Department of Physiology, Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2 Canada
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ABSTRACT |
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Martin-Caraballo, Miguel and John J. Greer. Electrophysiological properties of rat phrenic motoneurons during perinatal development. Past studies determined that there is a critical period at approximately embryonic day (E)17 during which phrenic motoneurons (PMNs) undergo a number of pivotal developmental events, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, contact with spinal afferent terminals, the completion of diaphragm innervation, and a major transformation of PMN morphology. The objective of this study was to test the hypothesis that there would be a marked maturation of motoneuron electrophysiological properties occurring in conjunction with these developmental processes. PMN properties were measured via whole cell patch recordings with a cervical slice-phrenic nerve preparation isolated from perinatal rats. From E16 to postnatal day 1, there was a considerable transformation in a number of motoneuron properties, including 1) 10-mV increase in the hyperpolarization of the resting membrane potential, 2) threefold reduction in the input resistance, 3) 12-mV increase in amplitude and 50% decrease duration of action potential, 4) major changes in the shapes of potassium- and calcium-mediated afterpotentials, 5) decline in the prominence of calcium-dependent rebound depolarizations, and 6) increases in rheobase current and steady-state firing rates. Electrical coupling among PMNs was detected in 15-25% of recordings at all ages studied. Collectively, these data and those from parallel studies of PMN-diaphragm ontogeny describe how a multitude of regulatory mechanisms operate in concert during the embryonic development of a single mammalian neuromuscular system.
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INTRODUCTION |
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Phrenic motoneurons (PMNs) and the diaphragmatic
musculature are the major components of the respiratory neuromuscular
system responsible for expanding the rib cage during inspiration.
Relative to other neuromuscular systems, the PMN and diaphragm
functional properties have to be in an advanced state of maturation by
birth to ensure viability of the newborn. In fact, the PMN-diaphragm system is operational before birth to generate fetal breathing movements in utero (Jansen and Chernick 1991). It is
known that the expansions of the rib cage associated with fetal
breathing movements in utero are essential for proper lung maturation
(Harding et al. 1993
; Kitterman 1996
).
There was further speculation that central respiratory drive influences
the maturation of the fetal respiratory neuromuscular system
(Jansen and Chernick 1991
). However, to critically test
this hypothesis, a fundamental understanding of respiratory
motoneuronal and muscle properties and their ontogenesis during the
perinatal period will be necessary. Toward this goal, the data outlined
in this paper represent the initial contribution toward examining the
correlation among PMN electrophysiological properties, the inception of
fetal respiratory drive, and the onset of continuous rhythmic breathing
at birth.
Previous studies using the perinatal rat model identified several key
stages of phrenic nerve-diaphragm development (gestational period is
21 days; Fig. 1A). Phrenic
axons emerge from the spinal cord at embryonic day (E) 11, migrate to
contact the primordial diaphragm musculature by E13, begin to form
intramuscular branches concomitantly with the initial formation of
diaphragm myotubes at E14, and branch within the full extent of the
developing diaphragm by E17-E18 (Allan and Greer
1997a). PMNs first receive descending inspiratory drive
transmission and synaptic contacts from spinal afferents at E17
(Allan and Greer 1997b
; Greer et al.
1992
). Interestingly, the time of target innervation and the
inception of functional recruitment coincide with the onset of a rapid
and profound morphological development of PMNs (Allan and Greer
1997b
). Thus, for the purpose of this study, we chose to
examine the electrophysiological properties of PMNs during the critical
period prior and subsequent to the major morphological reorganization,
the completion of target musculature innervation, the onset of afferent
and descending respiratory synaptic drive in utero, and continuous
rhythmic activation at birth. To test the hypothesis that there would
be a significant maturation of passive membrane properties, action
potential characteristics, and repetitive firing properties of PMNs
during this period, whole cell patch recordings of identified PMNs were
performed with a cervical slice-phrenic nerve preparation isolated
from perinatal rats ages E16, E18, and postnatal day (P) 0-1.
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Regarding the general issue of the ontogeny of motoneuron
electrophysiological properties, a number of experimental models was
used in the past, including cultured preparations of dissociated chick
motoneurons (McCobb et al. 1989, 1990
), rat spinal
explants (Xie and Ziskind-Conhaim 1995
), and acutely
isolated spinal cord segments (Di Pasquale et al. 1996
;
Fulton and Walton 1986
; Ziskind-Conhaim 1988
). Those studies provided valuable data demonstrating that, as motoneurons develop, there are typically changes in the action potential shape, firing patterns, and ionic conductances. This study of
PMN development complements the past work by providing two important
advantages. First, we are restricting our recordings to one class of
target-specific mammalian motoneurons rather than pooling data from
mixed populations within a given ventral horn region. We are analyzing
a population consisting of ~220 motoneurons that innervate one
muscle. In contrast, when examining the development of mixed
populations within cervical or lumbar ventral horns, one must contend
with thousands of functionally heterogeneous motoneurons that innervate
multiple muscles and develop at differing rates (Landmesser
1992
). Second, we are taking the perspective of examining
changes in PMN electrophysiological properties with respect to the
overall context of our past findings regarding phrenic nerve-diaphragm
development such as axon outgrowth, target innervation, functional
recruitment, and morphological changes. Information regarding these
aspects was not available in past studies of motoneuron development.
A preliminary account of this work appeared previously in abstracts
(Martin-Caraballo and Greer 1997a,b
).
