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Published in final edited form as: Respir Physiol Neurobiol. 2011 Oct 12;180(1):1–7. doi: 10.1016/j.resp.2011.10.002

Isolated in vitro brainstem-spinal cord preparations remain important tools in respiratory neurobiology

Stephen M Johnson 1, Sara M Turner 1, Adrianne G Huxtable 1, Faiza Ben-Mabrouk 1
PMCID: PMC3242829  NIHMSID: NIHMS331329  PMID: 22015642

Abstract

Isolated in vitro brainstem-spinal cord preparations are used extensively in respiratory neurobiology because the respiratory network in the pons and medulla is intact, monosynaptic descending inputs to spinal motoneurons can be activated, brainstem and spinal cord tissue can be bathed with different solutions, and the responses of cervical, thoracic, and lumbar spinal motoneurons to experimental perturbations can be compared. The caveats and limitations of in vitro brainstem-spinal cord preparations are well-documented. However, isolated brainstem-spinal cords are still valuable experimental preparations that can be used to study neuronal connectivity within the brainstem, development of motor networks with lethal genetic mutations, deleterious effects of pathological drugs and conditions, respiratory spinal motor plasticity, and interactions with other motor behaviors. Our goal is to show how isolated brainstem-spinal cord preparations still have a lot to offer scientifically and experimentally to address questions within and outside the field of respiratory neurobiology.

1. Strengths and limitations of in vitro brainstem-spinal cord preparations

The isolated in vitro brainstem-spinal cord preparation introduced by Suzue (1984) has been an instrumental part of studies that have made profound contributions to respiratory neurobiology in the past 25 years (>250 papers to date). This preparation, however, is highly criticized because of its experimental limitations. While acknowledging these limitations, the goal of this review is to recognize the importance of this preparation in the history of respiratory neurobiology and focus on its advantages and enduring versatility. We believe that this preparation is still useful, especially when combined with novel experimental techniques to address important scientific questions in respiratory motor control. Due to space limitations, the advantages of brainstem-spinal cord preparations will be discussed and supported by representative papers. A thorough review of all papers on a given topic using brainstem-spinal cord preparations is beyond the scope of this review.

Most isolated in vitro brainstem-spinal cord preparations are dissected from anesthetized pre- and postnatal rats or mice although brainstem-spinal cord preparations can also be isolated from ectothermic adult vertebrates (see Section 9). Brainstem-spinal cord preparations are typically placed in a recording chamber where hyperoxygenated artificial cerebrospinal fluid (aCSF) bathes the tissue at a fixed temperature (26-28°C). Spontaneous respiratory-related motor output is typically recorded from ventral spinal nerve roots on the cervical and thoracic spinal cord, or cranial nerve roots. One caveat is that brainstem-spinal cord preparations are superfused (instead of arterially perfused), which causes large PO2, K+, and H+ gradients to be established within the tissue (especially the brainstem) and in unstirred layers surrounding the tissue (Brockhaus et al., 1993; Okada et al., 1993). The hypoxic, hyperkalemic, acidic core in the brainstem is argued by some to transform the motor output of the respiratory network into gasping or a gasp-like rhythm (St. John, 1996; Wang et al., 1996). There is considerable controversy in the field with respect to whether rhythm produced by these preparations is related to normal breathing (Duffin, 2003; Ramirez and Lieske, 2003; Richter, 2003; St. John and Paton, 2003). Another limitation is that most brainstem-spinal cord preparations from older rodents either do not produce rhythmic respiratory-related motor output or only produce motor output for a short period of time. Postnatal changes in the developing respiratory control network are the main reasons for this age-dependent decrease in motor output, rather than age, mass, or the presence of an intact pons (Fong et al., 2008). Thus, neonatal rodent brainstem-spinal cord preparations are viable during a limited period of time during early development.

