J Appl Physiol 94: 795–810, 2003;
10.1152/japplphysiol.00847.2002.
highlighted topics
Plasticity in Respiratory Motor Control
Invited Review: The crossed phrenic phenomenon:
a model for plasticity in the respiratory pathways
following spinal cord injury
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HARRY G. GOSHGARIAN
Department of Anatomy/Cell Biology, Wayne State University, Detroit, Michigan 48201
Goshgarian, Harry G. Invited Review: The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal
cord injury. J Appl Physiol 94: 795–810, 2003; 10.1152/japplphysiol.
00847.2002.—Hemisection of the cervical spinal cord rostral to the level of
the phrenic nucleus interrupts descending bulbospinal respiratory pathways, which results in a paralysis of the ipsilateral hemidiaphragm. In
several mammalian species, functional recovery of the paretic hemidiaphragm can be achieved by transecting the contralateral phrenic nerve. The
recovery of the paralyzed hemidiaphragm has been termed the “crossed
phrenic phenomenon.” The physiological basis for the crossed phrenic phenomenon is as follows: asphyxia induced by spinal hemisection and contralateral phrenicotomy increases central respiratory drive, which activates
a latent crossed respiratory pathway. The uninjured, initially latent pathway mediates the hemidiaphragm recovery by descending into the spinal
cord contralateral to the hemisection and then crossing the midline of the
spinal cord before terminating on phrenic motoneurons ipsilateral and
caudal to the hemisection. The purpose of this study is to review work
conducted on the crossed phrenic phenomenon and to review closely related
studies focusing particularly on the plasticity associated with the response.
Because the review deals with recovery of respiratory muscles paralyzed by
spinal cord injury, the clinical relevance of the reviewed studies is highlighted.
hemidiaphragm; hemisection; motor recovery; paralysis
spinal cord rostral to
the level of the phrenic nucleus may interrupt the
descending bulbospinal respiratory pathways and
cause respiratory muscle paresis or paralysis. The resultant respiratory insufficiency is often treated in
human cases of cervical spinal cord injury by placing
the patient on long-term mechanical ventilator support. Such treatment is not always optimal and could
lead to serious complications, such as infection, pneumonia, atelectasis, or even death (20, 45). Moreover,
quadriplegic patients who are tethered to mechanical
TRAUMATIC INJURY TO THE CERVICAL
Address for reprint requests and other correspondence: H. G.
Goshgarian, Dept. of Anatomy/Cell Biology, Wayne State Univ.,
School of Medicine, 540 East Canfield Ave., Detroit, MI 48201 (Email: [email protected]).
http://www.jap.org
ventilators do not have the freedom to fully participate
in physical and occupational rehabilitative strategies.
It is important, therefore, to understand the underlying mechanisms related to plasticity and recovery of
the respiratory pathways after spinal cord injury, since
this information may lead to therapies directed toward
resolving the problem of spinal cord injury-induced
respiratory insufficiency in humans.
In 1895, Porter (107) provided one of the first demonstrations of functional restitution in the respiratory
system after spinal cord injury. He paralyzed the ipsilateral hemidiaphragm by spinal cord hemisection rostral to the level of the phrenic nucleus in dogs and
rabbits and showed that subsequent transection of the
contralateral phrenic nerve immediately restored func-
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society
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ANATOMIC SUBSTRATE FOR THE CPP
Porter (107) demonstrated that bulbospinal respiratory impulses cross the spinal cord at the level of the
phrenic nuclei during the induction of the CPP. He
hypothesized that some phrenic motoneuron dendrites
cross the spinal cord midline and receive uncrossed
bulbospinal synaptic connections from the opposite
side, thus suggesting an anatomic basis for the CPP.
Cameron et al. (11), after injecting horseradish peroxidase (HRP) intracellularly into phrenic motoneurons
of the adult cat, demonstrated phrenic dendrites that
cross the spinal cord midline, but Furicchia and Goshgarian (46) did not identify any crossing phrenic dendrites in the adult rat after injecting choleratoxin-HRP
into the diaphragm. However, Lindsay et al. (80) did
find that a significant proportion of phrenic dendrites
do cross the midline of the spinal cord in neonatal rats,
and, more recently, Prakash et al. (109) determined
that the percentage of phrenic dendrites crossing over
to the contralateral spinal cord was markedly higher in
21-day-old rats than in adult rats (26.3 ⫾ 2.0 vs. 3.1 ⫾
1.5%, respectively). These data suggest that contralaterally projecting rat phrenic dendrites may either retract back to their cell bodies of origin during postnatal
maturation (109) or there may be a higher growth rate
of motoneurons compared with the spinal cord (80).
J Appl Physiol • VOL
Thus there may be differences regarding the anatomic
substrate over which the CPP is mediated in different
species.
In an attempt to delineate the pathways over which
the CPP is mediated in adult rats, Moreno et al. (92)
injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into functionally recovered hemidiaphragm muscle during the CPP. When
WGA-HRP is injected into muscle or a peripheral nerve
it is retrogradely transported back to the motoneuron
cell bodies and also across synapses to other neurons
within the synaptic chain (63, 64, 70, 108). Furthermore, the transynaptic transport is selectively mediated across physiologically active connections rather
than silent or less active connections (63, 64, 73). The
results indicated that the neurons that drive phrenic
motoneurons in spinal-hemisected rats during the CPP
are located bilaterally in the rostral ventral respiratory
group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted despite careful
analysis of the spinal cord from C1 to C8 (92). The only
spinal motoneurons labeled were in the phrenic nucleus ipsilateral to the hemisection and hemidiaphragm injection. Thus the results suggested that both
crossed and uncrossed bulbospinal axon pathways
from the rVRG collateralize to both left and right
phrenic nuclei and that propriospinal neurons do not
relay respiratory drive to phrenic motoneurons (92). A
diagram of the bulbospinal pathways involved in the
CPP is shown in Fig. 1. The arrows in the figure
illustrate the pathway taken by respiratory impulses
during the CPP.
Before the Moreno et al. (92) study, it was known
that respiratory rVRG axons descend via both crossed
and uncrossed pathways to bilaterally innervate the
phrenic nuclei (39, 40, 42) as depicted in Fig. 1. What
was novel about the Moreno et al. study was the suggestion of spinal decussating rVRG axon collaterals,
which also bilaterally innervate the phrenic nuclei (i.e.,
the “crossed phrenic pathways”). Subsequently, Goshgarian et al. (51) provided direct anatomic evidence for
the decussation of bulbospinal respiratory axons at the
level of the phrenic nuclei in adult rats that were
injected with the anterograde neuronal tracer, Phaseolus vulgaris leucoagglutinin, into the rVRG. Thus, at
least in the adult rat, respiratory impulses transmitted
across the midline of the spinal cord during the CPP
are most likely mediated by bulbospinal axons rather
than phrenic dendrites as Porter (107) initially suggested. However, Prakash et al. (109) showed that a
small proportion of spinal decussating phrenic dendrites also exist in adult rats; therefore, it is possible
that respiratory impulses cross the midline over both
anatomic substrates.
