.9 bursts min−1) and durations (0.45 ± 0.04 vs. 0.36 ± 0.08 s) very similar to control (Fig. 3).
The mean boundary of six preparations with rostrally exposed preBötC was 0.39 ± 0.04 mm caudal to VIIc (Fig. 4). Initial burst rate was 9.3 ± 1.7 bursts min−1, but decreased monotonically until rhythm stopped spontaneously (mean longevity 71.5 ± 14.1 min, n= 6). In three preparations still active after 80 min of recording, burst rate at that time was 3.3 ± 0.3 bursts min−1 and burst duration was 0.17 ± 0 s (Fig. 4). K+ at 6.2 mm and rolipram (1 μm) reactivated rhythms with rates slightly lower than (7.5 ± 1.2 vs. 8.3 ± 0.5 bursts min−1) and durations similar to (0.32 ± 0.07 vs. 0.30 ± 0.08 s) the initial value (Fig. 4).
Four preparations with the rostral boundary ≥ 0.45 mm caudal to VIIc, and thus likely to be very close to the preBötC centre (Fig. 1), did not generate respiratory rhythm in either 3 mm or 6.2 mm K+. In three of these cases, elevated K+ induced < 0.3 s irregular cervical nerve bursts with > 15 events min−1 and a distinct activity consisting of a > 1 min massive discharge (supplemental Fig. S4).
Analysis of pooled data from the above groups of brainstem–spinal cord preparations revealed a significant correlation between rostral boundary and longevity of rhythm, but not between boundary and burst rate, or initial frequency and longevity (Fig. 5).
Pre/postinspiratory lumbar rhythm in transected brainstem–spinal cords
In a further approach, we studied the dependence of pre/postinspiratory lumbar nerve bursting (Janczewski et al. 2002) on the rostral boundary of en bloc brainstems with complete spinal cord. Ten preparations with boundaries < 0.6 mm rostral to VIIc, thus containing < 79% of VII, did not show pre/postinspiratory lumbar bursting, but generated regular cervical inspiratory rhythm (Fig. 6). On the contrary, seven preparations with boundaries > 0.6 mm rostral to VIIc showed both pre/postinspiratory lumbar and inspiratory cervical nerve bursting (Fig. 6).
Hypoglossal nerve activity in transected brainstem–spinal cords
Next, we assessed whether inspiratory hypoglossal rhythms in en bloc preparations also depend on the transection level. In five preparations cut between preBötC and VIIc (mean boundary 0.20 ± 0.04 mm caudal to VIIc), burst rates in 3 mm K+ of synchronous rhythms in cervical and most rostral hypoglossal nerve roots were identical. After an initial time period of 8.7 ± 1.1 bursts min−1, burst rate stabilized at 7.1 and 7.7 bursts min−1 between 20 and 120 min, but decreased to 4.8 ± 1.0 and 4.8 ± 1.3 bursts min−1 at 180 and 240 min, respectively (Fig. 7). The amplitude of rostral hypoglossal root bursts was stable during recording periods > 4 h in some cases (Figs 7 and 8). Also their duration remained stable at ∼0.4 s, whereas cervical burst duration decreased from ∼0.4 s to < 0.3 s after 1 h of recording (Fig. 7). Hypoglossal burst duration was also similar to control when rhythm was reactivated after spontaneous arrest by elevated K+ (6.2, 7, 9 mm) or rolipram (1 μm), all of which restored cervical burst duration to control values (Fig. 7). Only 9 mm K+ increased the burst rate of re-evoked rhythms above initial control values. Under all these conditions, hypoglossal and cervical rhythms showed 1 : 1-coupling (Fig. 7). Neither K+ nor rolipram affected the amplitude of hypoglossal or cervical bursting. In 12 additional preparations with similar boundaries caudal to VIIc, the amplitude of bursting of rostral hypoglossal roots remained constant or increased during the first hour of recording (Fig. 8).
