Spontaneous crossed phrenic activity in the neonatal respiratory network

Spontaneous crossed phrenic activity in the neonatal respiratory network

Experimental Neurology 194 (2005) 530 – 540 www.elsevier.com/locate/yexnr Spontaneous crossed phrenic activity in the neonatal respiratory network MR...

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Experimental Neurology 194 (2005) 530 – 540 www.elsevier.com/locate/yexnr

Spontaneous crossed phrenic activity in the neonatal respiratory network MR Beth Zimmer*, Harry G. Goshgarian Wayne State University, School of Medicine, Department of Anatomy and Cell Biology, 540 East Canfield, Detroit, MI 48201, USA Received 11 October 2004; revised 29 March 2005; accepted 29 March 2005 Available online 5 May 2005

Abstract Hemisection of the cervical spinal cord causes paralysis of the ipsilateral hemidiaphragm in adult rats. Activation of a latent crossed phrenic motor pathway can restore diaphragmatic function, although structural changes take place before the pathway can be activated. Since mechanisms are employed to eliminate non-functional projections during development, we predicted that this latent neural pathway might be active during development. Therefore, we examined the effect of spinal hemisection (C2) on respiratory-like activity bilaterally using the brainstem – spinal cord preparation from neonatal rats (0 – 4 days). Spontaneous crossed phrenic activity (respiratory-like activity recorded from the ipsilateral C4 or C5 ventral roots following C2 hemisection) was observed in an age-dependent manner; younger preparations exhibited more than older preparations. Increasing drive (increasing [K+] or superfusion of theophylline) either increased or induced crossed phrenic activity. Hemisection caused no change in the frequency, the burst area, duration or peak amplitude contralateral to hemisection. Unlike adult rats, this study shows that crossed phrenic activity is present in the in vitro respiratory network of neonatal rats suggesting that a crossed neural pathway may be functionally active in neonates. D 2005 Elsevier Inc. All rights reserved. Keywords: Respiration; Spinal cord injury; Neonatal development; Crossed phrenic pathway; Brainstem – spinal cord preparation; Theophylline

Introduction Injury of the cervical spinal cord results in a significant impairment of the respiratory system. This is due to damaged descending respiratory premotor axons which arise bilaterally in the medulla and run down the spinal cord to synapse with phrenic motoneurons (Dobbins and Feldman, 1994). Cervical spinal cord injury significantly reduces or eliminates the motor output from the phrenic motor nucleus resulting in diaphragmatic paralysis. Studies that have examined the effect of spinal cord injury on ventilatory function in adult animals (using a C2 hemisection model) have revealed the existence of a nonfunctional neural pathway that crosses the midline in the spinal cord at the level of the phrenic motor nucleus (Goshgarian and Guth, 1977; Porter, 1895). The crossed phrenic pathway described in young adult rats consists of

* Corresponding author. Fax: +1 313 577 3125. E-mail address: [email protected] (M.B. Zimmer). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.03.013

premotor respiratory axons that also arise bilaterally in the ventral respiratory group (Goshgarian et al., 1993; Moreno and Goshgarian, 1992) and descend into the spinal cord. The axons then cross below the site of injury (C2) to synapse with phrenic motor neurons on the opposite side of the spinal cord. If the latent crossed phrenic pathway is activated (through multiple means), the phrenic motor neurons below the site of injury become depolarized and effectively restore function to the previously paralyzed hemidiaphragm (Fuller et al., 2002, 2003; Ling et al., 1994; Nantwi and Goshgarian, 1998). The phrenic motor neuron output ipsilateral to hemisection has been referred to as ‘‘crossed phrenic activity’’ (Goshgarian, 2003). Research has shown, that immediately after spinal cord injury, the synaptic connection between the crossed phrenic premotor axons and phrenic motoneurons is ineffective in guinea pigs and adult rats, that is, they cannot depolarize phrenic motor neurons (Goshgarian and Guth, 1977; O’Hara and Goshgarian, 1991). However, within hours, there is a significant increase in the ability to induce crossed phrenic activity by asphyxia (O’Hara and Goshgarian, 1991). The