PLASTICITY ASSOCIATED WITH THE CPP
In most mammalian species, the CPP can be expressed within minutes after spinal hemisection by
contralateral phrenicotomy and is the result of an
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tion to the previously paralyzed hemidiaphragm. This
example of functional restitution was later termed the
“crossed phrenic phenomenon” (CPP; Ref. 113). Subsequently, several laboratories confirmed the presence of
the CPP in dogs (23, 79, 113), cats (23, 79, 111, 116),
rabbits (16, 111–113), rats (50, 102), guinea pigs (44,
59), and even woodchucks (113). There was disagreement, however, in the earlier studies (before 1951)
regarding the conditions and factors required to induce
the CPP. The disagreement may have been due in part
to the mechanical diaphragmatic recording techniques
used in the early studies, which were not sufficiently
sensitive to detect small movement and were inevitably affected by movements transmitted by the contralateral hemidiaphragm (79). The physiological basis
for the CPP was not elucidated until 56 years after
Porter’s (107) investigation. In 1951, Lewis and
Brookhart (79) used more sensitive diaphragm electromyographic recordings to show that the CPP was attenuated by artificial respiration and enhanced by conditions that resulted in an increase in central
respiratory discharge (e.g., a cold block of the phrenic
nerve contralateral to hemisection). The authors concluded “that the amount of crossed respiratory activity
is directly proportional to the intensity of the central
respiratory discharge and is entirely independent of
the state of conduction in the direct phrenic nerve. The
CPP occurs only when experimental conditions permit
an augmentation of central respiratory discharge to
take place as a secondary result of interruption of the
direct phrenic nerve” (79).
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INVITED REVIEW
increase in central respiratory discharge (79). Thus,
according to the definitions offered by Mitchell and
Johnson (90), the expression of the CPP actually does
not represent “plasticity” but rather “modulation” of
respiratory activity. Specifically, plasticity is defined
as “a change in future system performance based on
experience” and modulation is defined as “a neurochemically induced alteration in synaptic strength or
cellular properties, adjusting or even transforming
neural network function” (90). The term plasticity, as
used by these authors, implies a long-lasting change,
whereas modulation suggests a temporary change that
is reversed once the modulating stimulus is removed.
The fact that the expression of the CPP is an example
of modulation is exemplified by the work of Aserinsky
(1), who showed in cats that, when a local anesthetic
was applied to the contralateral phrenic nerve after
spinal hemisection, the CPP was induced, but, when
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Fig. 1. Surgical procedures and pathways involved in the crossed
phrenic phenomenon (CPP). Inspiratory drive to phrenic motoneurons is mediated by medullary neurons in the rostral division of the
medullary respiratory group (rVRG). These neurons project bilaterally to the phrenic nuclei. Moreno et al. (92) showed that both crossed
and uncrossed descending respiratory pathways have spinal decussating collaterals that project to both phrenic nuclei (i.e., the
crossed phrenic pathways). Hemisection rostral to the phrenic nucleus interrupts (dotted lines) the major bulbospinal drive to the
ipsilateral phrenic nucleus, which results in paralysis of the left
hemidiaphragm. Transection of the right phrenic nerve immediately
after hemisection paralyzes the right hemidiaphragm and induces
the CPP in most mammalian species. Arrows indicate the pathways
followed by respiratory impulses during the CPP to restore function
to the hemidiaphragm paralyzed by spinal cord injury. [Reprinted
from Ref. 14 with permission by Academic Press.]
the effects of the anesthetic block wore off, activity
returned to the blocked hemidiaphragm and crossed
phrenic activity in the hemidiaphragm ipsilateral to
hemisection was attenuated. The same results were
obtained when the experiment involving anesthetic
block of the contralateral phrenic nerve was repeated
in rats (50). Goshgarian (50) extended Aserinsky’s finding by carrying out an additional study that showed
that, even when the inducing stimulus is prolonged for
several weeks by crushing the contralateral phrenic
nerve, crossed phrenic activity disappears after the
crushed phrenic axons regenerate and reestablish
function in the denervated hemidiaphragm (50). Subsequent transection of the axons in the regenerated
phrenic nerve once again induced the CPP (50).
There are aspects of the CPP, however, that do
involve plasticity-related changes in the respiratory
pathways. Although the CPP was demonstrated in
several mammalian species (16, 23, 79, 113), it was not
readily elicited in guinea pigs (59, 113). When spinal
hemisection was followed within minutes by contralateral phrenicotomy, both sides of the diaphragm remained paralyzed and these animals always died of
asphyxia. Guth (59) found that the CPP could be induced in guinea pigs if an interoperative interval of
several months elapsed between the spinal hemisection and contralateral phrenicotomy; subsequently,
Goshgarian and Guth (53) showed that the response
could be induced if the interoperative interval was as
short as 3.5 h. It was concluded that plasticity-related
changes occurred within hours after spinal cord injury
that converted “functionally ineffective” synapses in
the crossed phrenic pathway to “functionally latent”
connections, named so because they do not restore
hemidiaphragm activity under normal conditions. The
“functionally latent” synapses, however, become “functionally effective” after contralateral phrenicotomy
(53). Thus specific plasticity-related alterations occurred in the respiratory neural circuitry within hours
after spinal cord injury that changed the system response to the modulatory stimulus (i.e., asphyxia).
Functionally ineffective synapses were also demonstrated in the respiratory pathway of young (5–9 wk
old) Osborn Mendel female rats (49). The synapses
could be converted to functionally effective ones in
those animals provided that 1–7 days elapsed between
the spinal hemisection and contralateral phrenicotomy
(49). This early study employed diaphragm electromyographic recording techniques in spontaneously breathing animals to document a yes/no presence of the CPP.
Using a more sensitive quantitative electrophysiological recording technique from the phrenic nerve, O’Hara
and Goshgarian (102) showed in Sprague-Dawley female rats that the time course for the conversion of
functionally ineffective synapses was similar to that of
guinea pigs. Specifically, the study showed a statistically significant increase in crossed phrenic activity at
2 h posthemisection compared with a weak expression
of activity recorded at 30 min posthemisection. There
were further smaller increases at 4 and 6 h post-
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INVITED REVIEW
MORPHOLOGICAL PLASTICITY ASSOCIATED
WITH THE CONVERSION OF FUNCTIONALLY
INEFFECTIVE SYNAPSES
Although the above studies were the first to associate functionally ineffective synapses with the unmasking of a latent respiratory motor pathway that restores
function to muscle paralyzed by spinal cord injury,
functionally ineffective synapses had been demonstrated physiologically by other investigators on sensory neurons throughout the central nervous system
(CNS). This type of sensory connection had been demonstrated in spinal cord (6, 24–28, 117, 125), visual
system (9), somatosensory cortex (44, 126), thalamus
(34, 110), trigeminal complex (33), and dorsal column
nuclei (32, 88, 89). Although several physiological
mechanisms underlying the rapid conversion of functionally ineffective synapses to a functionally effective
state have been hypothesized (22, 29), the precise
mechanism is still unknown.
An attempt was made to determine whether a morphological basis for the conversion of functionally ineffective synapses in the crossed phrenic pathway could
be demonstrated at the light or electron microscopic
level. Because there was little information available in
the literature pertaining to the morphological organization of the phrenic nucleus in the rat when this work
was initiated (78), a series of studies were published
that detailed the anatomy of the phrenic nucleus at the
light microscopic level (54), including the neurons that
give rise to the accessory phrenic nerve (30) and the
dendritic organization of phrenic motoneurons (46). In
addition, a study of the origin and distribution of
phrenic primary afferent nerve fibers (56) and a detailed electron microscopic analysis of the ultrastructure and synaptic architecture of phrenic motoneurons
in the spinal cord of the adult rat (55) were completed.