In contrast, burst amplitude, but not duration, decreased within 20–90 min after start of recording in caudal hypoglossal roots of brainstem–spinal cords without VII (Fig. 8). Bursting disappeared in 1 of 4 preparations and stabilized after ∼1 h in the other cases at 17 ± 6.9% of the initial value. In four preparations with VII (mean boundary 0.11 ± 0.07 mm rostral to VIIc), amplitude of rostral hypoglossal root bursts fell to 23 ± 4.7% of initial values after 20–50 min of recording. Furthermore, the depression was more pronounced (6.8 ± 2.8%) after similar time periods in four preparations with complete VII (Fig. 8).
Rhythm in preBötC slices
Next, we compared inspiratory-related rhythms in 1.2 mm Ca2+ and 3 mm K+ between en bloc medullas and 600 μm m-preBötC slices with a mean rostral boundary 0.24 ± 0.11 mm (n= 5) caudal to VIIc. The initial rate of bursting in the region of the ventral respiratory column containing the preBötC was 7.2 ± 1.0 bursts min−1, but decreased within 20 min to 5.2 ± 0.8 bursts min−1. Burst rate remained approximately at that value until rhythm stopped after 60–140 min (mean longevity 87.8 ± 16.8 min, n= 5) (Fig. 9). preBötC burst duration fluctuated between 0.5 and 0.8 s within the first 80 min of recording. As with cervical bursts in en bloc medullas, the rate of hypoglossal bursts was 1 : 1-coupled to preBötC activity, while their duration was shorter and decreased from an initial value of 0.39 ± 0.07 to 0.30 ± 0.14 after 1 h (Fig. 9). After spontaneous arrest, preBötC and hypoglossal rhythms were restored by 6.2 mm K+ or rolipram (1 μm) with rates comparable to those in 3 mm K+ 20–40 min after start of recording. While the duration of K+-induced preBötC and hypoglossal bursts was also similar to control, rolipram-induced bursts were shorter (Fig. 9).
The limited longevity of slice rhythms in standard 1.2 mm Ca2+ and 3 mm K+ solution contrasted with ∼4 h longevity of rhythm in m-preBötC slices with similar boundaries in our previous study (Ruangkittisakul et al. 2006). Thus, we tested next whether this difference may be due to use of 1 mm instead of 1.2 mm Ca2+ in that report. Indeed, rhythm in five slices with the rostral boundary 0.21 ± 0.08 mm caudal to VIIc lasted > 2 h and even > 3 h in three of these cases in 1 mm Ca2+. Thus, longevity was significantly greater (211.4 ± 39.2 min, P < 0.05) in 1 mm Ca2+ compared to slices in 1.2 mm Ca2+ (Fig. 10). Also in 1 mm Ca2+, hypoglossal bursts showed 1 : 1-coupling to preBötC bursting, but their amplitude fluctuated by ∼40% with a period of 20–40 min in two slices. Rhythm was effectively restored after spontaneous arrest by both K+ (6–9 mm) and rolipram (1 μm) (Fig. 10).
Next, we studied whether slice rhythms are modified by raising Mg2+ from 1 to 2 mm as was used by Ruangkittisakul et al. (2006). In four slices with mean rostral boundary 0.10 ± 0.07 mm caudal to VIIc, longevity of rhythm (216 ± 37.1 min, n= 4) was very similar in 1 mm Ca2+, 2 mm Mg2+ to that in 1 mm Ca2+, 1 mm Mg2+ slices and significantly (P < 0.05) greater than in 1.2 mm Ca2+, 1 mm Mg2+ slices. Also two of these slices showed fluctuations in hypoglossal burst amplitude (Fig. 10). In three slices, synchronous preBötC and hypoglossal rhythms were stable for > 3 h, in two cases for > 4 h (Figs 10 and 11). Burst rates during these time periods ranged from 7 to 9.4 bursts min−1, which was higher than in 1.2 mm Ca2+ (after 40–60 min). The duration of preBötC and hypoglossal bursts decreased from initially 0.75 ± 0.06 s to 0.49 ± 0.03 s and 0.45 ± 0.06 s to 0.21 ± 0.08 s after 3 h, respectively (Fig. 11). After spontaneous arrest, rhythm of similar burst duration was restored by K+ (6.2, 7, 9 mm) or rolipram (1 μm). K+ at 6.2 mm and 7 mm as well as rolipram restored bursting at rates similar to control, whereas 9 mm K+ induced a faster rhythm at ∼15 bursts min−1 (Fig. 11).