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increase in crossed phrenic activity coincides temporally with the retraction of astrocytic projections from in between phrenic motoneurons and an increase in the number of specialized synapses and dendrodendritic appositions in the phrenic nucleus (Goshgarian et al., 1989; Sperry and Goshgarian, 1993). Presumably, these modifications and synaptic reorganization convert the ineffective synaptic pathway to a functionally latent pathway which is then capable of being induced. Numerous studies have shown that mechanisms such as apoptosis and neural pruning eliminate unnecessary and non-functional neural projections during development in many systems (Buffelli et al., 2004; Raff et al., 2002), including the respiratory system (Cameron et al., 1991a,b; Redfern, 1970; Sieck and Fournier, 1991). Since the crossed phrenic neural pathway is present in adults, although ineffective and non-functional, we predicted that the pathway might have been functional during development when the pathway was being laid down; otherwise, it should have been eliminated. Therefore, we examined whether crossed phrenic activity was detectable in neonate rats using the brainstem –spinal cord preparation from neonates of different ages (postnatal days 0 – 4). We predicted that the youngest neonatal preparations would produce spontaneously active crossed phrenic activity and that increasing drive would further enhance the crossed activity. In older neonatal preparations, if crossed phrenic activity was not present, then increasing respiratory drive should elicit crossed phrenic activity. Preliminary data from this study have been presented previously in abstract form (Zimmer and Goshgarian, 2004).

Methods Timed pregnant, female Sprague – Dawley rats were purchased from Harlan Rodent Laboratories and allowed to give birth in the animal care facilities at Wayne State University, School of Medicine, Detroit, MI. Individual neonatal rats (postnatal days (P)0 n = 4; P1 n = 11; P2 n = 13; P3 n = 11; P4 n = 4) were brought to the laboratory and anesthetized with halothane. The brainstem and spinal cord were carefully dissected (pons removed, spinal cord cut at approximately T8) and the meninges removed under constant superfusion of artificial cerebral spinal fluid (ACSF composed of 113.0 mM sodium chloride, 3.0 mM potassium chloride, 1.2 mM sodium phosphate, 1.5 mM calcium chloride, 1.0 mM magnesium chloride, 30.0 mM sodium bicarbonate and 30.0 mM dextrose) bubbled with 95% oxygen and 5% carbon dioxide. The tissue was pinned to a wire mesh which separated the recording chamber into two compartments: a lower and upper compartment. Superfusion of a constant supply of ACSF (27-C, pH 7.4) was supplied simultaneously to both compartments to ensure adequate superfusion of the tissue. Bilateral respiratory-like motor activity was recorded in every brainstem preparation using

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suction electrodes placed on both the left and right ventral C4 or C5 roots. Electrical signals were amplified and filtered (300 – 3 K Hz, gain 20,000) using Grass amplifiers (Model P511) and data recorded continuously on computer using Spike2 data acquisition software (CED, Cambridge UK) (sample rate 3000 Hz). After baseline recordings of bilateral respiratory-like motor output were obtained (approximately 20 min), a hemisection just rostral to C2 was made on either the right or left side of the spinal cord using fine spring microscissors. Under a dissecting microscope, the tip of one blade was placed down the anterior median fissure through the midline of the spinal cord to the wire mesh and the other blade cut through one half of the spinal cord. The scissors were then used to scrape the wire mesh to ensure a complete cut was made through the tissue. Since the cut often dislodged nerve-electrode contacts, suction was reapplied to both nerves after every hemisection. Bilateral respiratorylike motor output continued to be recorded. In the experimental group, the K+ concentration in the ACSF was increased from 3 mM to 9 mM (to result in the general depolarization of neurons and excite the respiratory neural system) followed by a single application of theophylline (a known respiratory stimulant) (n = 25). Theophylline (44 AM) was acutely applied to the bath (in 9 mM K+ ACSF) for approximately 1 min and the respiratory-like motor output analyzed approximately 10 min after application. Untreated, uncut (no hemisection) brainstem – spinal cord preparations (n = 10) and hemisection alone preparations (n = 9) were run for up to 90 min to determine baseline frequency, burst peak amplitude, burst duration and burst area. Bilateral respiratory-like recordings were full-wave rectified and integrated using CED data analysis software. In controls, the frequency, burst peak amplitude, burst duration and burst area were measured every 5 min (all bursts within the full 5 min were analyzed). In hemisected tissue, data were analyzed for 10 min prior and 10 min after hemisection at 40 min (after 9 mM K had been superfused through the chamber) and at 60 min (after theophylline had been applied to the chamber). Every Fburst_ ipsilateral to hemisection was analyzed for its peak amplitude. The respiratory-like bursts contralateral to hemisection were used to define when a respiratory signal was sent down the spinal cord and the peak amplitude ipsilateral to the hemisection was measured and included in the analysis; even those preparations that did not produce crossed phrenic activity were included in the analysis. The peak amplitude data were normalized to the start of the recording session. Histological verification of a complete hemisection was completed. At the end of the experiment, all brainstem – spinal cord preparations were post-fixed in 10% neutral buffered formalin. Tissue was embedded in paraffin and 6 Am transverse sections cut. The sections were mounted on slides, stained with hematoxylin and eosin and examined with a microscope (Nikon).