J Appl Physiol • VOL
After completion of the studies of the normal anatomy of the rat phrenic nucleus, a detailed quantitative
morphometric analysis of the ultrastructure of the rat
phrenic nucleus was conducted in both normal animals
and in animals subjected to a C2 spinal cord hemisection ipsilateral to the analyzed nucleus (58). Phrenic
motoneurons were identified at the electron microscopic level by retrograde HRP labeling. On the basis of
the early guinea pig studies, which indicated that functionally ineffective synapses were converted by 3.5 h
posthemisection (53), the phrenic nucleus was analyzed in one group of rats at 4 h posthemisection. In
addition to 4 h, analyses were also made in separate
groups of rats at 1, 2, and 4 days after injury. Only the
morphological characteristics of the phrenic nucleus
pertinent to the conversion of functionally ineffective
synapses will be discussed here.
Briefly, HRP labeling of phrenic motoneurons in
noninjured rats revealed that the phrenic nucleus is a
column of cells at the C3 –C6 spinal levels (30, 58, 78).
Although there are dendrites that radiate out into the
spinal cord at almost right angles from the cell column
(46), the majority of dendrites run rostrocaudally in the
phrenic nucleus and surround phrenic motoneuron cell
bodies (58). These longitudinally oriented dendrites
are arranged in bundles that appear to be tightly
fasciculated at both light microscopic and low-power
electron microscopic levels. High-power electron microscopy of transverse sections through the phrenic
nucleus, however, revealed that the longitudinally oriented dendrites lying in the phrenic neuropil adjacent
to HRP-labeled cell bodies are not as tightly fasciculated as that suggested by the low-power observations.
In fact, the membranes of adjacent dendrites are seldom in direct apposition. Intervening between adjacent
dendrites are thin astroglial processes that separate
the dendrites and isolate them from one another in the
neuropil (Fig. 2A). Occasionally, direct dendrodendritic
membrane apposition in the phrenic nucleus occurs,
but the frequency of these appositions is low before
spinal cord hemisection (58).
The other morphological feature pertinent to the
conversion of functionally ineffective synapses relates
to the synaptic architecture in the phrenic nucleus
before spinal cord hemisection. Most of the axon terminals in the normal phrenic nucleus form single synapses with their postsynaptic targets. That is, each
terminal establishes one or more synaptic active zones
(the actual site of vesicle accumulation, membrane
densities, and transmitter release), with only one
postsynaptic profile in the same plane of section (Fig.
2A). Occasionally, a double synapse, i.e., a single terminal forming synaptic active zones with two postsynaptic profiles in the same plane of section, was found in
the normal phrenic nucleus (Fig. 2A).
MORPHOLOGICAL CHANGES IN THE PHRENIC
NUCLEUS 4 H POSTHEMISECTION
One of the first observations made within the
phrenic nucleus was a statistically significant increase
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hemisection, but thereafter the same activity level persisted at 12 and 24 h posthemisection (102). Interestingly, it was discovered that, in older adult Osborn
Mendel rats (6 mo old), the CPP could be induced
without any delay between the spinal hemisection and
the contralateral phrenicotomy (49). This suggested in
this case that the functional conversion of ineffective
synapses also occurred normally (i.e., without injury to
the spinal cord) as the animals aged from young maturity to older maturity. Once again, with the use of
more sensitive quantitative recording techniques from
the phrenic nerve, the experiment was repeated on
young (9–10 wk old) and older (9–10 mo old) female
Spraque-Dawley rats (132). In both groups, the CPP
was induced 30 min after spinal hemisection. The results indicated that there was almost a fourfold enhancement of crossed phrenic nerve activity generated
in older rats compared with young rats. On the basis of
this observation, it was concluded that as rats age from
young maturity to older maturity, there is a gradual
reorganization of the respiratory neural circuitry that
enables older animals to more strongly express the
CPP (132).
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in the number of double synapses occurring by 4 h
posthemisection (Fig. 2B). The double synapses consisted of clusters of spherical vesicles associated with
asymmetrical synaptic membrane densities. Synapses
with this characteristic morphology have been described as excitatory rather than inhibitory (105, 124).
The morphological features of the double synapse did
not change at later posthemisection survival periods.
Another morphological change noted in the phrenic
nucleus at 4 h posthemisection was a statistically significant increase in the length of phrenic dendroden-
Fig. 2. A series of schematic drawings illustrating the ultrastructural features of the normal rat phrenic nucleus (A) and the main
changes of the phrenic neuropil that occur 4 h (B) and 4 days (C) after
ipsilateral spinal cord hemisection at C2. A: normally, the longitudinally oriented phrenic dendrites (D1 –D6, shown here in cross section)
are not tightly fasciculated and for the most part are isolated from
each other by astroglial processes (dotted areas). Occasionally, short
dendrodendritic direct membrane appositions (e.g., between D1 and
D2 and between D5 and D6) with punctum adhaerens (open arrow,
top left) are seen. The majority of the synaptic terminals in the
normal phrenic nucleus (T1 –T4) form single synapses with postsynaptic dendritic profiles, but an occasional double synapse (between
T5 and D5, D6) is also observed. N, here and in the other drawings,
represents part of the nucleus of a phrenic motoneuron. B: by 4 h
after hemisection, the mean length of previously existing dendrodendritic appositions increases (i.e., between D1 and D2 and D5 and D6).
Such an increase in mean length of the appositions may result from
active retraction of glial processes (exemplified by the direction
indicated by the solid arrows). In addition, the number of double
synapses increases significantly over normal levels by this time (e.g.,
between T1 and D1, D2 and between T4 and D5, D6). C: at 4 days
posthemisection, the mean length of a dendrodendritic apposition
reverts back to normal levels, but the percentage of appositions
increases significantly over normal values. In addition, a slight
decrease in the number of double synapses is accompanied by a
corresponding increase in the number of triple and quadruple synapses (e.g., between T5 and D2, D3, D5, and D6). At this time, some
degenerated terminals (DT representing T2 in the previous drawings) were incorporated into glial processes. [Reprinted from Ref. 58
with permission by Alan R. Liss.]
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dritic membrane appositions (Fig. 2B). Although qualitative observations suggested that there may be a
higher number of appositions in the phrenic neuropil
at 4 h posthemisection, quantitative analysis revealed
only a trend toward increasing numbers at 4 h. There
was a statistically significant increase in the number of
dendrodendritic appositions, however, by 2 days posthemisection (58). It was suggested that the increases
in length and numbers of dendrodendritic appositions
were due to the active retraction of astroglial processes
normally intervening between adjacent dendrites in
the phrenic nucleus (58).
The morphological changes seen in the phrenic nucleus at 4 h posthemisection were also observed at later
posthemisection intervals. Thus the rapid changes
seen in the phrenic nucleus within hours after spinal
hemisection are not transient changes but can be observed at 1, 2, and 4 days posthemisection. Certain
aspects of these changes, however, were modified at
some of the later intervals (see Ref. 58 for details).
Other changes, not observed at 4 h posthemisection,
were detected at the later intervals. Specifically, in
addition to double synapses, triple and quadruple synapses, defined as the synaptic active zones formed by a
single presynaptic terminal with three and four
postsynaptic profiles in the same plane of section, respectively, were also observed in the phrenic nucleus at
the later posthemisection intervals (Fig. 2C). For quantitative purposes, double, triple, and quadruple synapses were combined into a single “multiple” synapse
category (58).