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METHODS |
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Cervical slice-phrenic nerve preparation
Embryos (E16-E18) were delivered from timed-pregnant
Sprague-Dawley rats anesthetized with halothane (1.2-1.5% delivered
in 95% O2-5% CO2) and maintained at 37°C by
radiant heat, following procedures approved by the Animal Welfare
Committee at the University of Alberta. To determine the timing of
pregnancy, the day in which a morning test revealed the appearance of
sperm plugs was designated E0. Fetal age was confirmed by comparing the
crown-rump length of the embryos with previously published values by
Angulo y Gonzalez (1932). Newborn rats (P0-P1) were anesthetized by
inhalation of metofane. Embryos and newborns were decerebrated, and the
brain stem-spinal cord with the phrenic nerve attached was dissected in artificial cerebrospinal fluid (CSF) equilibrated with
95%O2-5% CO2 (pH 7.4; 27 ± 1°C). A
spinal segment was then cut from within the brain stem-spinal cord
preparation with a vibratome (Pelco; Redding, CA) into a single slice
containing the C4 segment (E18, P0-P1) or C3-C4 segment (E16) with
the phrenic nerve and the dorsal root ganglia attached (
750 µm
thick). The dorsal roots were then cut to prevent reflex-mediated
synaptic stimulation of PMNs in response to antidromic stimulation of
the phrenic nerve used for identification of PMNs. The spinal cord
slice was transferred to a Sylgard-coated recording chamber and pinned
down (at the dorsal border of the white matter) and continuously
perfused with oxygenated artificial CSF solution at pH 7.4 and 27 ± 1°C (perfusion rate 2 ml/s, volume of the chamber 1.5 ml). The
slice was left to equilibrate for at
1 h before recording, and data
were typically acquired for
5 h after slice preparation. Previous
measurements of O2 tension within the tissue of similar in
vitro preparations determined that neuronal populations are well
oxygenated under these experimental conditions (Brockhaus et al.
1993
; Jiang et al. 1991
).
Whole cell recording
Recording electrodes were fabricated from thin-wall borosilicate
glass (A-M Systems, Everett, WA). The pipette resistances were between
4 and 6 M. To decrease capacitance transients, pipette tips were
coated with Sigmacote (Sigma Chemical; St. Louis, MO), and the level of
fluid submerging the slice was minimized during recordings. The
electrode was advanced with a stepping motor (PMC 100, Newport; Irvine,
CA) into the PMN pool located in the medial zone of the ventral horn
close to the border between the white and gray matter. To avoid
clogging of the recording electrode, positive pressure (
30 mmHg) was
applied while entering the tissue to a depth of
100 µm from the
slice surface. Pressure was then removed while advancing within the PMN
pool. Once the pipette made contact with a cell, negative pressure was
used to form a gigaohm (>1 G
) cell-pipette seal, and gentle
suction was then applied to rupture the patch membrane. Seal formation
and membrane breakthrough were monitored by observing the response to
hyperpolarizing current steps (0.3 nA). Whole cell recordings were
initially established in the artificial CSF solution and performed with
an AxoClamp 2B amplifier (Axon Instruments; Foster City, CA). Liquid
junction potentials were corrected before seal formation with the
compensation circuitry of the patch-clamp amplifier. Data were filtered
at 30 kHz, digitized via an A/D interface, and analyzed with pClamp (Axon Instruments) and Origin (Northampton, MA) software.
Seal formation and whole cell recordings were carried out in
current-clamp mode. Once the whole cell configuration was established motoneurons were identified as belonging to the PMN pool by antidromic stimulation of the phrenic nerve via a suction electrode. Rectangular pulses of 0.5-ms duration and 0.5-Hz frequency were delivered with a
pulse generator (Master 8, AMPI; Jerusalem, Israel), and pulse
amplitude was manually controlled with a stimulus isolation unit
(Iso-Flex, AMPI). Stimulation amplitudes for the phrenic suction
electrode were 0.2 mA. To minimize current spread, the ground wire
for antidromic stimulation was wrapped around the tip of the suction
electrode. Antidromic action potentials were recorded on tape with a
videocassette recorder (Sony, Tokyo) or captured with pClamp software
for subsequent analysis. Neurons not responding to antidromic
stimulation were not analyzed. Presumptive glial cells, characterized
by having resting membrane potentials hyperpolarized beyond
70 mV and
incapable of firing action potentials in response to antidromic or
orthodromic stimulation, were also encountered but not analyzed.
Electrophysiological properties of PMNs were typically recorded at 60
mV holding membrane potential. However, the effects of differential
holding membrane potentials on spike and firing properties were
investigated by continuous injection of hyperpolarizing or depolarizing
current as required. Independent action potentials were evoked by
antidromic stimulation of the phrenic nerve or by intracellular
injection of depolarizing current in the form of a 0.5-ms rectangular
pulse. Repetitive firing properties were investigated after injections
of 1 s-long depolarizing pulses of varying amplitudes. A schematic
illustration of the slice preparation and electrode positioning is
shown in Fig. 1B.