Despite these limitations, mammalian brainstem-spinal cord preparations still offer several experimental advantages. A key feature of these preparations (and other reduced in vitro preparations) is that spontaneous respiratory-related motor output can be recorded on nerves whose function is known. Respiratory motor output on cervical and thoracic spinal nerves pumps air in and out of the lungs, whereas respiratory motor output on cranial nerve roots (XII, X) control upper airway patency. Changes in cranial or spinal nerve respiratory motor output are physiologically interpretable and meaningful in terms of ventilation. Specific advantages of brainstem-spinal cord preparations are shown in Fig. 1 and discussed below in the following sections.

Fig. 1. Advantages of brainstem-spinal cord preparations.

Fig. 1

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A drawing of an isolated brainstem-spinal cord preparation illustrates several experimental advantages of this preparation. The circled numbers within the figure schematically highlight the advantages listed in the box below.

ADVANTAGES OF BRAINSTEM-SPINAL CORD PREPARATIONS

1. Central respiratory network in the pons and medulla is intact.

2. Monosynaptic descending bulbospinal connections can be electrically activated.

3. Brainstem and spinal cord can be bathed separately.

4. Phrenic, intercostal, and abdominal motoneuron physiology can be compared.

5. Interaction with other motor behaviors (locomotion) can be studied in vitro.

6. Fictive spinal respiratory motor output is correlated with ventilation.

7. Respiratory spinal plasticity can be studied in vitro.

2. Brainstem-spinal cord preparations reveal connectivity between respiratory-related neurons

One of the most important features of brainstem-spinal cord preparations is that a large portion of the central respiratory network is intact, which allows spatially separate pools of respiratory-related neurons to be revealed along with their connectivity. Interactions between pontine and medullary neurons can be studied in brainstem-spinal cord (and brainstem only) preparations in contrast to other less intact in vitro preparations, such as rhythmically active medullary slice (Smith et al., 1991), “tilted sagittal slab” (Mellen et al., 2003; Barnes et al., 2007), and medullary slice “island” preparations (Johnson et al., 2001).

The brainstem-spinal cord allows the timing of respiratory phases to be precisely determined, at least with respect to inspiration, because the inspiratory phase is defined by inspiratory-related cervical root discharge. Neuronal activity in the medulla can then be compared to cervical root discharge to classify neurons as inspiratory, expiratory, post-inspiratory, etc. For example, the pre-Bötzinger Complex (preBötC) and the para-facial respiratory group (pFRG)/retrotrapezoid nucleus (RTN) regions were first identified and characterized in newborn rodent brainstem-spinal cord preparations. The preBötC was discovered by sequentially sectioning the brainstem in a rostrocaudal direction using a newborn rat brainstem-spinal cord preparation (Smith et al., 1991). The preBötC is hypothesized to generate the inspiratory rhythm (Feldman and Del Negro, 2006) and an intact preBötC is necessary and sufficient for inspiratory rhythm generation (Gray et al., 1999, Wenninger et al., 2004; McKay et al., 2005).

Similarly, the pFRG was identified early on with its distinctive neurons that fire during the pre-inspiratory and post-inspiratory phases in relation to spinal nerve C4 inspiratory motor output in brainstem-spinal cord preparations (Onimaru and Homma, 1987). The pFRG wraps caudally and ventrally around the facial nucleus in the medulla and appears to overlap with RTN and the Bötzinger Complex, but its borders are relatively ill defined (Feldman and Del Negro, 2006). The pFRG is proposed to be the primary source of the respiratory rhythm (Onimaru et al., 1987; Onimaru 1995; Onimaru and Homma, 2006) because pFRG neurons fire prior to preBötC neurons as revealed in elegant imaging studies on brainstem-spinal cord preparations bathed in voltage-sensitive dyes (Onimaru and Homma, 2003). Using brainstem-spinal cord preparations was critical in identifying the importance of the pFRG/RTN and preBötC, but also in elucidating connectivity between these two regions (Mellen et al., 2003), which has led to a “dual oscillator” model of respiratory rhythm generation (Feldman and Del Negro, 2006; Janczewski and Feldman, 2006).