Finally, in addition to the above-described rapid
morphological changes, the well-known anterograde
degenerative changes of the injured spinal cord were
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ASSOCIATION OF PHRENIC NUCLEUS
ULTRASTRUCTURAL CHANGES TO THE CONVERSION
OF FUNCTIONALLY INEFFECTIVE SYNAPSES
The increase in synaptic active zone length may have
functional consequences, especially since other studies
show that neural injury induces plasticity that is manifest as a change in synaptic morphology. (17, 69).
Hillman and Chen (69) demonstrated change in the
size of postsynaptic densities following lesion of parallel fiber afferents to Purkinje cells in the cerebellum.
They found that the average length of postsynaptic
densities greatly increased when reduction of afferents
was between 50 and 70%. In their study, decreases in
the number of afferent connections onto Purkinje cells
caused the remaining sites to enlarge, thus suggesting
constancy in total contact area. The authors suggested
that the presynaptic area may enlarge to liberate sufficient transmitter to fully depolarize the neurons
without having to alter the threshold level. In a more
recent study, Chen and Hillman (17) demonstrated
that a reduction of cortical input to the neostriatum
generated a rapid and lasting increase in the average
contact area of synaptic sites on principal cells of the
J Appl Physiol • VOL
striatum. This study further supported conservation of
total synaptic contact area on target neurons, as demonstrated by the reciprocal relationship between synaptic size and number (17).
On the basis of the above, it is possible that lengthening of active zones in the phrenic nucleus after spinal cord hemisection could enhance the effectiveness of
preexisting synapses, resulting in more stable and efficient connections. It is possible that lengthening of
the synaptic active zones after spinal cord hemisection
may play an important role in mediating the CPP and
thereby restoring function to the previously paralyzed
hemidiaphragm.
A significant increase in double and multiple synapses in the phrenic nucleus was not detected at 2 h
posthemisection (119), but an increase in these specialized synapses was detected by 4 h posthemisection (58). Thus, between 2 and 4 h after hemisection
are required for full differentiation of double and
multiple synapses. It was hypothesized that the ongoing process of synaptic active zone elongation in
terminals that remain in the phrenic nucleus after
spinal cord hemisection is causally related to the
documented increase in the number of double and
multiple synapses. Figure 3 illustrates this relationship. Briefly, there may be several synaptic active
zones contacting different postsynaptic targets in
rVRG axon terminals in the normal phrenic nucleus.
However, because the synaptic active zones are relatively short, a random section (Fig. 3, dotted lines)
through the terminal may reveal only one of the
active zones, thus identifying the terminal as a single synapse. After hemisection interrupts the main
ipsilateral bulbospinal pathways, synaptic active
zones in terminals of the crossed phrenic pathway
elongate. Thereafter, random sections are more
likely to reveal terminals with synaptic active zones
contacting more than one postsynaptic target (i.e., a
“multiple” synapse) because the zones are longer.
The above hypothesis was strengthened by two additional ultrastructural studies (52, 121). In the first
(52), phrenic motoneurons ipsilateral to a C2 spinal
hemisection were retrogradely labeled with HRP,
whereas respiratory bulbospinal terminals in the
phrenic nucleus were anterogradely labeled after the
injection of a mixture of WGA-HRP and HRP into the
rVRG. This study provided direct anatomic evidence
that bulbospinal respiratory neurons from the rVRG
provide a source of double synapse formation in the
phrenic nucleus after C2 hemisection. In the second,
ultrastructural study (121), terminals in the phrenic
nucleus were immunochemically stained for glutamate and GABA, the main excitatory and inhibitory
neurotransmitters projecting to the phrenic nucleus.
It has been shown that glutamate is the excitatory
amino acid that depolarizes phrenic motoneurons
during inspiration (84, 86), whereas the termination
of the inspiratory burst and the subsequent quiescent expiratory phase of respiration are mediated by
a disfacilitation of phrenic motoneurons and their
active inhibition by GABA (8, 41). The study indi-
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also observed. Beginning at 2 days posthemisection,
electron-dense degenerating myelinated axons and terminals were observed with frequency in the phrenic
nucleus (Fig. 2C).
Subsequent to the above quantitative morphometric
study of the phrenic nucleus (58) and the early physiological studies (49, 50, 53), O’Hara and Goshgarian
(102) used more sensitive quantitative recording methods from the phrenic nerve in rats and showed that
there was an initial weak expression of the CPP that
was significantly enhanced as early as 2 h posthemisection. Thus, in rats, the shortest time required for the
conversion of functionally ineffective synapses to a
more effective state was 2 h posthemisection. In light of
this finding, a second morphometric study was conducted to determine whether ultrastructural changes
could be detected in the rat phrenic nucleus as early as
2 h posthemisection (119).
The results of the second ultrastructural study indicated that, by 2 h posthemisection, there was a statistically significant increase in the mean percentage of
phrenic dendrodendritic membrane appositions compared with normal controls. Thus astroglial processes
retract from their normal intervening position between
adjacent phrenic dendrites as early as 2 h after hemisection (119). Furthermore, although a significant
increase in the number of double synapses could not be
demonstrated, there was a documented significant increase in the length of the synaptic active zones in the
terminals of the phrenic nucleus at 2 h posthemisection
compared with controls (119). These observations led
to the development of a hypothesis that related the
morphological alterations observed in the phrenic nucleus within hours after spinal hemisection to the conversion of functionally ineffective synapses in the
crossed phrenic pathway.
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cated that there were fewer glutamate and GABAlabeled terminals in the phrenic nucleus 30 days
after C2 hemisection compared with controls (121).
However, among the terminals that remained, the
average number of active zones per terminal was
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Fig. 3. Diagram suggesting a role of synaptic active zone enlargement leading to a greater number of multiple synapses in the
phrenic nucleus neuropil. A and B represent a bundle of dendrites
in the phrenic neuropil and their contact with synaptic terminals
from rVRG neurons in the medulla. A: characteristics of the
synaptic morphology of a control (nonhemisected) animal. B: modifications occurring in synaptic active zones in animals subjected
to hemisection. The blue ovals correspond to the synaptic active
zones in rVRG terminals. The terminals are depicted at the end of
axons descending either ipsilaterally (the 2 axons projecting down
from the top of each panel) to the phrenic dendrites or contralaterally from axons of the crossed phrenic pathway (the 2 axons
projecting to the bundle from the left of each panel). Random
transverse sections through the dendrite bundle (dotted lines at
S1 and S2) are depicted diagrammatically to the right of each
panel. Before hemisection (A) the synaptic active zones (blue
ovals) in each terminal are relatively short. Thus random sections
through the dendrite reveal a predominance of single synapses (S1
and S2 in A). Within hours after hemisection (B), there is an
elongation of synaptic active zones in the crossed phrenic pathway. Because of the synaptic active zone enlargement, random
transverse sections through the dendrite bundle now reveal a
predominance of multiple synapses (S1 and S2 in B).
greater in the hemisection group than in the control
group, with the active zones in the glutamate terminals mainly accounting for the difference. Moreover,
the length of the active zones in the glutamate terminals was significantly longer in the hemisection
group compared with that in controls. Interestingly,
the length of the active zones in the GABA terminals
was also found to be longer in the hemisection group
compared with that in controls (121). These data
taken together support the notion that respiratory
bulbospinal synaptic input to the phrenic nucleus
lost by spinal cord injury is compensated for in part
by an increase in contact area of the remaining
synaptic input.