Resting membrane potential was determined immediately after breaking
the seal between the membrane and the pipette. Action potential
duration was measured at half-maximal amplitude. Input resistance
(Rin) was determined from the voltage deflection
on injection of a series of 400 ms-long hyperpolarizing current pulses (0.05 nA). The membrane time constant (
) was calculated by fitting the membrane voltage response to injection of negative current. The
membrane response could be closely fit by a single exponential. Threshold potential (Vth) was determined as the
absolute membrane potential at the onset of an action potential. The
mean rheobase (Irh) was calculated as the
depolarizing voltage required to elicit an action potential
(Vth) divided by its input resistance, assuming an ohmic membrane (DiPasquale et al. 1996
). The duration
of the afterhyperpolarization (AHP) was measured from the falling phase of the action potential to the point in the AHP membrane potential trajectory that returned to the holding membrane potential. The AHP
amplitude was measured at the point of maximum voltage deflection relative to the holding membrane potential. Sag depolarization and
rebound excitation were examined after injection of hyperpolarizing currents of increasing amplitudes. All data values are presented as
means ± SE. Significant differences between values before and after a drug treatment within a given age were calculated by using paired Student's t-test, whereas differences between
various age groups were tested with analysis of variance (Origin).
Intracellular and extracellular solutions
Artificial CSF contained (in mM) 128 NaCl, 3 KCl, 0.5 NaHPO4, 1.5 CaCl2, 1 MgCl2, 23.5 NaHCO3, and 30 glucose (pH 7.4) when bubbling with 95%O2-5% CO2. In the calcium-free solution, calcium ions were replaced by an equimolar concentration of cadmium chloride, and NaHPO4 was removed to avoid precipitation. The sodium-free solution (HEPES-based buffer) contained (in mM) 139 choline chloride, 10 HEPES, 3 KCl, 0.5 NaHPO4, 1.5 CaCl2, 1 MgCl2, and 30 glucose (pH 7.4) with TrisOH and bubbled with 100%O2. The standard pipette solution contained (in mM) 130 potassium gluconate, 10 NaCl, 1 CaCl, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 10 HEPES, 5 Mg ATP, and 0.3 NaGTP (pH 7.3) with KOH. The composition of the pipette solution with a lower calcium buffer capacity was similar to that of the standard solution except for the following changes. BAPTA was decreased to 0.1 mM, calcium was not added, and potassium gluconate was increased to 155 mM.
The osmolarity of all the external solutions was kept between 320 and 325 mosm, and the osmolarity of the pipette solutions was ~315 mosm as measured with a freezing point osmometer (Advanced Instruments; Needham, MA).
Drugs
Stock solutions of drugs were prepared as ×100-1,000
concentrates. All drugs were added into the perfusate by switching to reservoirs containing the appropriate test solution. A waiting period
of 5 min was used to allow for equilibrium before data were
collected. TTX (Sigma; St. Louis, MO) and lidocaine N-ethyl bromide (QX 314) (RBI; Natick, MA) were used.
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RESULTS |
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Electrical properties of PMNs were determined from neurons meeting
the criteria of having a stable resting membrane potential more
negative than 45 mV and action potential amplitudes of
50 mV. There
are two points that should be taken into consideration when
interpreting the results obtained with the cervical slice preparation.
First, PMNs were recorded at a depth of 100-200 µm from the surface
of the slice, which meant that, although large portions of their
dendritic trees were intact, the distal rostrocaudally projecting
dendrites were typically severed. Second, the normal endogenous
synaptic drive received in vivo, which could influence PMN properties
and behavior, will obviously be absent in our experimental conditions.
Membrane properties
Passive properties of developing motoneurons, in particular the resting membrane potential, firing threshold, and input resistance, are important factors in determining electrical excitability in response to synaptic inputs. Thus the first component of our study was to characterize age-related changes in PMN passive properties. Results from the population data are summarized in Table 1.
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Between ages E16 and E18, approximately the time of the inception of inspiratory drive transmission, the resting membrane potentials became hyperpolarized by ~8 mV. Between E18 and birth there was a slight further hyperpolarization of ~2 mV in the resting membrane potential. Despite these changes in the resting membrane potential, the threshold for generating an action potential did not change substantially at any age studied. An ~32% reduction in the input resistance occurred between E16 and E18. A further ~48% decrement in input resistance occurred by birth. With no significant variation in threshold potential, the threefold increase in the mean rheobase from E16 to P0-P1 was most likely due to the reduction in the input resistance of PMNs. An analysis of the voltage responses to hyperpolarizing currents (Fig. 2) also revealed that the membrane time constants of PMNs were significantly reduced (by ~29%) during the transition from E16 to P0-P1 (Table 1).
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Subthreshold membrane responses to hyperpolarizing current injections
were studied to assess whether there were age-related changes in inward
rectification and rebound depolarization (Bayliss et al.
1994; Dekin and Getting 1987
). The voltage
deflections of membrane potentials after injections of prolonged
hyperpolarizing current pulses (400 ms) are shown in Fig.
2A. Plots of the voltage responses at the onset
(Vinitial) and end (Vsteady
state) of the current pulses resulted in superimposed linear
graphs (Fig. 2B), indicating the lack of sag depolarization
and inward rectification at all ages examined. The level of inward
rectification was also determined as the percentage ratio of
Vsteady state/Vinitial at
100 ± 10 mV membrane potential. The values obtained were
similar at all ages [E16, 99 ± 0.4% (n = 18);
E18, 99 ± 0.3% (n = 16); P0-P1, 100 ± 0.1% (n = 10)].
Rebound depolarizations after the completion of hyperpolarizing
current pulses were observed in a subpopulation of PMNs, with the
presence decreasing with age. Approximately 65% of E16 PMNs exhibited
rebound depolarizations (11/17); this value decreased to 45% in E18
(5/11) and to 12% in P0-P1 (3/26) PMNs. As the strength of the
hyperpolarizing pulse was increased, the depolarizing rebound potential
eventually reached threshold, triggering an action potential (Fig.