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Statistical analysis A multivariate repeated measures ANOVA followed by post hoc analysis and the nonparametric Kolmogorov – Smirnov two sample test were applied. All data are presented as means T SD. P < 0.05 was considered significant.

Results The frequency of respiratory-like activity significantly decreased over time in both non-treated controls and hemisected controls (Fig. 1). There was also a significant increase in the peak amplitude, burst duration and burst area of the respiratory-like bursts recorded from C4 and C5 roots

of brainstem – spinal cord preparations from neonatal rats (aged 0– 4 days) (Figs. 1 and 2). There was no effect of age on these time-dependent changes. Effect of spinal cord hemisection (C2) on respiratory-like motor output After baseline measurements were recorded, a spinal hemisection on the left or right side was made rostral to the ventral C2 root and suction was quickly reapplied to the roots since the cut either usually dislodged or weakened the seal between the nerve and the electrode. There were no differences between a hemisection made on the left and right side and thus the data were combined. Hemisection of the spinal cord did not alter the frequency nor the burst duration, burst area (Fig. 1)

Fig. 1. The respiratory-like discharge frequency (A) of brainstem spinal cord preparations of neonatal rats (0 – 4 days) steadily decreased over time (*) in all preparations. Exposure to 9 mM K+ (at 40 min) increased the frequency above control values (**), and theophylline (at 60 min) showed a further increase in frequency (***). The burst area (B) as well as the burst duration (C) showed a steady increase (*) over time in all preparations. No effect of K+ or theophylline was observed on the burst area or duration of the nerve contralateral to hemisection. Note that hemisection did not alter these parameters. Non-treated controls (o), hemisected controls (g) and hemisected experimental group (O).

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Fig. 2. The peak amplitude of the respiratory-like bursts in neonatal brainstem spinal cord preparations (0 – 4 days) significantly increased over time in nontreated controls (o) (*) denotes a significant increase from start value). Hemisection did not alter the peak neural output contralateral to hemisection in hemisected controls (g) or in the experimental group (O). Hemisection did, however, significantly reduce the peak amplitude ipsilateral to hemisection below starting values (*). Exposure to 9 mM K+ caused a significant increase in the peak amplitude in the ipsilateral nerve above control values (**) but not in the contralateral nerve, and theophylline did not alter the peak amplitude further but remained elevated above control values (**).