There may also be a functional significance to the
astroglial process retraction observed in the phrenic
nucleus after hemisection. It has been suggested
that glial process retraction from in between adjacent neurons in other CNS centers, such as the
supraoptic and paraventricular nuclei of the hypothalamus, would result in a rise in the extracellular
potassium concentration because the glial processes
would no longer be available to absorb the potassium
that is released from the physiologically active cells
(66–68). Elevated extracellular potassium would increase neuronal membrane excitability by partial
depolarization (66, 67). In addition, the increase in
neural membrane apposition resulting from glial retraction may create the development of electrical
field effects around groups of neurons. The result of
these field effects would be to synchronize the firing
of cells, especially if electrical excitability were already elevated by depolarizing extracellular ion concentrations (66).
Enhanced phrenic motoneuron excitability, augmentation of crossed phrenic presynaptic input, and
synchronization of phrenic motoneuron output are
all manifestations that would predispose to the conversion of functionally ineffective synapses in the
crossed phrenic pathway after spinal cord injury. It
is possible that functionally ineffective synaptic terminations from the crossed phrenic pathway may be
physiologically normal but quantitatively insufficient to fully depolarize phrenic motoneurons in spinal-hemisected young adult rats. Thus, when the
animals are subjected to contralateral phrenicotomy
within minutes after spinal cord hemisection, the
summated action potentials of the crossed phrenic
input may not be able to fire the cells. Within hours
after spinal cord injury, however, astroglial process
retraction and increases in phrenic membrane apposition could result in enhanced excitability of phrenic
motoneurons. The postsynaptic changes resulting in
enhanced phrenic motoneuron excitability coupled
with the rapid presynaptic augmentation of input
mediated by synaptic active zone elongation and the
associated double and multiple synapse formation
could be the mechanism enabling previously ineffective synapses of the crossed phrenic pathway to
activate the phrenic motoneurons (58, 119).
802
INVITED REVIEW
IS THE PLASTICITY ASSOCIATED WITH THE
CONVERSION OF FUNCTIONALLY INEFFECTIVE
SYNAPSES INJURY INDUCED OR ACTIVITY
DEPENDENT?
Fig. 4. Electromyogram (EMG) recordings
from the right (R) and left (L) hemidiaphragm of a control and experimental animal subjected to 4 h of cold block of the
bulbospinal respiratory drive at C2 on the
left. A: before cold block. B: 1 h after cold
block was initiated. C: 4 h after the initiation of cold block and 1 min after the cold
probe was removed from the surface of the
spinal cord. No major change in EMG activity is observed in the control animal. In
B, the right EMG signal contralateral to
cold block is enhanced, whereas the left
EMG response is absent. After the cold
probe was removed (C), activity in the
previously quiescent left hemidiaphragm
is enhanced, but the enhanced activity on
the right seen in B returns to baseline
levels (compare with A). (Reprinted from
Ref. 13 with permission by Academic
Press.)
J Appl Physiol • VOL
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Although very specific, rapidly occurring morphological alterations were documented in the phrenic nucleus after spinal cord hemisection, it was not clear
whether the observed plasticity was the result of the
complex multifactoral events associated with the physical trauma to the spinal cord (i.e., edema, ischemia,
local tissue hypoxia, and so forth) or whether it was the
result of the more specific effect of interruption of the
descending respiratory drive onto phrenic motoneurons. To address this issue, the descending bulbospinal
respiratory drive to the phrenic nucleus was blocked
for 4 h by cold application at the C2 level of the spinal
cord while avoiding major damage to the cord (13, 14).
Complete block of axon transmission of the respiratory
pathways running unilaterally in the ventral as well as
in the lateral funiculus was achieved by approximation
of a cold probe to the ventral surface of the spinal cord.
The spinal cord surface temperature was lowered to
7°C and was maintained by a cold recirculated alcohol
system. The efficacy of the reversible block was assessed by continuous bilateral electromyogram (EMG)
activity recorded from the hemidiaphragms ipsilateral
and contralateral to the cold application. Sham-operated control animals were employed to confirm that the
surgical exposure of the cord and/or the chronic placement of the probe and the administration of intravenous dopamine given to maintain stable blood pressure
did not affect respiration. An example of the results of
the reversible hemispinalization of the rat spinal cord
by a cooling device is shown in Fig. 4. No significant
change occurred in EMG hemidiaphragmatic activity
in any of the sham-operated control animals. The descending pathway from the rVRG to the phrenic nucleus was completely and continuously blocked for 4 h
in all the experimental animals as demonstrated by
abolition of the EMG hemidiaphragmatic signal ipsi-
lateral to cold block (Fig. 4). In all experimental animals, hemidiaphragmatic activity returned when the
cold block was removed, demonstrating the reversibility of the block and the fact that no significant damage
to the spinal cord occurred at the site of the cold-block
application (13). The recovered EMG activity was significantly greater than the preblock values (Fig. 4).
This suggested that persistent plasticity-related
changes occurred in the ipsilateral respiratory neural
circuitry within 4 h of cold block. Interestingly, EMG
activity contralateral to the block did not change significantly from preblock values after the block was
removed but was significantly enhanced during cold
block (Fig. 4). The temporary enhancement of activity
contralateral to cold block most likely reflects an increase in central respiratory discharge during cold
block as expected when one-half of the diaphragm
becomes paralyzed. In addition, there is an increase in
both burst frequency and burst duration in the right
hemidiaphragm during cold block, further suggesting
that cold block had altered respiratory drive (Fig. 4).
Immediately after the 4 h application of the probe at
the C2 level of the spinal cord in the experimental and
sham-operated animals, the phrenic nucleus ipsilateral to probe placement was prepared for quantitative
morphometric analysis using the same techniques employed in the C2 hemisection studies (58, 119). The
following morphological alterations were documented
in cold-block animals compared with controls: 1) a
significant increase in the number of multiple synapses, 2) a significant increase in the number of dendrodendritic appositions, and 3) a significant increase
in the length of symmetric and asymmetric synaptic
active zones. The above changes are similar to the
changes induced in the phrenic nucleus following C2
hemisection. It was concluded, therefore, that injury to
the spinal cord is not a requirement for the morphological plasticity thought to underlie the conversion of
functionally ineffective synapses in the crossed phrenic
pathway. Rather, the induced changes are activity
803
INVITED REVIEW
EFFECTS OF SEROTONIN ON THE PLASTICITY
ASSOCIATED WITH THE CONVERSION OF
FUNCTIONALLY INEFFECTIVE SYNAPSES
It is generally agreed that 5-hydroxytryptamine (5HT, serotonin) is a neuromodulator that has an excitatory effect on spinal motoneurons, including those in
the phrenic nucleus (5, 81, 91, 127, 128). However,
there have been studies that have shown that 5-HT
may have inhibitory effects on phrenic motoneurons
(31). Moreover, there is a disagreement regarding the
mechanism of 5-HT-enhanced excitability of spinal motoneurons. Some studies suggest that 5-HT directly
stimulates the spinal motoneurons by depolarizing
J Appl Physiol • VOL
membrane potential beyond threshold via a decrease in
potassium conductance (81, 123). Other studies have
shown that the application of 5-HT alone does not
evoke spinal motoneuron action potentials (128). Nevertheless, excitatory effects on spinal motoneurons mediated by glutamate or ventral/dorsal root stimulation
are augmented by iontophoretic application of 5-HT,
whereas systemic injection of the 5-HT antagonist,
metergoline, reduces the ability of 5-HT to enhance
glutamate-evoked motoneuron activity (127, 128).