2A). Rebound depolarizations were not inhibited in the
presence of the intracellular sodium channel blocker QX 314 (1.5 mM)
(Connors and Prince 1982) or after blockade of potassium
channels with intracellular TEA and cesium ions (Fig.
3C). Incubation of the spinal
slice in a calcium-free solution eliminated the rebound depolarizations, thus indicating that a calcium-mediated conductance generates the rebound depolarizations (Fig. 3A). As also
shown in Fig. 3, activation of the rebound depolarization was dependent on the amplitude (B) and duration (C) of the
hyperpolarizing potential. Hyperpolarizations greater than
75 mV and
duration of
200 ms were required for the expression of rebound
depolarization. Further increases in the strength and duration of the
hyperpolarizing stimulation were followed by larger depolarizing
responses.
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Action potential characteristics
Previous studies demonstrated that age-related changes in action
potential parameters are often influenced by the holding membrane
potential (McCobb et al. 1990; Spigelman et al.
1992
). Thus age-dependent comparisons between PMNs were
standardized by holding the membrane potential at approximately
60 mV
at all ages, before investigations of the influence of depolarizing and hyperpolarizing holding potentials. From E16 to P0-P1, action potential amplitude increased by ~12 mV (Table 1). There was a
particularly striking decrease of ~34% in the action potential duration pre- and postinception of the inspiratory drive transmission between E16 and E18 (Table 1). From E18 through to P0-P1, the action
potential duration underwent a further ~27% decrease (Table 1).
The action potential spike was followed by afterpotentials of
various shapes, depending on age (Fig.
4). At E16 the action potential spike was
followed by a slowly decrementing afterdepolarization (ADP), with no
clear indication of an AHP. Through ages E18 to P0-P1, a hump-like ADP
and a medium-duration afterhyperpolarizing potential (mAHP) developed.
Further, a fast AHP (fAHP) separated the repolarizing component of the
spike from the following ADP in a subpopulation of P0-P1 PMNs (Fig.
4). The fAHP was expressed as an early-peaking, brief duration
potential, whereas the mAHP peaked later and had a more prolonged time
course (50 ms; Fig. 4, P0).
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A further analysis of the age-dependent changes in the expression of
the mAHP was performed as a result of potential concerns with the
standard pipette solutions used during our recordings. Previous results
from a variety of neuronal recordings demonstrated that the mAHP is
often due to the activation of a calcium-activated potassium
conductance (Viana et al. 1993b; Walton and
Fulton 1986
). Our data showing that incubation of the neonatal
spinal slice in calcium-free buffer (discussed in IONIC
CONDUCTANCES UNDERLYING COMPONENTS OF ACTION POTENTIAL) resulted
in the inhibition of the mAHP among neonatal PMNs supported this idea
(Fig. 6C). However, we thought it possible that a
significant component of the mAHP may have been blunted with our
standard pipette solution, which contained 10 mM of the calcium
chelator BAPTA (e.g., a small mAHP may have been present but obscured
in E16 PMNs). Thus we repeated our recordings with a pipette solution
with a lower calcium buffer capacity (reduced from 10 to 0.1 mM BAPTA;
Fig. 5). The exact intracellular calcium concentrations reached during
an action potential in the presence of the two levels of calcium
chelation cannot be determined without direct measurements of the
spatiotemporal distribution of calcium levels within the neuron during
the course of an action potential. However, by using a software package
designed to calculate the relative buffering capacities of whole cell
patch solutions (Sol I. D.; E. A. Erter, Univ. of Washington)
and taking into consideration what was estimated regarding calcium
influx during an action potential (Jassar et al. 1994
;
Lockery and Spitzer 1992
) we approximated the
intracellular calcium concentrations that would be reached with the two
solutions during an action potential. We estimated that in the presence
of 1- to 50-µM range of calcium influx the calcium concentrations
would range from 8.4 to 221.1 nM with the low BAPTA solutions compared
with 24.3-25.3 nM with the standard solution. Thus we were confident
that any calcium-mediated conductances would be amplified with the low BAPTA solution. However, even with the low BAPTA conditions, a mAHP was
still not evident in any of the E16 PMNs recorded from (n = 5; Fig. 5). A mAHP
was observed in 2 of 5 E18 PMNs in the modified low calcium-buffering
solution compared with 5 of 19 PMNs in the standard solution. The
amplitude (1.5 ± 0.4 vs. 2.5 ± 0.5 mV, P
0.05) and duration (69 ± 8 vs. 93 ± 18 ms,
P
0.05) of the mAHPs in E18 PMNs also differed with
the low intracellular calcium buffering. In P0-P1 PMNs, a clear mAHP
was observed in all 5 PMNs tested with the modified low
calcium-buffering solution compared with 8 of 30 PMNs in the standard
recording solution. The amplitude (1.9 ± 0.4 vs. 4.6 ± 0.4 mV, P
0.05) but not the duration (124 ± 20 vs.
117 ± 21 ms) of the mAHPs in P0-P1 PMNs differed under the
modified conditions. It should be noted that, at all ages studied, the
action potential amplitude tended to deteriorate (as much as 50%)
within ~30 min after establishing the whole cell configuration when
using the 0.1 mM BAPTA recording solution. Thus, although the low BAPTA
solution was useful for delineating the presence and amplifying the
effects of calcium-mediated conductances in the shaping of the action
potential, it was not suitable for routine long-term stable recordings.