3. Spatial separation of accessible respiratory spinal motoneurons from brainstem rhythm-generating neurons is exploited experimentially

In brainstem-spinal cord preparations, spinal motoneurons that control pump muscles are spatially separated from rhythm-generating neurons in the brainstem, which confers several experimental advantages. A barrier placed at the spinomedullary border allows spinal respiratory neurons to be bathed with drugs or chemicals while the brainstem is bathed in normal solutions to maintain rhythmic motor activity. For example, neuromodulation of bulbospinal excitatory glutamatergic synaptic transmission can be studied with respect to pre- vs. postsynaptic sites of action. Bath application of an adenosine analog drug to the cervical spinal cord decreases inspiratory burst amplitude on cervical spinal roots without altering phrenic motoneuron input resistance or responsiveness to exogenous glutamate application (Dong et al., 1996). Instead, the adenosine analog drug decreases the frequency of miniature EPSPs, suggesting that adenosine acts presynaptically to reduce glutamate release (Dong et al., 1996). A similarly designed study shows that serotonin increases phrenic motoneuron excitability while reducing inspiratory synaptic currents most likely by a presynaptic mechanism (Lindsay and Feldman, 1993).

In addition, phrenic motoneurons in the cervical spinal cord are large and readily accessible, thereby permitting long-lasting whole-cell recordings that provide insights into the nature of monosynaptic glutamatergic excitatory synaptic transmission from brainstem premotor neurons (reviewed in Monteau and Hilaire, 1991). For example, exquisite low-noise whole-cell recordings of spontaneous excitatory postsynaptic potentials in phrenic motoneurons from neonatal rat brainstem-spinal cord preparations provided one of the first demonstrations of quantal synaptic transmission in the mammalian CNS (Liu and Feldman, 1992). Likewise, inspiratory currents recorded from phrenic motoneurons in neonatal rat brainstem-spinal cords were cleverly injected back into the phrenic motoneurons to evaluate the impact of high-frequency (>10 Hz) oscillations on action potential frequency (Parkis et al., 2003). The major finding was that high-frequency oscillatory inputs increase the efficiency with which motoneurons transduce synaptic inputs into action potentials.

Finally, respiratory spinal motor output provides a “window” into the rhythm-generating network located rostrally in the brainstem. For example, expiratory motor activity is produced by intercostal and abdominal spinal motoneurons in the caudal thoracic and lumbar spinal cord (reviewed in Iizuka, 2011). Since the pFRG is hypothesized to be a primary source of expiratory activity within the medullary dual oscillator (Pagliardini et al., 2011), studying spinal expiratory motor activity may provide insights into mechanisms that contribute to the expiratory phase. Neonatal rodent brainstem-spinal cord preparations are useful in this regard because spinal expiratory motor activity can be recorded on ventral thoracic spinal roots (Iizuka, 1999). For example, bath-applied bicuculline (GABAA receptor antagonist) causes expiratory-related spinal motor activity to overlap temporally with inspiratory motor activity, suggesting that tonic inhibition via GABAA receptors (and not glycine receptors) plays a role in regulating the timing of the expiratory phase (Iizuka, 2003). Thus, the separation of readily identified and accessible respiratory spinal motoneurons from rhythm-generating brainstem neurons is advantageous for a variety of experimental approaches addressing many different scientific questions.

4. Perinatal brainstem-spinal cord preparations reveal insights into early motor network development

The central respiratory control network must be functional at birth, but still undergoes substantial postnatal development in parallel with developing airway, lungs, and muscles (Chatonnet et al., 2002, Borday et al., 2003, Chatonnet et al., 2003). Brainstem-spinal cords isolated from prenatal rodents are ideal for studying early development of the respiratory control system because these preparations are: (1) large enough to be isolated from embryos with minimal mechanical damage; (2) smaller and contain less myelin compared to postnatal preparations, thereby minimizing the diffusion limitations of nutrients and gases to the center of the tissue; (3) viable throughout the prenatal period; and (4) spontaneously produce respiratory-related motor activity for hours.