These results suggest that 5-HT may act as a neuromodulator that decreases the threshold for glutamateevoked action potentials and thus enhances the effects
of excitatory inputs to motoneurons. Indeed, our
laboratory as well as others have demonstrated the
neuromodulatory effects of 5-HT on either revealing
subthreshold ineffective respiratory pathways or enhancing the expression of the CPP (82, 134–136). Because these studies focus more on the excitatory neuromodulatory role of 5-HT and do not directly implicate
5-HT in the plasticity of the respiratory pathways after
spinal cord injury, they will not be discussed here.
There are three other studies, however, that do suggest
two different mechanisms involving 5-HT directly with
the conversion of functionally ineffective synapses in
the crossed phrenic pathway (61, 62, 122). The first
(122) suggests that plasticity of 5-HT synaptic terminals after spinal cord injury may be part of the morphological substrate leading to the functional conversion of these specialized synapses in the respiratory
pathways, whereas the other two (61, 62) suggest that
normal levels of serotonin must be present in the
spinal cord to initiate the plasticity underlying the
conversion of ineffective synapses.
It was hypothesized that other neurotransmitters in
the phrenic nucleus such as 5-HT may help to compensate for the loss of glutamatergic and GABAergic terminals after spinal cord injury and thus contribute to
the conversion of functionally ineffective synapses in
the crossed phrenic pathway (122). The conversion of
these synapses then leads to the recovery of the hemidiaphragm paralyzed by an ipsilateral C2 hemisection.
The above hypothesis is particularly salient because
5-HT has been implicated in playing an important role
in the process of functional recovery of other motor and
sensory systems after spinal cord injury (65, 114, 133).
In light of the above, an electron microscopic morphometric immunochemical analysis of 5-HT-labeled
terminals in the phrenic nucleus was conducted before
and 30 days after an ipsilateral C2 spinal cord hemisection to document injury-induced synaptic architectural
changes involving this neurotransmitter (122). The
results indicated that, although the total number of all
types of terminals in the phrenic nucleus was significantly less 30 days after hemisection compared with
nonhemisected controls, the number of 5-HT-immunoreactive terminals in the hemisected group well exceeded that of the control group. Although 5-HT-immunoreactive terminals contained both symmetrical
(presumably inhibitory) and asymmetrical (presumably excitatory) synaptic membrane densities, the
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dependent and are likely caused by the interruption of
the descending bulbospinal respiratory drive to the
phrenic nucleus (14).
One additional experiment was conducted to verify
the above conclusion. Both hemisection and hemispinalization by cold block at the C2 spinal level cause not
only a loss of the ipsilateral descending respiratory
drive, (i.e., a “functional deafferentation” of the ipsilateral phrenic nucleus; Ref. 14) but also an increase in
the remaining contralateral descending respiratory
drive. The contralateral respiratory pathways connect
with phrenic motoneurons ipsilateral to cold block or
hemisection by decussating collateral axons that cross
the spinal cord midline below the hemisection/coldblock site. Thus the observed phrenic nucleus plasticity
after hemisection/cold block could be caused by a loss of
the ipsilateral descending drive, an enhancement of
the contralateral respiratory drive, or both. To differentiate between these two possible inducers of the
plasticity, both the glial and synaptic cytoarchitectures
of the phrenic nucleus were assessed morphometrically
in nonoperated rats exposed to 48 h of hypoxia in an
atmosphere chamber. The hypoxia exposure produces
an increased descending respiratory drive without
functional deafferentiation. The 48-h time interval was
based on the work of Hatton (66). He noted morphological alterations in the supraoptic and paraventricular
hypothalamic nuclei of noninjured rats 48 h after dehydration similar to those observed after C2 hemisection (58, 119). Phrenic nucleus morphometric analysis
revealed that there were alterations in the astroglial
cytoarchitecture similar to those induced by hemisection and cold block, but there were no alterations of the
synaptic cytoarchitecture (14, 57). From these results,
it was concluded that the enhancement of descending
respiratory drive by hypoxia will induce astroglial process retraction, but the retraction does not necessarily
lead to synaptic remodeling in the phrenic nucleus (57).
Additionally, it was concluded that no synaptic plasticity occurs in the phrenic nucleus without functional
deafferentation, despite an increase in descending respiratory drive. Therefore, functional deafferentation
or, in other words, a loss of ipsilateral descending
respiratory drive may be the primary inducer of
phrenic nucleus synaptic plasticity occurring after hemisection or cold block (14).
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J Appl Physiol • VOL
(74), substance P (131), and thyrotropin-releasing hormone (131), it is also possible that 5-HT may coexist
with glutamate in synaptic terminals terminating on
phrenic motoneurons. By employing a combination of
retrograde tracing and tricolor immunohistofluorescence techniques, Nicholas et al. (101) showed that
glutamate immunoreactivity is present in virtually all
substance P and 5-HT immunoreactive neurons in the
medulla that have projections to the spinal cord. In
addition, the triple immunoreactive terminals were
demonstrated in an area immediately adjacent to motoneurons in the spinal cord (101). Furthermore, Johnson (74) showed in intracellular recording studies from
5-HT neurons in microculture that 40% of the neurons
evoked excitatory glutamatergic potentials in themselves or in target neurons. Finally, in the study by Tai
et al. (122), a comparison of the percentage of 5-HTlabeled terminals (47% of control) with the percentage
of both glutamatergic and GABAergic terminals in the
phrenic nucleus (81% combined in the control group,
Ref. 121) suggests a major overlap between these two
populations. Thus it is feasible that 5-HT and glutamate may colocalize in the same terminal in the
phrenic nucleus.
Because previous physiological studies have shown
that the conversion of functionally ineffective synapses
in the crossed phrenic pathway is likely to occur within
2 h after C2 hemisection in rats (102), injury-induced
changes in the 5-HT innervation of the phrenic nucleus
would also have to occur within hours after injury
before the changes could be related directly to the
conversion of the synapses. In the quantitative morphometric study by Tai et al. (122), it was necessary to
employ a 30-day posthemisection interval to clearly
differentiate between the terminals of injured 5-HT
axons and the terminals of uninjured axons, which
presumably sprout across the midline of the spinal cord
(114) to innervate the deafferented phrenic motoneurons. A 30-day posthemisection interval was selected to
allow for the complete degeneration of the injured
terminals. However, as already suggested, the enhanced 5-HT innervation of phrenic motoneurons documented by the study of Tai et al. (122) at 30 days
posthemisection could be related to an enhanced release of glutamate from the surviving terminals of the
medullary respiratory centers (i.e., in the crossed
phrenic pathway). What needs to be stressed here is
that, based on the morphology of the terminals undergoing the most prominent alterations in the phrenic
nucleus within hours after C2 hemisection (58, 119), it
is likely that many of the synaptic terminals contain
5-HT. Specifically, Sperry and Goshgarian (119)
showed that the mean length of both asymmetrical and
symmetrical synaptic active zones in unlabeled terminals elongated significantly in the phrenic nucleus 2 h
after hemisection compared with control. The same
finding was made for labeled 5-HT terminals at 30 days
posthemisection (122). In the study by Sperry and
Goshgarian (119), however, because of the short posthemisection interval, it was unclear whether bulbospinal connections interrupted by hemisection or the
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5-HT terminals with asymmetrical synapses mainly
accounted for the difference between the two groups.