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Figure 5 demonstrates that the amplitudes of the afterpotentials observed in PMNs at each age were dependent on the holding potential. In E16 PMNs, the slowly decrementing ADP was enhanced with hyperpolarizing holding potentials. In P0-P1 PMNs, the hump-like ADP was also enhanced with hyperpolarizing holding potentials and diminished by depolarizing potentials. In contrast, the mAHPs observed in E18 and P0-P1 PMNs were enhanced by depolarizing holding potentials. Thus the repetitive firing frequencies and patterns, which are modulated by the afterpotential characteristics of the individual action potentials, will be influenced by the resting membrane potential.
Ionic conductances underlying the components of the action potential
IONIC DEPENDENCE OF ACTION POTENTIAL CHARACTERISTICS.
A detailed analysis of the ionic conductances associated with
age-dependent changes of the action potential characteristics is
currently being analyzed with voltage-clamp techniques and is beyond
the scope of this study. However, we did examine the relative
contributions of sodium and calcium currents in the generation of
action potential characteristics (Spitzer and Baccaglini
1976; Ziskind-Conhaim 1988
). As early as E16,
the action potentials of PMNs were sodium dependent, as demonstrated by
blocking sodium channels externally with TTX (0.5-1 µM;
n = 5; Fig.
6A). Incubation of the spinal
slice in sodium-free buffer (n = 4) or addition of the
intracellular blocker of sodium channels QX314 (1.5 mM, n = 10) to the pipette solution was also effective in
preventing action potential generation in E16 PMNs (data not shown). A
calcium component contributed to prolonging the duration of the action potential of E16 PMNs, as indicated by the reduction of the spike duration after incubation in a calcium-free buffer (Fig.
6B). The action potential duration of E16 PMNs decreased by
30 ± 8% (n = 5) in calcium-free buffer. However,
in E18 PMNs, elimination of external calcium did not interfere
significantly with spike duration. In four of seven P0-P1 PMNs, there
was a slight increase of 15 ± 6% in spike duration after
incubation in calcium-free buffer (Fig. 6C), suggesting that
a calcium-dependent potassium current is involved in spike
repolarization in neonatal PMNs. This idea was further supported by the
fact that in P0-P1 PMNs the duration of the action potential was
significantly lower when recorded with a modified low-BAPTA-pipette
solution [2.0 ± 0.2 (n = 6) versus control
high-BAPTA solution 3.2 ± 0.2 ms (n = 26), P
0.05).
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IONIC DEPENDENCE OF AFTERDEPOLARIZING POTENTIALS.
Previous work indicated that ADPs observed in other motoneuronal
populations are calcium dependent (Walton and Fulton
1986; Viana et al. 1993a
). Thus we tested
whether this applies to both the slowly decrementing and the hump-like
ADPs observed in PMNs. As expected, both the slowly decrementing and
hump ADPs were reduced by bathing the slice in a calcium-free solution
(Fig. 6, B and C, respectively). We also
considered whether the ADPs could be in part because of passive
spreading of current through gap junctions. However, if this were the
case, the amplitude of the ADPs would not have been voltage dependent
(Walton and Navarrete 1991
). As illustrated in Fig. 5,
ADP amplitudes were enhanced at hyperpolarizing potentials and reduced
at depolarizing potentials.
Electrotonic coupling among PMNs
Our initial intent was to use antidromic stimulation of the
phrenic nerve to verify the identity of PMNs. However, rather unexpectantly, we noted that in a subpopulation of PMNs subthreshold antidromic stimulation of the phrenic nerve resulted in the generation of low-amplitude depolarizing potentials (Fig.
7A; observed in 3/22 E16, 5/19
E18, and 8/30 P0-P1 PMNs). This depolarizing potential had a very
short latency with respect to the onset of the antidromic action
potential, referred to as a short latency depolarization (SLD)
(Walton and Navarrete 1991). In accordance with previous work (Walton and Navarrete 1991
), the SLDs are
indicative of electrotonic coupling among PMNs because of the presence
of gap junctions. We classified SLDs as electrotonic if at least three
of the following criteria were met: 1) depolarizing
potentials had a short latency with respect to the antidromic action
potential, 2) an increase in stimulation strength evoked a
graded response of the SLD, 3) SLDs were resistant to
high-frequency stimulation (20 Hz), 4) SLDs were not
affected by collision of an orthodromic action potential (generated by
somatic current injection) with the antidromic spike, 5)
SLDs were insensitive to holding potential, and 6) SLDs were unaffected by removal of external calcium or blockage of calcium conductances. It should be noted that reflex-mediated synaptic activity
in the form of excitatory or inhibitory potentials (i.e., excitatory or
inhibitory postsynaptic potentials) was not observed during recording
of electrical activity of PMNs as the dorsal roots were cut. Further,
no clear evidence for the presence of Renshaw neurons, in the form of
high-frequency firing neurons, after antidromic stimulation was found
(Hilaire et al. 1986
).