and burst peak amplitude (Fig. 2) recorded from the contralateral nerve (same as control values), but the activity ipsilateral to hemisection was significantly reduced (Fig. 2). A small amount of respiratory-like activity coincident with the contralateral motor output did persist in the ipsilateral nerve (Fig. 3). This spontaneous crossed phrenic activity observed in brainstem spinal cord preparations was age-dependent; younger preparations (0– 2 days old) showed a significantly higher percentage exhibiting crossed phrenic activity than older preparations (3 –4 days old) (Fig. 4A). Immediately after hemisection, the background activity recorded from both nerves increased dramatically and then slowly returned back to control conditions (Fig. 5A). Along with the decrease in background activity was a decrease in the peak amplitude of the crossed phrenic activity before stabilizing at a new low level of activity (Fig. 5B). Examination of the spinal cord tissue revealed a complete hemisection of the spinal cord at C2 (Fig. 5C) indicating

that the crossed phrenic activity is arising from the side of the spinal cord contralateral to the hemisection. Effect of increasing central drive on respiratory-like motor output after spinal cord hemisection In hemisected control animals, the frequency, burst peak amplitude, area and duration of the contralateral nerve were not different than controls, whereas the ipsilateral peak amplitude was significantly reduced by hemisection. The burst peak amplitude recorded from the ipsilateral nerve did not increase over time like the contralateral phrenic output. Increasing the [K+] in the ACSF from 3 mM to 9 mM resulted in a significant increase in the frequency of respiratory-like bursts (Fig. 1) and a significant increase in the peak amplitude of the crossed phrenic activity over control values, whereas the peak amplitude on the side contralateral to hemisection remained unchanged (Figs. 2 and 3). Burst area and burst duration were not affected by

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Fig. 3. Raw (bottom) and integrated (top) signals from a 1-day-old brainstem – spinal cord preparation showing the respiratory-like neural output both contralateral and ipsilateral to a left hemisection. Note the marked reduction in neural activity after the hemisection, but respiratory-like activity still persisted and was correlated with the respiratory-like activity on the contralateral (right) nerve. Note the significant increase in the peak amplitude after 9 mM K+. While it may appear that burst duration and burst area increased after theophylline administration, the mean data show no significant effect of theophylline (see Fig. 1).

changing the [K+]. Increasing the [K+] concentration also activated crossed phrenic pathways in those preparations that did not spontaneously express crossed phrenic activity after initial hemisection (Fig. 4B). Acute theophylline administration caused an initial, nonspecific increase in background nerve discharge recorded in both nerves followed by periodic ‘‘bursts’’ of non-specific (non-respiratory) increased neural discharge (Fig. 6). These bursts were noted for the entire duration of the study (at least another 1/2 h after theophylline was administered). Theophylline caused a significant increase in the burst frequency above 9 mM K+ alone (Fig. 1) but no change in the peak amplitude (Figs. 2 and 3). Theophylline did not alter the burst area or duration. During the periodic bursts of increased neural discharge, however, the peak amplitude of the crossed activity appeared greatly enhanced (Fig. 6B). Theophylline also caused those few remaining preparations that did not exhibit any crossed activity to start producing some crossed activity (Fig. 4B). This activity, however, was not present on every burst but was present only when the background neural activity increased (data not shown).

Discussion Crossed phrenic activity in the neonatal brainstem – spinal cord preparation Fig. 4. Brainstem – spinal cord preparations from 0 – 2-day-old neonates almost always produced spontaneous crossed phrenic activity, whereas 3 – 4-day-old neonatal preparations showed spontaneous activity only 50% of the time (A, * denotes significance). In the 3 – 4-day-old preparations, increasing respiratory drive via increasing the [K+] and theophylline significantly (*) increased the number of preparations exhibiting crossed phrenic activity (B).

In adult rats, hemisection of the spinal cord rostral to the phrenic motor nucleus results in a cessation of activity in the ipsilateral phrenic nerve and complete paralysis of the ipsilateral hemidiaphragm. The ability to activate latent crossed phrenic pathways increases over the first 2 h after hemisection which is coincident with synaptic remodeling