Ca2+ sensitivity of inspiratory rhythms in transected brainstem–spinal cords
As one major finding, inspiratory-related rhythms in en bloc medullas with distinct rostral boundaries shared various features with those in preBötC slices. These features included a similar limited longevity of rhythms in brainstem–spinal cords with exposed preBötC and m-preBötC slices. In the slices, longevity and burst rate were greatly enhanced by lowering superfusate Ca2+ from 1.2 mm to 1 mm. This suggests that, conversely, raising Ca2+ above 1.2 mm may depress preBötC-related (motor) rhythms. In the Introduction, we hypothesized that raised Ca2+ may be responsible for block of cervical rhythms in transected brainstem–spinal cords. The studies reporting such block (Onimaru & Homma, 1987; Onimaru et al. 2006) used ‘Suzue-type’ solution containing 2.4 mm Ca2+ and also higher K+ (6.2 mm) and Mg2+ (1.3 mm) compared to the standard solution used here.
We tested the effects of this solution on five brainstem–spinal cord preparations that were transected between preBötC and VII. Raising first K+ from 3 to 6.2 mm changed neither the rate nor the duration of cervical bursts, whereas subsequent elevation of Ca2+ (from 1.2 to 2.4 mm) and Mg2+ (from 1 to 1.3 mm) blocked rhythm within < 10 min (Fig. 12). Rhythm with a rate and burst duration similar to control was reactivated in that solution by 10 mm K+, whereas 8 mm K+ was less effective (Fig. 12). Six preparations cut more caudally (at the rostral preBötC border) responded similarly. However, 10 mm K+ reactivated rhythm with a burst duration similar to controls, but at a lower rate, and 8 mm K+ did not restore bursting (Fig. 12). This high Ca2+/K+ (Mg2+) solution also modified non-respiratory activity in three preparations with boundaries ≥ 0.45 mm caudal to VIIc that did not generate respiratory rhythm (supplemental Fig. S4).
The fact that Ca2+ was doubled, whereas Mg2+ was raised by only 30%, suggests that Ca2+ is responsible for depression of cervical rhythm. To test this, eight preparations with exposed preBötC were kept in 6.2 mm K+ to maintain stable rhythm and were superfused with 1.8 mm Ca2+ at constant Mg2+ (1 mm). This modest Ca2+ rise by 0.6 mm decreased burst rate within < 10 min from 5.5 ± 0.9 to 1.1 ± 0.4 bursts min−1 (P < 0.01; block of rhythm in 3 cases) (Fig. 13). The inhibition by Ca2+ of cervical rhythm was accompanied in > 50% of cases by non-respiratory bursting that was absent, or less pronounced, in hypoglossal recordings (Fig. 13). Thus, Ca2+ effects were quantified by analysing only synchronous cervical and hypoglossal bursts, which were 1 : 1-coupled during both control and raised Ca2+ (Fig. 13).
In four of the preparations exposed to 1.8 mm Ca2+, a subsequent Ca2+ rise to 2 mm depressed rhythm further to 0.3 ± 0.3 bursts min−1 (P < 0.01; block of rhythm in 3 cases) (Fig. 14). Rhythm reappeared at 4.8 ± 0.5 bursts min−1 with burst duration similar to control in 10 mm K+ after block by 2 mm Ca2+, whereas 8 mm K+ was less effective (2.4 ± 1.1 bursts min−1).
In contrast to preparations transected between VII and preBötC, cervical rhythm in 6.2 mm K+ was not blocked by 1.8–2.4 mm Ca2+ in preparations with (portions of) VII (Figs 13 and 14). The [Ca2+] to effectively depress or abolish rhythm increased significantly with increasing amount of tissue rostral to the preBötC (Fig. 14).