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As shown in Fig. 7A, subthreshold stimulation of a P0 PMN evoked low-amplitude depolarizations that closely followed the onset of the antidromic action potential. In the particular PMN presented in Fig. 7A, subthreshold stimulation evoked two SLDs of increasing amplitude, indicating the presence of coupling among three PMNs. Removal of external calcium to prevent synaptic release did not diminish the graded SLD (Fig. 7B). Collision experiments were carried out to clearly visualize the SLDs after eliminating the contribution of the antidromic action potential during the invasion of the soma. When a soma-generated action potential was elicited before antidromic stimulation, an SLD (Fig. 7C, continuous line) was evoked where the antidromic action potential would be expected (Fig. 7C, discontinuous line). Further, high-frequency stimulation of the phrenic nerve did not reduce the expression of the SLD, indicating further that this depolarization is independent of synaptic activity (Fig. 7D). When the pipette solution contained the intracellular inhibitor of sodium channels QX 314 the antidromic action potential was eliminated as expected within a few minutes of membrane rupture. However, the SLD, which was insensitive to the holding membrane potential, persisted (Fig. 7E), suggesting that this potential does not involve synaptically mediated postsynaptic events or the activation of voltage-sensitive ionic conductances within the neuron being recorded from (i.e., SLD resulted from passive spread of current from coupled neuron).
The coupling among PMNs was also reflected in the relatively lower input impedance and higher mean rheobase measured in neurons where coupling was detected compared with those where it was not. For E18 PMNs the input impedances in PMNs with and without SLDs were 387 ± 108 (n = 5) versus 555 ± 61 (n = 16), and the mean rheobases were 76 ± 32 (n = 5) versus 42 ± 6 (n = 16). For P0-P1 PMNs the input impedances in PMNs with and without SLDs were 106 ± 14 (n = 7) versus 266 ± 33 (n = 26), and the mean rheobases were 259 ± 86 (n = 7) versus 89 ± 14 (n = 26). Only one of three E16 PMNs showing SLDs were recorded with an intracellular solution that did not contain QX-314, and thus insufficient numbers were available for a statistically meaningful comparison of passive properties among neurons with and without SLDs at that age.
Repetitive firing properties
Changes in the passive and action potential properties have a
critical effect on the firing pattern of differentiating neurons. Thus
we investigated age-dependent changes in the firing properties of PMNs
by injection of 1 s-long depolarizing pulses of increasing strength.
The population data illustrating age-dependent changes in firing
properties are listed in Table 2.
Representative data for PMNs of differing ages and firing
characteristics are shown in Figs. 8 and
9. The majority of PMNs at all ages was
able to generate sustained trains of action potentials with spike
frequency adaptation (SFA) (~72% of E16, 73% of E18, and 63% of
P0-P1 PMNs; Fig. 8, A-C). At ages E16 and E18,
however, ~28% of PMNs fired only one or a few action potentials
during the 1 s-long depolarizing pulse (not shown). It is possible that
these PMNs were damaged after electrode attachment or are among that
population of PMNs that undergoes apoptosis during this period
(Harris and McCaig 1984). However, these ideas are
contradicted by the fact that the motoneurons had healthy membrane
potentials, high-input impedances, and overshooting action potentials.
Thus these embryonic PMNs with minimal firing capabilities may simply
reflect a population with a relatively underdeveloped complement of
ionic conductances. In P0-P1 PMNs, although the majority of neurons
were able to generate continuous discharges with frequency adaptation,
two further populations of PMNs with different firing patterns were
observed. A second group of neonatal PMNs generated a burst of action
potentials at the beginning of the depolarizing pulse (20%; Fig.
8D). Bursting seemed to arise from an afterdepolarizing
potential that followed the first spike. Bursting firing was enhanced
by hyperpolarizing holding potentials (not shown). A third group of
P0-P1 PMNs (17%) with a very low input resistance (113 ± 6 M
) required strong depolarizations (
0.9 nA) to evoke firing (Fig.
8E). Of the seven PMNs within this group, SLDs indicative of
electrical coupling were detected in five neurons. Thus the
high-threshold and low-input impedance may be a direct result of
coupling (Getting 1974
; LoTurco and Kriegstein
1991
). Although coupling was also observed among embryonic
PMNs, the lack of a clear indication of a high-threshold population may
have been due to the fact that the differences among PMN passive
properties among those neurons with and without SLDs were not as
exaggerated as was the case at P0-P1.
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In E16 but not in older PMNs repetitive firing significantly altered the duration of the following spikes when compared with the first spike of the train (Fig. 8, A vs. C and E). Thus during repetitive firing in E16 PMNs action potentials became longer in duration, and their amplitude decreased over the first few intervals. Values for the duration of the first versus the last action potential in a 1 s-long train of spikes were as follows: E16 (6.4 ± 0.5 vs. 9.7 ± 1.3, n = 15, P < 0.05); E18 (4.0 ± 0.2 vs. 4.3 ± 0.3, n = 14); P0-P1 (3.7 ± 0.3 vs. 3.7 ± 0.4, n = 21).
As revealed by the firing threshold (Table 2) and frequency-current plots (Fig. 8F), embryonic PMNs required lower levels of depolarizing current than neonatal PMNs to generate repetitive firing. The significant decrease in the input resistance of PMNs from E16 to P0-P1 meant that there was an age-dependent increase in the strength of the depolarizing current required to generated repetitive firing. For example, P0-P1 PMNs required an approximately twofold increase in the threshold current to evoke repetitive firing compared with E16 and E18 PMNs (Table 2). Among neonatal PMNs, the high-threshold motoneurons (likely electrotonically coupled neurons) required the highest levels of current stimulation to elicit repetitive firing (Fig. 8F).