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Fig. 5. Hemisection resulted in an increase in the background neural discharge of the whole phrenic nerve which slowly decreased back to control levels (A; 1day-old preparation). During the first 5 min after the hemisection, there was a significant reduction in the peak amplitude of the spontaneous crossed phrenic activity coincident with the decrease in background activity (B). The graph (B) shows the mean peak amplitude of the first 40 bursts after hemisection. There were no significant differences between hemisected controls and the experimental group, so the data were averaged together. Panel C shows an example of a paraffin section through one brainstem – spinal cord preparation demonstrating a complete left hemisection; the arrow indicates the actual position of the cut that was made. The tissue present on the left side is just rostral to the hemisection and, under high power (data not shown), neurons are pyknotic and cells are already changing shape, indicating that cells are already beginning to die within the hour after the cut was made in vitro.

within the phrenic motor nucleus (Sperry and Goshgarian, 1993). Unlike in adult rats, this study shows that respiratorylike activity persists in the ipsilateral phrenic nerve immediately after complete hemisection in the in vitro neonatal brainstem – spinal cord preparation. This activity was greatly reduced and sometimes only detectable via an audio monitor feed from the amplified signal, but its presence in the in vitro respiratory network suggests that a crossed phrenic neural pathway is active and functional in the neonate. The mechanism or neural pathway underlying the spontaneous expression of crossed phrenic activity in the neonatal respiratory network is unknown. In adult rats, normal resting ventilation is controlled via respiratory bulbospinal premotor neurons which arise bilaterally in the VRG and run down the lateral and ventral funiculi of the spinal cord to synapse with phrenic motoneurons (Dobbins and Feldman, 1994). The latent crossed phrenic pathways that can be activated after spinal cord hemisection in adult rats consist of respiratory premotor axons that also arise bilaterally in the VRG and run down the spinal cord. Once they arrive at the level of the phrenic motor nucleus, axons

cross the midline of the spinal cord in both the anterior gray and anterior white commissure (Goshgarian et al., 1991) to synapse onto phrenic motoneurons ipsilateral to hemisection. The neural organization of the respiratory pathway in neonates, however, may be different than adults. Using cross-correlogram methods, Peever and Duffin (2001) and Li and Duffin (2004) have proposed that the trajectory of the active respiratory impulse underlying normal respiration in neonates also arises from bulbospinal inspiratory neurons situated bilaterally in the medulla. All of these neurons, however, send axons across the midline in the medulla at the level of the hypoglossal roots. The axons then project down the cord to synapse onto the phrenic motor neurons contralateral to the cell soma in the medulla (solid lines, Fig. 7A). Their data suggest no functional connection between VRG bulbospinal neurons and ipsilateral phrenic motor nuclei, indicating that this major respiratory pathway of adults has not been fully formed yet (dotted lines, Fig. 7A). Li and Duffin (2004) did find common excitatory inputs arriving at both right and left phrenic motor nuclei. They suggest that this comes from the common excitation of VRG premotor neurons (possibly

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Fig. 6. This is an example showing the initial effect of theophylline (A) and the long lasting effect of theophylline (B); integrated neural recordings showing the neural output ipsilateral to hemisection (top) and contralateral to hemisection (bottom). Application of theophylline lasted approximately 1 min, after which a large burst in background activity was observed (A; 3-day-old preparation). Along with the increase in background activity was an observed increase in the peak amplitude of the bursts from the ipsilateral nerve (top trace, A) which was not evident in the contralateral nerve (bottom, A). Panel B (1 day old preparation) shows a ‘‘periodic burst’’ of neural discharge that occurred approximately 10 min after the theophylline was applied. Again, note the increase in peak amplitude of the ipsilateral nerve which coincided with the increased background activity.

through gap junction coupling) and not from premotor neurons that bifurcate and send projections to both phrenic nuclei. At first, these data appear contradictory to the

finding of spontaneous crossed phrenic activity in this study. However, a close examination of the data suggests several possible pathways by which spontaneous crossed phrenic

Fig. 7. This figure shows diagrammatic sketches of the proposed functional trajectory of the respiratory bulbospinal pathways in the neonate adapted from Peever and Duffin (2001) (A) and three possible sources of inputs which may have contributed toward the spontaneous crossed phrenic activity observed after hemisection using the neonatal rat brainstem – spinal cord preparation in this study (B – D). First, age-dependent functional bulbospinal projections may arise from the contralateral VRG, descend the same side of the spinal cord and cross the midline at the level of the phrenic motor nucleus (B). Secondly, pontine inputs were removed in this study, thereby removing a known source of respiratory inhibition. This may have allowed more inspiratory neurons to fire and thus initiate crossed phrenic activity mediated by pathways found in the adult (C). Finally, phrenic motor neuron dendrites may be functional early during development and thus may turn on crossed phrenic activity (D).