Ca2+ block of preBötC slice rhythm
The latter results in brainstem–spinal cord preparations suggested that the capability of the preBötC to generate rhythm in moderately elevated K+ is substantially impaired by raising Ca2+ by only 0.6 mm. In a final approach, we investigated whether preBötC bursting and inspiratory motor rhythm in slices with robust longevity in 1 mm Ca2+ and 3 mm K+ have a similarly high sensitivity to acutely elevated superfusate Ca2+.
In 10 slices with a mean rostral boundary 0.19 ± 0.10 caudal to VIIc, raising Ca2+ from 1 to 1.5 mm depressed within 10–15 min the rate of 1 : 1-coupled preBötC and hypoglossal bursting from 8.1 ± 0.8 to 1.2 ± 0.4 bursts min−1 (P < 0.01; block of rhythm in 4 cases) (Fig. 15). Both rhythms started to recover within 10–20 min after return to 1 mm Ca2+, but mean recovery was < 50% of control even after 30 min of washout of 1.5 mm Ca2+. The incomplete recovery of burst rates was not caused by time-dependent spontaneous slowing of rhythm in 3 mm K+ as inspiratory frequency was stable during comparable time periods in seven control slices with a mean rostral boundary 0.18 ± 0.11 mm caudal to VIIc (Fig. 15).
For comparison with our findings in brainstem–spinal cords, the effects of 1.8 mm Ca2+ in 6.2 mm K+ were tested in six m-preBötC slices with a mean rostral boundary 0.26 ± 0.12 mm caudal to VIIc. Synchronous preBötC and hypoglossal burst rates were depressed by 1.8 mm Ca2+ from 6.1 ± 0.8 to 0.2 ± 0.2 bursts min−1 (P < 0.01; block of rhythm in 5 cases). Elevating K+ to 8 mm and 10 mm reactivated both types of rhythms at 3.2 ± 0.7 and 10.1 ± 1.9 bursts min−1, respectively, with burst durations similar to control (Fig. 15).
Inspiratory-related rhythms in newborn rat brainstem–spinal cords containing the preBötC inspiratory centre (plus more rostral respiratory regions) and preBötC slices were similar in physiological Ca2+/K+ solution. The reduced longevity of inspiratory rhythms in en bloc preparations with exposed preBötC and slices is caused by a strong extracellular Ca2+/K+ antagonism. We discuss whether the absence of pre/postinspiratory rhythms in brainstems lacking a major portion of VII may be due to reduction of the RTN/pFRG, whereas block of hypoglossal bursting in preparations with (portions of) VII may possibly result from interstitial accumulation of inhibitory neuromodulator(s).
Implications from defined sectioning of isolated brainstem–spinal cords
In early postnatal rats, the constancy of rostrocaudal extensions of marker brainstem nuclei enables the generation of preBötC slices with defined boundaries (Ruangkittisakul et al. 2006). Based on a similar constancy of ventral brainstem surface landmarks, we generated here brainstem–spinal cords with a defined rostral boundary. Such preparations with rostrally exposed preBötC allow the study of how the preBötC drives hypoglossal and spinal motor networks with as little as possible interference from the more rostral BötC and RTN/pFRG. Also preBötC interactions with the RTN/pFRG and/or BötC should be studied in defined preparations where the extension of the rostral ventral respiratory column can be determined within a few tens of micrometres. The finding of stable cervical rhythm in en bloc preparations cut between VII and preBötC is very similar to our previous report (Smith et al. 1991). As discussed below, use of 2–2.4 mm Ca2+ (instead of 1.5 mm Ca2+ in the latter study) explains depression/block of rhythm observed by others upon similar transection (Onimaru & Homma, 1987; McLean & Remmers, 1994; Onimaru et al. 2006).