Embryonic PMNs also tended to have a more limited firing range compared with neonatal PMNs (Fig. 8F, Table 2). The firing frequency during the 1 s-long stimulation pulse increased steadily between E16 and P0-P1 at the minimal level of depolarizing current required for repetitive firing and at twice that level (Table 2). The initial firing rate, i.e., the inverse of first interspike interval (ISI) duration, also increased steadily from E16 to P0-P1 as a function of the injected current (Table 2). The slope of the f-I plot for the first ISI did not change significantly among the age groups studied, although the slope of the last ISI decreased because of the decline in the firing rate with time (Table 2). SFA (Figs. 8 and 9) and a marked increase in the amount of SFA as a function of firing frequency (Fig. 9) were observed at all ages studied.
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DISCUSSION |
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There are number of critical events associated with PMN
development that occur at E17, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, arrival of spinal afferent terminals within the PMN pool, and the
completion of intramuscular innervation of the diaphragm (Allan and Greer 1997a,b
; Greer et al. 1992
). During
the ensuing 3- to 4-day period there is also a major transformation of
PMN morphology (Allan and Greer 1997b
) and the
continuous rhythmic activation at birth. The current results
demonstrate that PMNs undergo pronounced changes in their passive and
active electrical properties in association with these developmental events.
Development of membrane properties
During the period of PMN development spanning from E16
to P0-P1, the resting membrane potential becomes more hyperpolarized without a significant change in threshold, whereas the input resistance and time constant decreased. Thus PMNs are electrically more excitable at the inception of inspiratory drive (E17) compared with more mature
states. Functionally, the increased propensity for firing will
compensate for a relatively weak descending inspiratory drive from the
medullary respiratory center and thus facilitate the production of
fetal breathing movements (Di Pasquale et al. 1996; Greer et al. 1992
). It was interesting to note that
there was an ~8 mV change in the resting membrane potential of PMN
spanning the 2-day period immediately before and postinception of
inspiratory drive transmission (E16-E18). Future studies reexamining
PMN electrophysiological properties at these ages when the descending
drive transmission was experimentally eliminated will be useful for
evaluating the relative importance of activity-dependent
transformations of PMN resting properties.
This trend for changes in PMN passive properties is similar to that
described during embryonic and postnatal development of spinal and
brain stem motoneurons (Fulton and Walton 1986;
Nunez-Abades et al. 1993
; Viana et al.
1994
; Xie and Ziskind-Conhaim 1995
; Ziskind-Conhaim 1988
) and hippocampal neurons
(Spigelman et al. 1992
). Further, a similar trend
continues postnatally in PMNs (Cameron et al. 1990
,
1991a
,b
). Several mechanisms could mediate the age-related
changes in passive properties of motoneurons. A maturation in the
relative permeability to sodium and potassium ions would account for
some of the hyperpolarizing of the resting membrane potential during
PMN differentiation (Spigelman et al. 1992
). Further,
the regulation of the sodium-dependent chloride cotransporter is
different in embryonic motoneurons, resulting in an increase in the
intracellular concentration of chloride ions and a subsequent raising
of the chloride equilibrium potential compared with more mature states
(Rohrbough and Spitzer 1996
; Wu et al.
1992
). This idea is supported for PMNs by the fact that application of the classical inhibitory neurotransmitter GABA induces a
depolarization of the neuronal membrane at E16 rather than a
hyperpolarization typically observed in mature neurons (Martin-Caraballo and Greer, unpublished observations).
The reduction in the time constant and input resistance with age seems
to reflect an increased density of ionic channels and cell size. An
increase in the density of ionic conductances, as previously observed
in developing chick motoneurons (McCobb et al. 1990),
was suggested by the reduction of the membrane time constant and thus
specific membrane resistance, (Rsp =
m/Csp, assuming no age-dependent
changes in the specific membrane capacitance of ~1
µF/cm2). Previous morphological studies demonstrated that
PMNs undergo a significant increase in cell size and elaboration of the
dendritic branching from E16 up to birth (Allan and Greer
1997b
).
Presence of electrical coupling among perinatal PMNs
We detected the presence of electrical coupling among
subpopulations of PMNs between ages E16 and P0-P1 (ranging from 14 to 26% of PMNs). The evidence to date suggests that at most two or three
cells are coupled via gap junctions. Previous findings from recordings
of neonatal (P0-P3) rat thoracic motoneurons indicated coupling in as
many 77% of lumbar motor neurons, with the mean number of cells per
coupling being ~4.5 (Walton and Navarrete 1991). It
may be that a similar degree of coupling is present in PMNs but at an
earlier stage of development than we studied (i.e., pre-E16), as there
is a clear rostrocaudal gradient of development within spinal neurons
(Nornes and Das 1974
). However, there are at least two
reasons the presence of gap junctions may have been underestimated in
our study. First, we only looked for SLDs that appeared subthreshold to
the antidromic currents necessary to generate an antidromic action
potential in the PMN being recorded from. Thus, in the event that the
PMN being recorded from was coupled to a neuron with a higher threshold
for antidromic activation (e.g., smaller-diameter axon), the coupling
would remain undetected. Second, one cannot be assured that all axons
within the nerve are stimulated by the suction electrode and/or conduct
the antidromic action potentials to the full extent of the PMN pool.