For example, brainstem-spinal cord preparations show that phasic contractions of the diaphragm and abdomen (fetal breathing movements) begin at embryonic day 15 (E15) for the mouse (Thoby-Brisson et al., 2005) and E17 for the rat (Pagliardini et al., 2003, Huxtable et al., 2009). Respiratory neuron activity in the brainstem at these early stages is demonstrated by calcium imaging and electrophysiological recordings from brainstem-spinal cord and rhythmic medullary slice preparations (Di Pasquale et al., 1992, Greer et al., 1992, Abadie et al., 2000, Pagliardini et al., 2003, Thoby-Brisson et al., 2005). The primary source of the underlying rhythm appears to be correlated with the onset of activity in the preBötC (reviewed in Thoby-Brisson and Greer, 2008). These fetal breathing movements are postulated to facilitate lung maturation and growth, and influence the development of respiratory motoneurons and muscles. Thus, prenatal brainstem-spinal cord preparations provide novel information into how the respiratory motor network develops prior to birth.

5. Brainstem-spinal cord preparations can be isolated from animals with genetic mutations or prenatal exposures to toxins

Since brainstem-spinal cords can be isolated prenatally or immediately after birth, studies can be performed on mutant animals that otherwise wouldn’t survive following birth (e.g., lethal knockouts). Demonstrating that a brainstem-spinal cord preparation from a mutant animal produces a normal respiratory motor output argues against respiratory failure as the cause of death. For example, mutant mice lacking the NMDAR1 gene die within 24-48 h after birth (Forrest et al., 1994), but the respiratory rhythm in P0 brainstem-spinal cord and rhythmic slice preparations is identical to the respiratory rhythm in control wild-type mouse preparations, suggesting that the lethality of the NMDAR1 knockout is not due to disruption of respiratory rhythm generation (Funk et al., 1997).

In addition, respiratory motor output can be analyzed and compared in brainstem-spinal cord preparations from mutant versus wild type rodents to gain insights as to the potential functions of specific genes. For example, murine brainstem-spinal cord preparations from Phox2a mutants have poorly developed adrenergic A6 regions and increased respiratory cycle variability compared to control mice (Morin et al., 1997; Viemari et al., 2004), thereby linking the loss of adrenergic modulation with irregular breathing. Respiratory disturbances (e.g., apneas and irregular breathing) can be characterized in brainstem-spinal cord preparations from Prader-Willi mice (Ren et al., 2003; Pagliardini et al., 2005; Zanella et al., 2008), or mice deficient in serotonin (Hodges et al., 2009) or deficient in the expression of Lbx-1 (Pagliardini et al., 2008), tachykinin-1 receptors (Berner et al., 2007), and monoamine oxidase (Bou-Flores et al., 2000; Bou-Flores and Hilaire, 2000).

Finally, pregnant rodents can be exposed to toxins and viable brainstem-spinal cords can be isolated and tested for long-lasting changes or damage to the respiratory control system. Infants born to mothers that smoke while pregnant have abnormal breathing patterns (reduced tidal volume and increased respiratory rate), higher frequency of apnea, blunted responses to hypoxia, and decreased ability to autoresuscitate during severe hypoxia (reviewed in Hafström et al., 2005). Isolated brainstem spinal-cord preparations allow investigators to detect similar changes in respiratory function and explore underlying mechanisms in rodents. For example, prenatal nicotine exposure causes neonatal rat brainstem-spinal cord preparations to be less responsive to nicotine (Pilarski and Fregosi, 2009), and more responsive to GABAA and glycine agonist drugs (Luo et al., 2004, 2007) and AMPA (Pilarski and Fregosi, 2009). Similarly, prenatal nicotine exposure causes neonatal murine brainstem-spinal cord preparations to produce slower, more irregular respiratory-related motor bursts, and the bath acidification response switched from being muscarinic to a nicotinic-based mechanism (Eugenin et al., 2008; Coddou et al., 2009). Thus, brainstem-spinal cord preparations provide viable tissue from newborn animals that may not survive long following birth due to respiratory-related disturbances.