Despite caveats (Wilson et al. 2006), sectioning experiments can increase understanding of structure–function relationships of respiratory networks. For example, the RTN/pFRG in the rostral aspect of transected newborn rat medullas remained functional without the preBötC (Onimaru et al. 2006). Furthermore, transection close to VIIc abolished expiration in juvenile rats in vivo (Janczewski & Feldman, 2006). Those authors hypothesized that breathing is generated by anatomically separate rhythm generators, one generating active expiration located close to VII in the region of the RTN/pFRG, the other generating inspiration located more caudally in the preBötC. The latter study supported the conclusion from a previous combined in vivo/in vitro report that the pFRG ultimately drives expiratory abdominal muscles in vivo via ipsilateral medullary premotoneurons located caudal to the preBötC that project to contralateral pre/postinspiratory lumbar motoneurons (Janczewski et al. 2002). The findings of Janczewski & Feldman (2006) also support the hypothesis, based on a differential sensitivity in vivo and in vitro of preBötC- and pFRG-related rhythms to opioids, that these rhythmogenic respiratory groups form a functionally coupled dual respiratory centre (Mellen et al. 2003). We found that the brainstem level critical for pre/postinspiratory lumbar bursting is close to the rostral and not caudal end of VII, at least in isolated neonatal rat brainstems. It cannot be concluded though that pFRG networks, which presumably generate this expiratory motor behaviour, are only located rostral to that sectioning level. For example, if the axons of caudal RTN/pFRG interneurons or premotoneurons project rostrally first, cutting would affect this pathway.
Modulation of preBötC rhythms by neighbouring brainstem regions
Burst rates of cervical rhythm in physiological Ca2+/K+ did not substantially differ between en bloc medullas with VII and those cut close to the rostral preBötC boundary. This suggests that rhythmogenic preBötC networks are not subjected to a major specific frequency modulation by rostrally neighbouring portions of the BötC or RTN/pFRG, in line with our findings in en bloc medullas (Smith et al. 1991) and, more recently, in preBötC slices (Ruangkittisakul et al. 2006). In extension of these studies, we show that the longevity of cervical and hypoglossal rhythms is limited to < 1.5 h, when the transection is at, or removes some portion of, the rostral preBötC. As one explanation, long-term preBötC rhythm in en bloc medullas may depend on drive from more rostral respiratory networks (Feldman & Janczewski, 2006; Onimaru & Homma, 2006). However, rhythm in 1.2 mm Ca2+ and 3 mm K+ lasted for considerably shorter time periods in 600 μm m-preBötC slices (∼1.5 h) than in en bloc medullas (∼4 h) with similar rostral boundaries between preBötC and VIIc. This would suggest that caudal structures also drive the preBötC. But, since longevity of rhythms is greater in 600 μm compared to 500 μm m-preBötC slices (Ruangkittisakul et al. 2006), it is more likely that the amount of neighbouring tissue, rather than a specific rostral or caudal (respiratory) structure, determines the strength of drive to the preBötC. In that regard, one of several possible explanations for spontaneous arrest of rhythm in 3 mm K+ is a washout of excitatory neuromodulator(s), which would occur more rapidly in preparations with exposed preBötC (Ruangkittisakul et al. 2006).
Sectioning at the preBötC border removes distal dendrites of preBötC neurons that extend 0.3–0.4 mm rostrocaudally (S. W. Schwarzacher & K. Ballanyi, unpublished observations). Sectioning thus reduces the connectivity and recurrent excitation within and between the bilaterally organized preBötC regions, which is presumably pivotal for rhythm generation at physiological K+ (Ramirez et al. 2002; Del Negro et al. 2005). This would explain why rhythm stops after shorter time periods in highly reduced en bloc preparations. Inspiratory rhythms were reactivated by raised K+ or the blocker of cAMP-specific phosphodiesterase-4 rolipram. The stimulatory action of rolipram supports previous assumptions of the pivotal role of cAMP for maintaining respiratory rhythm (Ballanyi et al. 1997, 1999; Richter et al. 1997; Ruangkittisakul et al. 2006). In addition to the spontaneous arrest of preBötC rhythm in physiological Ca2+/K+ solution, we showed a time-dependent decrease in the duration of preBötC and cervical/hypoglossal nerve bursts. Such ‘rundown’ of burst duration (which was moderate for hypoglossal rhythm in en bloc preparations) was reversed by raised K+ (and in most cases by rolipram) as a further indication of a proposed washout of endo-genous excitatory neurostimulator(s). This phenomenon has to be taken into account for studies analysing neuromodulator effects on the strength of inspiratory activities in slice and en bloc models. Elevating superfusate K+ for a stable burst duration may not be adequate as this would eventually decrease the sensitivity of preBötC networks to neuroactive agents such as opioids (Onimaru et al. 2006; Ruangkittisakul et al. 2006).