Similarly, the mean number of asymmetrical synaptic
active zones in all 5-HT terminals increased significantly 30 days after hemisection over the control level.
Furthermore, the mean length of both asymmetrical
and symmetrical synaptic active zones in 5-HT terminals was significantly longer in the hemisection group
compared with that in controls. Finally, the total number of 5-HT terminals with multiple synapses was
significantly greater at the 30-day postlesion survival
time than the value observed in control animals (122).
The above data indicate that serotonergic axons and
terminals possess a remarkable capacity for plasticity
after spinal cord injury that results within 30 days
after C2 hemisection in a greater than 100% replacement of the serotonergic projections lost by the cord
injury (122).
Spinal cord injury, however, is not the only stimulus
that can induce plasticity of serotonergic projections to
the phrenic nucleus. Cervical dorsal rhizotomy (CDR)
also enhances serotonergic innervation of phrenic motoneurons and serotonin-dependent long-term facilitation of respiratory motor output in rats (76). Kinkead
et al. (76) showed that the number of serotonin-immunoreactive terminals within 5 ␮m of labeled phrenic
somata and primary dendrites increased more than
twofold ⬃1 mo after CDR vs. that shown after sham
operation. Moreover, the authors showed that CDR
enhances serotonin-dependent long-term facilitation of
phrenic motor output following periods of intermittent
isocapnic hypoxia (76).
It has been shown that administration of 5-HT in
cultured sensory neurons or in excised pleural ganglion
cell clusters of Aplysia triggers the adenylate cyclase
cascade by acting on G-protein-coupled 5-HT receptors.
The activation of the receptors causes a significant
elevation of the second-messenger cAMP concentration
in the presynaptic terminal of the neuron (4). The
elevated level of cAMP in the terminals subsequently
activates protein kinase A, which phosphorylates and
closes potassium channels. The closing of potassium
channels blocks potassium influx into the presynaptic
terminals and thus elevates the extracellular potassium concentration, which increases the excitability of
the terminal (21, 77, 104, 120). The stimulated terminals would thus increase the amount of neurotransmitter released in the synaptic cleft and thereby enhance
the postsynaptic receptor response. It is possible that
the enhanced 5-HT innervation documented in the
phrenic nucleus after spinal hemisection (122) may be
related to an enhanced release of glutamate from surviving presynaptic terminals originating from neurons
in the medullary respiratory centers. If this were true,
this would explain in part the mechanism for the conversion of functionally ineffective synapses to effective
ones in the crossed phrenic pathway. This proposed
mechanism is even more likely if 5-HT were colocalized
with glutamate in the same terminal.
Because 5-HT has been shown to colocalize in the
CNS with several neurotransmitters, including GABA
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INVITED REVIEW
J Appl Physiol • VOL
hemisection is directly related to the attenuation of
the synaptic plasticity also noted in these animals. It
may be that the reduction of 5-HT in terminals of the
phrenic nucleus before hemisection is responsible for
the overall reduction in synaptic plasticity. Furthermore, it needs to be stressed that p-CPA also decreases norepinephrine and dopamine levels. Thus
the effects described above may not be due to the
depletion of 5-HT alone (61).
Correlative morphological/physiological studies indicated that, when the morphological substrate believed to underlie the conversion of functionally ineffective synapses was present in the phrenic
nucleus (i.e., within hours after C2 hemisection or
cold block), there was an augmentation of the expression of the CPP (13, 14, 58, 102, 119). Administration
of p-CPA before hemisection attenuated the morphological alterations in phrenic nucleus cytoarchitecture that typically occur within hours after hemisection as described above (61). A correlative
physiological study tested the effects of p-CPA administration on the expression of the CPP 4 h after
C2 hemisection in rats (62). p-CPA was administered
3 days before hemisection as in the morphological
study. Eight of eight saline-injected control rats expressed crossed phrenic nerve activity at 4 h posthemisection, whereas only four of eight rats in the
p-CPA-treated group expressed recovery in the
phrenic nerve ipsilateral to hemisection (62). Quantification of integrated phrenic nerve waveforms indicated that the mean amplitude of respiratory-related activity in the ipsilateral phrenic nerve was
significantly lower in p-CPA-treated rats than that
in controls. Moreover, saline controls demonstrated
significant increases in mean respiratory frequency
and mean amplitude of contralateral phrenic nerve
activity during asphyxia, compared with normocapnia. However, p-CPA-treated rats did not express
significant differences in either frequency or amplitude during asphyxia compared with normocapnia.
These results suggest that the depletion of normal
levels of 5-HT by p-CPA before hemisection attenuates the enhanced expression of phrenic nerve activity seen within hours after hemisection. Coupled
with the morphological data (61), the results further
suggest that the attenuation of crossed phrenic activity in p-CPA-treated rats may be due to the failure
of the hemisection to induce the plasticity in the
phrenic nucleus thought to underlie the enhanced
expression of activity. However, the fact that p-CPAtreated animals do not show the characteristic increase in frequency during asphyxia suggests that
5-HT depletion may also affect brain stem respiratory centers (62). Moreover, the absence of an increase in phrenic nerve amplitude in p-CPA-treated
rats during asphyxia may reflect either spinal or
supraspinal mechanisms. Another interpretation is
that p-CPA blunted chemoreception, which could, in
this case, have a peripheral (e.g., carotid body) component.
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remaining crossed bulbospinal connections were undergoing synaptic modification, an issue resolved by
the study of Tai et al. (122). Finally, it should be
stressed that in both studies the majority of the elongating synapses were asymmetrical and presumably
excitatory (119, 122).
In summary, elongation of synaptic active zones in
5-HT terminals after spinal cord hemisection may be a
form of functional compensation for spinal cord injuryinduced synaptic loss. As 5-HT is capable of reducing
the threshold for glutamate-evoked action potentials
on spinal motoneurons (127, 128), more neurotransmitter released by elongated 5-HT synaptic active
zones in the phrenic nucleus could significantly augment phrenic motoneuron excitability mediated by glutamate after injury.
The issue of the role of serotonin in the conversion
of functionally ineffective synapses of the crossed
phrenic pathway was approached in another way.
Although it was clear that 5-HT levels were significantly altered in time after spinal cord injury, it was
unclear whether normal levels of 5-HT must be
present before spinal cord injury to help initiate the
morphological plasticity thought to be related to the
conversion of functionally ineffective synapses. To
address this issue, the effects of para-chlorophenylalanine (p-CPA), a serotonin synthesis inhibitor, on
plasticity in the rat phrenic nucleus 4 h after C2
hemisection were investigated (61, 62). Hemisected
control rats (vehicle-injected) demonstrated typical
morphological changes in the ipsilateral phrenic nucleus, including: 1) an increased number and length
of synaptic active zones and 2) an increased length
and number of dendrodendritic membrane appositions. p-CPA treatment 3 days before hemisection
reduced 5-HT levels and resulted in an attenuation
of the above changes in the ipsilateral phrenic nucleus 4 h after hemisection compared with hemisected controls (61). In addition, p-CPA treatment
attenuated injury-induced alterations in immunohistochemical staining of glial fibrillary acid protein
(GFAP) in astrocytes, although Western blot analysis demonstrated that overall levels of GFAP did not
differ significantly between groups (61).