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activity may be transmitted while still being consistent with available literature (Li and Duffin, 2004; Peever and Duffin, 2001). One possible neural pathway underlying spontaneous crossed phrenic activity observed in the neonatal in vitro preparation consists of neurons that arise in the medulla, send axons down the ipsilateral cord and then cross the midline at the level of the phrenic motor nucleus (Fig. 7B). The reason that these neurons were not accounted for in the Peever and Duffin (2001) model or by Li and Duffin (2004) was that a complete mid-sagittal section of the spinal cord did not appear to alter phrenic motor output significantly and did not alter the central peaks in cross correlograms of right and left phrenic nerve discharges. Peever and Duffin (2001), however, used neonates that were mostly 4 days old; an age at which we show reduced levels of spontaneous crossed phrenic activity. Another major difference is that the pons remained intact in Peever and Duffin’s study whereas this study removed the pons. It is well-known that pontine inputs have an inhibitory influence on inspiratory firing in neonatal brainstem – spinal cord preparations (Hilaire et al., 1989). Removal of the pons may have disinhibited a population of inspiratory neurons which may have contributed to the expression of spontaneous crossed phrenic activity in the present study. Furthermore, pontine inhibition in neonatal preparations could potentially mask crossed phrenic pathways that arise bilaterally from the VRG and extend to both phrenic nuclei (Fig. 7C); the anatomical substrate thought to mediate crossed phrenic activity in adult rats (Moreno and Goshgarian, 1992). Finally, Song et al. (2000) found that the injection of dye into the phrenic motor nucleus, retrogradely labeled VRG neurons bilaterally, as early as E15, and they also found that the projection was predominantly to the ipsilateral VRG. These potential pathways of spontaneous crossed phrenic activity (Fig. 7) are not the only sources of possible crossed phrenic activity. In neonates, the dendritic field of phrenic motor neurons is large. Some dendrites cross to the contralateral side (Allan and Greer, 1997; Song et al., 2000) indicating that phrenic motor neurons on the side ipsilateral to hemisection may interact with phrenic neurons on the contralateral side or with descending axons on that side (Fig. 7D). However, retraction of crossed phrenic motor dendrites is observed postnatally (Prakash et al., 2000), suggesting that they do not receive active motor input and therefore retract. In addition to the possible differences in neural organization of the neonatal respiratory network, the cellular physiology of neurons is different in neonates; perhaps contributing to the ability of the network to produce spontaneous crossed phrenic activity. In general, neurons of neonates, including respiratory neurons, are relatively more depolarized thus making them more excitable; that is, it takes less current to reach threshold in phrenic motor neurons to generate an action potential (Cameron and Nu´n˜ez-Abades, 2000). This is due to many factors including