Ca2+/K+ antagonism of preBötC rhythms
One major finding of this study explains the limited longevity in physiological Ca2+/K+ of rhythms generated by the preBötC after rostral exposure (brainstem–spinal cords) or isolation (slices). In highly reduced en bloc medullas, raising Ca2+ by only 0.6 mm from 1.2 mm greatly depressed cervical and hypoglossal rhythms. This Ca2+ sensitivity of preBötC-driven motor systems is likely to be even higher as these preparations were kept at 6.2 mm instead of 3 mm K+ for stabilization of rhythm, while further elevation of K+ to 8–10 mm reversed the Ca2+ block. In line with that view, both the longevity and long-term burst rate of preBötC slice rhythms in 3 mm K+ were substantially augmented in 1 mm instead 1.2 mm Ca2+, whereas raising Ca2+ from 1 to 1.5 mm in 3 mm K+ depressed burst rate to < 20% of control. This supports our hypothesis that raised superfusate Ca2+ is responsible for depressed cervical rhythms in transected brainstem–spinal cords (Onimaru & Homma, 1987; McLean & Remmers, 1994; Onimaru et al. 2006). First, our findings suggest that preBötC slices are not active in 2.4 mm Ca2+‘Suzue-type’ superfusate used by numerous groups (see Methods for references). Also, we propose that robust slice rhythm in 3 mm K+ in our recent study (Ruangkittisakul et al. 2006) is, at least partly, related to use of 1 mm superfusate Ca2+. As a third implication, longevity of rhythms in en bloc preparations with exposed preBötC will likely be substantially greater in 1 mm instead of 1.2 mm Ca2+.
The finding that the Ca2+ block of rhythm is also revealed in preBötC recordings in the slices excludes the possibility that raised Ca2+ primarily affects (pre)motor circuits. The Ca2+ block of primary preBötC rhythm can, nevertheless, be indirect as suggested by the finding that block of slice rhythm by bath-application of persistent Na+ channel blockers is mimicked by local injection of the agents into the raphe, but not the preBötC (Pace et al. 2007). Performing a similar test was beyond the scope of this study, as was to elaborate the mechanism of Ca2+ block. As one possibility, Ca2+ may inhibit preBötC cells or tonic neurons driving the preBötC via a more pronounced stimulatory effect on (tonic) inhibitory than on excitatory synapses (Jefferys, 1995). In fact, GABAA and glycine receptors depress inspiratory bursting in newborn rat brainstem–spinal cords (Onimaru et al. 1990; Brockhaus & Ballanyi, 1998, 2000). Ca2+-induced block of rhythm could also be caused by Ca2+ screening of negative membrane surface charges (Hille, 2001; Somjen, 2002). However, if this was the main mechanism, raising Mg2+ from 1 to 2 mm should have a similar blocking effect on slice rhythms, while returning to normal Ca2+ should have reversed the depression. Alternatively, raised Ca2+ may have long-term effects on Ca2+ homeostasis in respiratory (drive) neurons with low Ca2+ buffering capacity (Alheid et al. 2002).