The change in GFAP immunohistochemical staining noted in hemisected control rats was related to
GFAP filaments undergoing depolymerization and
redistribution toward astroglial soma during hemisection-induced astroglial process retraction (61).
The extent to which GFAP is polymerized is related
to the phosphorylation state of GFAP filaments (71).
Several neurotransmitters are known to alter the
phosphorylation of GFAP (85, 130). Because p-CPA
treatment attenuates alterations in GFAP immunohistochemical staining after spinal cord injury, 5-HT
may influence GFAP phosphorylation through a receptor-mediated mechanism that alters the phosphorylation of GFAP (61). Thus astroglial process
retraction in p-CPA-treated animals is attenuated. It
is not known whether the attenuation of astroglial
process retraction in p-CPA-treated animals after
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OTHER EXAMPLES OF PLASTICITY IN THE
RESPIRATORY PATHWAYS FOLLOWING
SPINAL CORD INJURY
J Appl Physiol • VOL
BEHAVIORAL EFFECTS OF SPINAL CORD INJURY AND
CLINICAL RELEVANCE
Although this review is focused on functional restitution after cervical spinal cord injury mediated by
latent pathways in the respiratory system, it should be
pointed out that functional recovery of other motor
systems possibly mediated by pathways not unlike the
latent respiratory pathway may be common in mammals, including humans. For example, midthoracic
hemisection in rats results in locomotor impairment of
the ipsilateral hindlimb (43). However, spontaneous
recovery from impaired locomotor capability mediated
by descending contralateral pathways that cross the
spinal cord midline in the lumbosacral region occurs
within days to weeks (35, 36, 100). The studies of Little
and co-workers (83) confirmed that locomotor recovery
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The majority of the work involving the underlying
mechanisms associated with the conversion of functionally ineffective synapses in the crossed phrenic
pathway focused on events that occur rapidly within
hours after spinal cord injury. Control studies from
this early work indicated that there was no spontaneous recovery of the hemidiaphragm paralyzed by C2
hemisection out to 30 days posthemisection; therefore,
it was concluded that the hemidiaphragmatic paralysis
was permanent (50). More recently, it was discovered
that this is not the case (95). Female Sprague-Dawley
rats subjected to C2 spinal cord hemisection and allowed to survive for prolonged posthemisection periods
(4–16 wk) show spontaneous recovery of the paralyzed
hemidiaphragm, which occurs in a time-dependent
manner. Specifically, spontaneous recovery did not occur in any of the animals tested 4 wk after injury.
However, spontaneous recovery in the phrenic nerve
and hemidiaphragm ipsilateral to hemisection was evident at 6 wk postinjury. The number of animals showing recovery increased progressively with time. By 12
and 16 wk, all animals demonstrated spontaneous recovery (95). The amount of recovery was expressed
quantitatively in two ways: 1) as a percentage of activity in the contralateral phrenic nerve in the same
hemisected animal and 2) as a percentage of activity in
the homolateral phrenic nerve of noninjured animals.
The recovery amounted to 56.76 ⫾ 5.10% of the activity
in the contralateral phrenic nerve of hemisected rats or
28.75 ⫾ 3.19% of the activity in the homolateral nerve
of noninjured rats (95). Ongoing studies in the Goshgarian laboratory are designed to determine what the
underlying mechanisms of this newly discovered aspect of plasticity in the respiratory pathways may be.
Another very recent study has suggested a druginduced aspect of plasticity in the respiratory pathways that has heretofore not been described (93). Eldridge et al. (37, 38) first demonstrated in intact cats
that the systemic administration of the methylxanthine, theophylline, enhances respiratory drive by
blocking adenosine receptors. Subsequently, several
studies have been published that show that the systemic administration of theophylline into C2 spinalhemisected rats also increases central respiratory discharge, activates the crossed phrenic pathway, and
thus restores function to the paralyzed hemidiaphragm
(94, 96–99). The shortened phrenic burst duration and
increased burst amplitudes in the neurograms reported by Nantwi et al. (94) suggest that respiratory
drive was increased. The above studies, however, involve straightforward modulatory influences of this
drug on the respiratory system and do not involve
plasticity. Recently, however, an aspect of theophylline-induced plasticity in the respiratory pathways was
discovered (93) and will be briefly described here. Rats
subjected to a left C2 hemisection received chronic oral
theophylline administration (3 times/day) for 3, 7, 12,
or 30 days. Some rats were assessed for recovery of left
phrenic nerve activity at the end of the drug administration period, whereas others were weaned from drug
administration for an additional 7, 12, or 30 days
before assessment of respiratory recovery. After an
electrophysiological recording, theophylline serum
analyses were conducted and the spinal cord of each
animal was removed for in situ hybridization and immunochemistry to assess adenosine A1 receptor mRNA
levels in the phrenic nucleus.
The results indicated that chronic theophylline administration induced recovery in the phrenic nerve
ipsilateral to hemisection over a theophylline serum
range of 1.2–1.9 ␮g/ml (93). These data are in accord
with the previous studies involving theophylline-induced activation of the crossed phrenic pathway (94,
96, 97–99). Interestingly, theophylline-induced recovered activity persisted for as long as 30 days after the
animals were weaned from theophylline and serum
levels of the drug were virtually undetected. Previous
work indicated that theophylline mediates its effects in
the C2 spinal hemisection model by blocking adenosine
A1 receptors (99). There were no significant changes,
however, in adenosine A1 receptor mRNA expression
after chronic theophylline administration (93). Thus it
was concluded that the persistent recovery of respiratory activity that follows chronic theophylline administration and weaning from the drug is not related to
changes in adenosine A1 receptor mRNA expression
(93). This interesting aspect of drug-induced plasticity
in the respiratory pathways is in marked contrast to
the modulatory actions of asphyxia induced by placing
a topical anesthetic on the phrenic nerve contralateral
to hemisection (1, 50) or crushing the phrenic nerve
(50). Regardless of how long the modulatory effects
were induced (i.e., several hours or even weeks), when
the stimulus was removed, the CPP was attenuated (1,
50). However, weaning a rat from theophylline after as
little as only a 3-day exposure to the drug will not
attenuate activity transmitted over the crossed phrenic
pathway (93). The mechanism underlying this druginduced plasticity is unknown, but ongoing studies are
directed toward resolving this issue.
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CLOSING REMARKS
The respiratory system is endowed with a significant
capacity for compensation and motor recovery after
spinal cord injury. The majority of ventilator-dependent tetraplegies can be eventually weaned from ventilator support (3, 12), but the process is a slow one and
there is disagreement as to the changes occurring in
the patient undergoing the weaning. Because it is
possible that the spontaneous unmasking of initially
latent respiratory pathways may contribute to the
chronic recovery of respiratory function in spinal cordinjured humans, it may also be possible to use theophylline or another adenosine A1 receptor antagonist
early after injury to accelerate the recovery process
and thus eliminate the deleterious effects of chronic
ventilator support. At any rate, more work needs to be
conducted at both the basic science and clinical levels
to reveal the underlying mechanisms and to test the
feasibility of this approach.
The author gratefully acknowledges the work of his former students and postdoctoral fellows as well as collaborators who over the
past 25 years have contributed significantly to many of the studies
outlined in this review.
This work was supported by National Institute of Child Health
and Human Development Grants HD-35766 and HD-31550.
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