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differences in ion channel composition, a higher resting membrane potential, excitatory rather than inhibitory GABA inputs, and so on (Ballanyi et al., 1999). It has even been suggested that, in adult rats, the functionally ineffective synapses between VRG neurons and phrenic motor neurons within the crossed phrenic pathway may be physiologically normal but quantitatively insufficient to fully depolarize phrenic motor neurons (Goshgarian, 2003). Thus, in the neonate, any crossed phrenic input might be translated into phrenic motor neuron firing, whereas in the adult relatively more crossed phrenic input may be needed to generate an action potential. Increasing respiratory drive and crossed phrenic activity Increasing the [K+] from 3 mM to 9 mM in the extracellular fluid resulted in a significant increase in the frequency of the respiratory-like bursts in the neonatal brainstem –spinal cord preparation, similar to other studies (Okada et al., 2005). Interestingly, the increase in [K+] caused a significant increase in the peak amplitude of the bursts from the nerve ipsilateral to hemisection, but not the contralateral nerve. It also activated crossed phrenic activity in some preparations that were not expressing any previously. This could simply be the result of depolarizing phrenic motor neurons. It has been shown that, during quiet breathing, >90% of phrenic motor neurons fire during each inspiratory effort in neonates (Cameron et al., 1991b). Since most phrenic motor neurons are already firing on the side contralateral to hemisection, an increase in [K+] may not change the contralateral phrenic motor output. However, an increase in [K+] might result in more motor neurons reaching threshold on the ipsilateral side, causing a significant change in the peak amplitude. Interestingly, it has been postulated that the functional significance of glial retraction and remodeling observed in the phrenic nucleus after spinal hemisection in adult rats is a resulting increase in local extracellular [K+]. The increased [K+] would lead to increased neuronal membrane excitability by depolarizing phrenic motor neurons (Goshgarian et al., 1989) and thus increased the ability to activate crossed phrenic pathways. Theophylline also increased respiratory frequency, but it did not cause a substantial increase in peak amplitude (crossed or uncrossed). It did, however, stimulate crossed phrenic activity in those few preparations that were not producing crossed activity. This may have been due to the general excitatory effects of theophylline. Indeed, it appears as if anytime background neural activity increased, the peak amplitude of the spontaneous crossed phrenic activity also increased; such as immediately after hemisection (see Fig. 5) or during the initial excitatory effect of theophylline (see Fig. 6A). Similar studies using methyxanthines in vitro have found similar effects on the respiratory-like neural output (Kawai et al., 1995; Wilken et al., 2000) and indicate that the effect of theophylline changes throughout early development (Wilken et al., 2000). Theophylline has been used in

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adult rats to activate crossed phrenic pathways after spinal hemisection (Nantwi and Goshgarian, 1998; Nantwi et al., 2003). The mechanism, however, by which theophylline activates crossed phrenic pathways in adults or neonates is not completely understood. Why and how might spontaneously active crossed phrenic inputs become latent? Brainstem – spinal cord preparations from older neonates exhibited less spontaneous crossed phrenic activity than younger preparations indicating that the efficacy of the crossed phrenic synapse may decrease with neonatal age. Indeed, crossed phrenic pathways are non-functional in adult rats (Goshgarian and Guth, 1977; Porter, 1895). During development, changes occur which may contribute toward the reduction in crossed phrenic activity; crossed dendritic processes retract (Prakash et al., 2000, dendrodendritic connections decrease, gap junction coupling disappears (Greer et al., 1999), the resting membrane of neurons becomes more hyperpolarized (Martin-Caraballo and Greer, 1999) and the number of motor neurons activated during each inspiratory effort is reduced (Cameron et al., 1991a) along with a reorganization of motor units (Sieck and Fournier, 1991). Even the change underlying synchronization of the left and right phrenic motor nuclei (Li and Duffin, 2004; Peever and Duffin, 2001) may contribute toward a weakening of the crossed phrenic input. According to Hebb’s rule, the firing of a pre-synaptic neuron leads to a strengthening of a synapse, but only if the firing of the presynaptic neuron is coincident with the firing of the postsynaptic neuron (Hebb, 1949). Alternatively, if the activity between the pre- and post-synaptic neurons becomes noncoincident, then the synapse becomes weakened (Stent, 1973). In the case of the respiratory system, synchronization of left and right phrenic motor neuron pools in neonates occurs in the medulla between left and right VRG premotor neurons (Shen et al., 2002), pre-Bo¨tzinger neurons and other unknown sources (Li et al., 2003). In adult rats, however, synchronization occurs because individual premotor neurons bifurcate in the medulla and innervate both right and left phrenic nuclei. So, in the context of Hebb’s rule, crossed phrenic pathways may weaken during development as synchronization changes; the firing of the pre-synaptic neuron (the crossed axonal projection) may become asynchronous with the post-synaptic neuron which is now primarily driven by the descending ipsilateral inputs. Accordingly, in the adult, when the major descending ipsilateral respiratory drive is removed due to spinal cord injury, any respiratory impulses traveling over crossed phrenic pathways might then lead to the strengthening of the involved synapses since there is no other asynchronous input, and this could lead to a functional connection. Indeed, the latent crossed phrenic pathway does become spontaneously active several weeks to months after injury (Nantwi et al., 1999).