In line with our findings, early in vivo studies showed that Ca2+ injection into the ventriculo-cisternal space depresses breathing (Berndt et al. 1969; Leusen, 1972; Berkenbosch & Adan, 1974). In that regard, a Ca2+/K+ antagonism was proposed to compensate for respiratory depression by Ca2+ when K+ was simultaneously injected (Leusen, 1972). Moreover, our results show that isolated preBötC rhythms at physiological K+ are stimulated by decreasing superfusate Ca2+. We chose 1.2 mm for our standard solution as a major number of in vivo studies reported values of 1.1–1.3 mm for interstitial Ca2+ in diverse brain tissue, including the ventral respiratory column (Heinemann et al. 1977; Nicholson et al. 1978; Richter & Acker, 1989; Trippenbach et al. 1990; Nilsson et al. 1993; Puka-Sundvall et al. 1994; Somjen, 2002). Respiratory rhythm was also revealed in 0.8 mm Ca2+ and 6.2 mm K+ superfusate in ∼1 mm thick mouse brainstem slices (Rekling et al. 1996), while hypothalamic slices generate in vivo-like spontaneous activity in 0.75 mm, but not > 1 mm Ca2+ (Pittman et al. 1981). Accordingly, ‘real’in vivo extracellular Ca2+ levels may be close to, or slightly less than, 1 mm. However, there may be a critical minimal Ca2+ level as ≤ 0.5 mm superfusate Ca2+ is a common epilepsy model (Konnerth et al. 1986; Jefferys, 1995). We propose that 1 mm Ca2+ is best suited to study isolated preBötC functions in 3 mm K+.
Relation of hypoglossal bursting with cervical and preBötC rhythms
Simultaneous recordings revealed 1 : 1-coupling of hypoglossal bursting with either cervical nerve activity (in en bloc medullas) or preBötC population activity (in slices). This argues against concerns that hypoglossal recordings may not necessarily be indicative of respiratory activity in vitro, in particular at hypothermia (St-John et al. 2004). However, the amplitude of hypoglossal bursts fluctuated periodically in 3 mm K+, 1 mm Ca2+ and 1–2 mm Mg2+ in some slices. Elevating K+ to 6.2–9 mm stabilized, and sometimes increased, the amplitude of hypoglossal (and preBötC) bursts and restored their durations to, or even beyond, the values during the initial phase of recordings. In contrast to a stable hypoglossal rhythm in the slices and (in rostral roots of) en bloc preparations transected close to or into the preBötC, rhythm was greatly depressed in all hypoglossal roots within ∼1 h in preparations containing (portions of) VII (for reasons, see below). This finding is important for studies devoted to the analysis of the function of hypoglossal neurons in en bloc medullas. For example, it was proposed that serotonin depresses hypoglossal, but not phrenic, motor output in brainstem–spinal cords (Monteau et al. 1990). In such studies, it is pivotal to test first for stable control hypoglossal nerve amplitudes.
Influence of restricted diffusion on respiratory activities in en bloc medullas
Notably higher superfusate Ca2+ levels were necessary to block rhythm in less-reduced en bloc preparations, e.g. ≥ 3.6 mm in brainstems with complete VII. This is in line with the observation that 4 mm Ca2+ depressed cervical bursting in such preparations (Kuwana et al. 1998). As one explanation for this phenomenon, (respiratory) structures in the rostral medulla may be less sensitive to Ca2+, thus being capable of driving the preBötC in high Ca2+. Alternatively, the decreased Ca2+ sensitivity of inspiratory rhythms may result from the larger dimension of en bloc preparations, constituting a diffusion barrier for build-up, e.g. of a modest interstitial K+ gradient (and a pH gradient) by an anoxic core that does not, however, appear to contain pivotal O2- or pH-sensitive inspiratory structures (Ballanyi et al. 1992, 1999; Brockhaus et al. 1993; Ballanyi, 2004). It may, though, be argued that the pre/postinspiratory activity pattern of newborn rat pFRG neurons in vitro results from hypoxia/anoxia in en bloc preparations with more rostral tissue as evoked hypoxia activated preinspiratory bursting in postinspiratory ventral respiratory column neurons of adult rats (Schwarzacher et al. 1991). It may further be speculated that transection 0.6 mm or less rostral to VIIc reduces the anoxic core, inactivates these neurons and, consequently, silences lumbar pre/postinspiratory bursting. However, several findings argue against this hypothetical scenario. First, preinspiratory activity was also induced in postinspiratory neurons of the latter in vivo study by prolonged lung inflation or deflation during normoxia. Second, pre/postinspiratory abdominal muscle activity is seen in normoxic juvenile rats in vivo (Janczewski & Feldman, 2006). Furthermore, experimentally induced anoxia blocks the preinspiratory and greatly augments the postinspiratory component of pFRG-related activities (Ballanyi, 2004), opposite to the above in vivo findings by Schwarzacher et al. (1991). Finally, rostral pFRG neurons are located within < 300 μm from the ventral brainstem surface (Onimaru & Homma, 2003), whereas the anoxic core is restricted to tissue depths > 700 μm (Brockhaus et al. 1993).