6. Brainstem-spinal cord preparations permit rapid screening of drugs and pathological conditions

The neonatal rodent brainstem-spinal cord preparation is relatively easily to isolate and use for experiments compared to other relatively intact in situ preparations, such as the working heart-brainstem preparation (Paton, 1996), and perfused rat and guinea pig semi-intact preparations (Piantadosi et al., 1985; Richerson and Getting, 1987; Hayashi et al., 1991; Morin-Surun et al., 1992). Thus, neonatal rodent brainstem-spinal cord preparations provide the opportunity to rapidly screen for the effects of drugs of abuse, anesthetics, or pathological conditions on a spontaneously active, vertebrate rhythmic motor network. Results from these studies may have clinical relevance and lead to new insights with respect to underlying mechanisms.

For example, ethanol application to neonatal rat brainstem-spinal cords has no effect on phrenic motoneuron activity but significantly reduces hypoglossal activity, suggesting that pathways to upper airway motoneurons are selectively disrupted (Di Pasquale et al., 1995). In contrast, barbiturates, such as sodium pentobarbital, decrease respiratory burst frequency, suggesting that rhythm-generating neurons are altered (Tarasiuk et al., 1991; Fregosi et al., 2004). Brainstem-spinal cord preparations are also used to examine the effects of different anesthetic drugs on brainstem respiratory neuron function, such as propofol (Kashiwagi et al., 2004), enflurane, halothane, and isoflurane (Otsuka 1998), and sevoflurane (Takita and Morimoto, 2010). The results with sevoflurane are particularly interesting because sevoflurane (at a specific concentration) inhibits pre-I neurons within the pFRG with minimal effects on other brainstem respiratory neurons, which may permit testing whether pre-I neurons are necessary for rhythm generation (Takita and Morimoto, 2010). Each drug appears to alter specific components of the brainstem respiratory control network and does not simply cause a global depression within the entire network. Finally, experiments using brainstem-spinal cord preparations and rhythmically active medullary slice preparations show that ampakine drugs reverse opioid-induced respiratory depression without altering antinociception (Ren et al., 2006).

Pathological conditions, such as central hypothermia, can be studied under in vitro conditions using brainstem-spinal cord preparations because the temperature of the tissue can be controlled without confounding compensatory responses to counteract hypothermia. For example, the mechanisms underlying the abrupt decrease in respiratory motor output with hypothermia and subsequent autoresuscitation upon rewarming are poorly understood. Brainstem-spinal cord preparations from neonatal rats show that hypothermia-induced respiratory arrest is due to reversible failure in the network and not to irreversible neuronal damage in respiratory neurons (Mellen et al., 2002; Zimmer and Milsom, 2004). The ability to resuscitate upon rewarming is riluzole-dependent in hamster, but not rat, brainstem-spinal cord preparations (Fong et al, 2009), which reveals important species differences and the value of a comparative experimental approach with respect to hypothermia. Thus, the low cost and ease of setting up rodent brainstem-spinal cord preparations makes these preparations attractive for rapidly testing the effects of drugs, toxins, or different conditions on respiratory motor control.

7. Brainstem-spinal cord preparations permit in vitro studies on respiratory motor plasticity

Respiratory plasticity is defined as an alteration in future system performance based on experience (Mitchell and Johnson 2003). Brainstem-spinal cord preparations are used to study various forms of respiratory spinal plasticity, such as activity-dependent plasticity, pattern-sensitive serotonin-dependent plasticity, and changes in the brainstem during recovery from acute spinal cord injury. In these studies, brainstem-spinal cord preparations take advantage of the spatial separation of the brainstem from the respiratory spinal motoneurons, the ability to stimulate primarily monosynaptic bulbospinal synaptic inputs to respiratory spinal motoneurons, the ability to record from different pools of respiratory spinal motoneurons that serve different functions, and the ability to control the timing of drug application to the spinal cord.