Critique of the method The earliest descriptions of the crossed phrenic phenomenon were described in vivo (Porter, 1895) and most, if not all, of the work since have been performed in vivo. To our knowledge, this is the first observation of crossed phrenic activity observed in vitro. The brainstem – spinal cord preparation is a highly reduced preparation: pontine – medullary transection, T8 spinal transection and a complete cervical and thoracic dorsal root section. What if any of this might affect crossed phrenic activity? In adults, both acute (Goshgarian, 1981) and chronic (Fuller et al., 2002) cervical dorsal root rhizotomy have been shown to activate crossed phrenic pathways after upper cervical spinal cord injury. Both of these studies, however, performed the experimental recordings days after the initial hemisection; long after synaptic and cytoarchitecture changes had already taken place in the phrenic motor nucleus. It is not known whether a dorsal root rhizotomy can initiate crossed phrenic pathways if the rhizotomy is performed at the time of hemisection. It is also not known whether crossed phrenic activity can be observed in a decerebrate, unanesthetized rat immediately after hemisection. T8 transection does alter total ventilation (Teng et al., 2003), but it would probably not affect crossed phrenic activity, since the pathways that innervate the diaphragm are not altered during a T8 transection. Finally, studies have shown that the morphological changes observed in the phrenic motor nucleus after spinal cord injury are activity dependent (i.e., the removal of the descending respiratory motor drive) and not due to a general effect of CNS injury (Castro-Moure and Goshgarian, 1996, 1997). Therefore, we do not believe that the spinal cord injury per se contributed to the observation of crossed phrenic activity in this study. The neonatal brainstem –spinal cord preparation has been widely used to evaluate respiratory neural function, and many believe that the results obtained from such studies have provided valuable information regarding respiratory neural control (Ballanyi et al., 1999), however, the validity of the preparation still remains in question (St. John et al., 2002). While we do agree that it is important to eventually validate in vitro experimental data with data obtained in vivo, the mere fact that we see spontaneous crossed phrenic activity despite the highly reduced nature of the preparation (lack of peripheral input, reduced temperature, etc. . .) suggests that this may be a strong neural component underlying normal respiratory drive in neonates. In fact, under in vivo conditions in which body temperature is elevated and the descending respiratory drive is elevated, we would predict that the crossed phrenic activity might be even stronger. The pathway by which this crossed activity occurs in the neonate, however, is not known. It is also not known whether any crossed respiratory neural signal which can be detected in the phrenic nerve would actually be translated into diaphragmatic contraction. This, too, remains to be seen.

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Alternatively, if the preparation is exhibiting gasping mechanisms (either hypoxia driven or mechanistically as St. John et al. (2002) hypothesize), this added ‘‘drive’’ may actually contribute toward activating crossed phrenic pathways, similar to what occurs in the adult. If this were the case, however, then we would predict that the older preparations would also be exhibiting a similar crossed phrenic activity; in fact, the effect of hypoxia should have been greater in the older, much larger preparations. We found the opposite; younger neonates showed more crossed phrenic activity than older preparations suggesting that gasping mechanisms are not the source driving the spontaneous crossed phrenic activity observed in this study.

Summary Spontaneous crossed phrenic activity was observed in neonatal brainstem – spinal cord preparations from the rat. While the source and pathway mediating the activity remain unknown, its presence strongly indicates that this pathway may be active initially in vivo contributing toward normal respiratory neural output. As development progresses, crossed phrenic activity becomes non-functional and latent in the adult.

Acknowledgment This study was supported by NIH grant HD 31550 (H.G. Goshgarian).

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