In preparations with a major amount of rostral tissue, extracellular [K+] is several millimolar higher in the vicinity of preBötC neurons than in the superfusate, due to the modest K+ gradient (Brockhaus et al. 1993; Okada et al. 2005). These elevated interstitial K+ levels may antagonize the depressing action of elevated superfusate Ca2+ as suggested by the finding that preBötC rhythm persisted in ‘Suzue-type’ solution after transection of brainstem–spinal cords between the preBötC and VII, but was abolished upon subsequent removal of the transected rostral aspect of the preparation (Onimaru et al. 2006). Consequently, lower K+ (e.g. 2 mm), may be used instead of 3 mm in en bloc preparations with more rostral tissue to compensate for changes in the interstitial Ca2+/K+ ratio. Hampered diffusion of raised Ca2+ into the tissue could also explain the attenuated blocking effect. However, this is not likely as millimolar superfusate Ca2+ concentrations equilibrate within minutes in this en bloc preparation, which does not show an interstitial Ca2+ gradient in control (Völker et al. 1995; Ballanyi et al. 1996; Kuwana et al. 1998). Also the longevity of hypoglossal rhythms depended on the rostrocaudal extension of en bloc medullas. We hypothesize that arrest of hypoglossal bursting in preparations with VII is caused by accumulation of inhibitory neuromodulator(s) and/or metabolite(s) depressing hypoglossal (pre)motoneurons, but not preBötC interneurons and cervical premotoneurons.
The isolated inspiratory centre generates robust rhythm in physiological cation solution. preBötC rhythms are blocked by minor extracellular Ca2+ rises, but can be restored by additional notable elevation of extracellular K+. The dependence of inspiratory-related rhythms on this strong Ca2+/K+ antagonism decreases in en bloc preparations with increasing amount of tissue rostral to the preBötC. Based on these findings, we recommend the use of superfusate Ca2+ levels at the lower end of the physiological spectrum, i.e. 1 mm, for the study of functions of the isolated preBötC in physiological K+. In addition, one may consider the use of reduced superfusate K+ (e.g. 2 mm) for studies of respiratory rhythms in en bloc preparations with extended rostral tissue. It should be considered further that hypoglossal rhythm is blocked in such less-reduced brainstem–spinal cords, whereas pre/postinspiratory lumbar bursting is absent in more reduced en bloc medullas. Our findings suggest that discrepant results using en bloc preparations with varying rostral boundaries may be partly explained by different dimensions, affecting diffusion of endogenous neuromodulators, rather than changes in specific structures present in more rostral respiratory regions. We hypothesize that the preBötC is more sensitive in such solutions to CO2/H+, hypoxia/anoxia or neuromodulators such as (endogenous) opiates or substance P, all of which determine respiratory rhythm in vivo.
This work was supported by the Canadian Institutes of Health Research (CIHR), the Canadian Foundation for Innovation (CFI) and the Alberta Heritage Foundation for Medical Research (AHFMR). K.B. is an AHFMR Scientist. A.R. has been awarded a CIHR studentship (MFN training grant). We thank Dr G. F. Alheid for critical comments on the histological sections of the manuscript.