For example, activity-dependent spinal plasticity is the alteration in synaptic efficacy due to previous activity at that synapse. Short-term (0-15 min) activity-dependent changes occur in bulbospinal synaptic inputs to respiratory spinal motoneurons (reviewed in Johnson and Mitchell, 2002), but long-term (>1 h) changes are also observed in adult turtle brainstem-spinal cord preparations (Johnson and Mitchell, 2000). In the turtle brainstem-spinal cord preparation, it is possible to record from separate expiratory and inspiratory nerves and there is sufficient spinal cord length to allow reversible inactivation of spontaneous respiratory-related bulbospinal synaptic inputs (Johnson and Mitchell, 2000). These experiments show that descending synaptic inputs to spinal motoneurons following high or low frequency stimulation are biased towards potentiation in inspiratory motoneurons, but depression in expiratory motoneurons, suggesting the importance of preserving inspiration for survival (Johnson and Mitchell 2000, 2002).

With respect to serotonin-dependent spinal plasticity, long-lasting augmentation of cervical and thoracic spinal motor output is induced by three spinal serotonin applications (Lovett-Barr et al., 2006). This demonstrated that episodic spinal serotonin receptor activation in vitro was sufficient to increase spinal respiratory motor output in a manner similar to intermittent hypoxia-induced phrenic long-term facilitation (Fuller et al., 2000; Mitchell et al., 2001). Surprisingly, sustained spinal serotonin application for 9 min induced a long-lasting increase in thoracic, but not cervical, spinal respiratory motor output (Lovett-Barr et al., 2006), demonstrating that serotonin-dependent plasticity in the cervical spinal cord is pattern-dependent whereas thoracic plasticity is pattern-independent.

Finally, after two days of recovery from a cervical C2 spinal cord lesion, brainstem-spinal cord preparations from neonatal rats respond differently to changes in bath pH compared to preparations from sham-operated and control neonatal rats (Zimmer et al., 2007). When bath pH is increased, cervical spinal respiratory burst duration and area increases in sham control preparations but decreases in preparations from spinally injured rats. Furthermore, there are significant changes in critical proteins related to respiratory motor control (e.g., glutamate receptor subunits, adenosine receptors, and neurokinin-1 receptors) in the medulla rostral to the spinal cord injury (Zimmer et al., 2007). Thus, neonatal rat brainstem-spinal cord preparations provide a novel experimental approach for studying changes in respiratory function following spinal cord injury in neonatal animals.

8. Interactions between respiration and other rhythmic motor behaviors

Respiratory motor activity is coordinated with other rhythmic motor activities, such as locomotion or swallowing. Recent comparisons between respiratory and locomotor networks are intriguing because critical interneurons within their central pattern generators appear to share similar genetic phenotypes and possibly similar functions (reviewed in Grossman et al., 2010; see also Gray 2008, Gray et al., 2010; Champagnat et al., 2011). In addition, insights can be gained into neuronal mechanisms that coordinate rhythmic motor behaviors because isolated neonatal rodent brainstem-spinal cord preparations contain other motor central pattern generators. Mammalian locomotor-respiratory coupling is hypothesized to prevent a lack of oxygen during exercise but the mechanisms underlying the coupling are poorly understood (Daffertshofer et al., 2004). Brainstem-spinal cord preparations from neonatal rodents show that respiratory activity can be reset and entrained by stimulating ascending lumbar or cervical propriospinal pathways that originate from the hindlimb or forelimb muscles (Morin and Viala, 2002).

In the brainstem, respiration and swallowing are highly coordinated because disruption of the interaction between respiratory and swallowing central pattern generators can cause lethal aspiration pneumonia. In neonatal rodent brainstem-spinal cord preparations, electrical stimulation of the superior laryngeal nerve activates the swallowing central pattern generator and increases the expiration phase duration (Yamanishi et al, 2010). The swallowing-induced inhibition of the respiratory rhythm generator is blocked by an alpha-2 adrenergic agonist and enhanced by and alpha-2 adrenergic antagonist, thereby revealing a key role of alpha-2 adrenergic receptors in the interaction between swallowing and respiratory motor networks (Yamanishi et al., 2010). Thus, brainstem-spinal cord preparations are valuable tools to begin unraveling complex neuronal interactions between different rhythmic motor networks.

9. Brainstem-spinal cords from other non-mammalian vertebrates – a comparative perspective

Brainstem-spinal cords from reptiles, such as the red-eared slider turtle, can be used to address similar questions in respiratory motor control. However, turtle brainstem-spinal cord preparations have unique advantages over neonatal rodent preparations. First, turtles are extremely hypoxia-resistant, which means that isolated brainstems (and presumably brainstem-spinal cords) from fully mature adult turtles can produce respiratory motor output under in vitro conditions for days (Wilkerson et al., 2003). Second, adult turtle brainstem-spinal cords can produce active expiratory and inspiratory activity on their appropriate spinal nerves that is similar to intact turtles (Johnson and Mitchell, 1998) at a physiological temperature (i.e., room temperature) and normal bath [K+] (Fig. 2; Johnson et al., 1998). Third, turtle brainstems appear to produce all three phases of breathing: inspiratory, expiratory, and post-inspiratory (post-inspiratory activity = unpublished observations in S.M. Johnson laboratory). To our knowledge, no other isolated adult vertebrate brainstem-spinal cord preparation has these advantages. Finally, studying mechanisms underlying respiratory motor control in turtles may provide insights into which mechanisms are evolutionarily conserved compared to amphibians and mammals.

Fig. 2. Expiratory and inspiratory spinal motor activity produced by an adult vertebrate brainstem-spinal cord preparation.

Fig. 2

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(A) A drawing of an isolated brainstem-spinal cord preparation from an adult turtle shows that respiratory-related motor activity is produced on pectoralis (expiratory) and serratus (inspiratory) nerves. (B) Rhythmic expiratory and inspiratory motor activity on nerves is correlated with respiratory activity on hypoglossal nerve roots in the brainstem. (C) Expiratory activity on pectoralis is typically bell-shaped while inspiratory activity on serratus is slowly-incrementing and rapidly decrementing. The resistance of turtle brain and spinal cord to hypoxia allows this preparation to produce spinal respiratory activity similar to intact adult turtles (discussed in Johnson et al., 1998). In contrast, the nature of respiratory-related motor output produced by neonatal rodent brainstem-spinal cord preparations is controversial. Abbreviations: KF = Kölliker-Fuse nucleus; PB = parabrachial nucleus; pFRG = para-facial group; preBötC = pre-Bötzinger Complex; VRC = ventral respiratory column.

10. Summary and conclusions

It is said that “no preparation is perfect”, which means that every experimental preparation has its limitations and shortcomings. The choice of which experimental preparation to use depends, of course, on the question to be addressed. The in vitro isolated vertebrate brainstem-spinal cord preparation has had its share of legitimate criticism over the years. Nevertheless, this preparation holds a prominent place in the history of respiratory neurobiology research, and still has a lot to offer scientifically and experimentally to address questions within and outside the field of respiratory neurobiology.

Acknowledgements

This work was supported by the following NIH grants: HL07654 (S.M.F. Turner), HL69064 (A.G. Huxtable, F. Ben-Mabrouk), and HL80209 (A.G. Huxtable).

Footnotes

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These preparations have several experimental advantages despite their limitations.

This review describes these advantages and highlights examples in the literature.

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