Despite these limitations for quantifying the degree of electrical
coupling, these are the first data demonstrating that there is in fact
coupling among PMNs. Neuronal coupling among rat PMNs is clearly not
present in the adult (Lipski 1984), and there is no
evidence to date for dye coupling among postnatal PMNs (>P1) (Cameron, personal communication). We propose that the
presence of neuronal coupling early in development and their removal
with maturation would be functionally appropriate. The CNS utilizes two
fundamental strategies for increasing the force produced by a muscle.
First, the firing frequency of a given motor unit can be increased.
Second, additional motor units can be recruited. In the case of fetal
PMNs, at the inception of fetal respiratory movements, our data
demonstrate that there are limits on the firing capabilities of PMNs.
However, the presence of neuronal coupling facilitates the second
strategy of increasing the number of motor units recruited for a given
descending synaptic drive. Thus, although the descending drive may be
relatively weak and the maximum discharge frequency of PMNs limited,
the presence of coupling among the neuronal population will ensure
adequate synchronous drive to the diaphragm for the purposes of
generating perinatal breathing movements. As the animal matures, the
situation changes to one where <30% of the PMN pool is recruited
during an inspiratory effort at rest (Cameron et al.
1991
; Torikai et al. 1996
). Therefore it would
be inappropriate and disadvantageous for neuronal coupling to persist
among the PMN pool at a time when precise, graded recruitment is desired.
Development of action potential characteristics and associated ionic conductances
By E16, PMNs are capable of generating overshooting action
potentials after intracellular injection of depolarizing current or
antidromic stimulation. As observed during the embryonic (Di Pasquale et al. 1996; Spitzer and Baccaglini
1976
) and postnatal (Fulton and Walton 1986
;
Viana et al. 1994
; Ziskind-Conhaim 1988
) maturation of other motoneurons, PMN action potentials increase in
amplitude and decrease in duration between E16 and P0-P1. As demonstrated by McCobb et al. (1990)
, the increase in action potential amplitude during motoneuron differentiation results from an increase in
the density of voltage-gated sodium channels. The maturation of
potassium conductances is important for spike repolarization and
voltage-dependent changes in action potential duration in other
neuronal systems (McCobb et al. 1990
; Spigelman
et al. 1992
). Although voltage-clamp experiments are required
to characterize the potassium conductances involved in shaping the
action potential and firing properties of PMNs, the current findings
suggest age-dependent changes in the expression of calcium-activated
potassium conductances. First, removal of extracellular calcium
contributes to prolonging the duration of action potential in neonatal
PMNs only, whereas the use of a pipette solution with low calcium
buffer capacity causes the oppositive effect on action potential
duration. This is consistent with the presence of a calcium-activated
potassium conductance (likely of the maxi-type), which is involved in
spike repolarization of neonatal motoneurons (Takahashi
1990
; Viana et al. 1993b
). Second, at E18, a
calcium-dependent conductance generating the AHP (or small-type
conductance) starts to develop and is fully expressed in the majority
of neonatal PMNs. This conductance is prominent in neonatal
motoneurons, where it plays a role in regulating repetitive firing
frequencies and modulating SFA (Viana et al. 1993b
;
Walton and Fulton 1986
).
We tested whether developing PMNs were capable of generating
calcium-dependent action potentials, as was reported for developing amphibian spinal neurons (Spitzer and Baccaglini 1976).
However, in PMNs, sodium ion influx is essential for action potential
generation at the earliest ages studied (E16) and throughout further
development. It remains to be determined whether calcium-mediated
action potentials occur in PMNs at earlier ages (
E15). Nevertheless,
whereas calcium spikes do not occur at E16, calcium ions do contribute
significantly to prolonging the action potential at this age. The
calcium-induced broadening of the action potential in PMNs is
diminished rapidly after the inception of inspiratory drive
transmission at E17 as no calcium component was seen to prolong the
action potential spike at E18. There was also an age-dependent decrease
in the presence of a calcium-dependent rebound depolarization, likely mediated by T-type conductances (Onimaru et al. 1996
;
Viana et al. 1993a
), among PMNs. Functionally, beyond
affecting firing properties, the influx of calcium ions is thought to
be important for promoting neurite growth in developing neurons
(Holliday and Spitzer 1990
; Komuro and Rakic
1996
; McCobb et al. 1989
). This would be
particularly important for PMNs during the period spanning E16-E19,
when they undergo rapid growth and reorganization of axons and
dendrites (Allan and Greer 1997b
). Further, calcium fluctuations are important for regulating the maturation of
voltage-gated ion channels during periods of electrophysiological
maturation (Gu and Spitzer 1995
).
Development of repetitive firing properties
The developmental changes of the passive properties and the ionic
conductances shaping action potentials were responsible for a major
change in the repetitive firing properties of PMNs. With the
age-dependent reduction in the duration of individual action
potentials, there was an increase in the overall repetitive firing
abilities of PMNs. Further, by P0, a second group of PMNs emerged that
fired bursts of action potentials at the onset of a depolarizing pulse
and may be related to early-recruited PMNs (Cameron et al.
1991b; Di Pasquale et al. 1996
). A third group of PMNs required significantly stronger depolarizations to be activated. This likely reflected a group of PMNs that remained electrotonically coupled rather than the emergence of PMNs
corresponding to high-threshold neurons observed at later stages of
development (Cameron et al. 1991b
; Di Pasquale et
al. 1996
). It will be interesting to examine the age-dependent
changes in diaphragm contraction properties to see how they correlate
with concomitant changes in PMN firing properties. Our preliminary
observations indicate that the half-decay time of a single muscle
twitch is two to three times longer at E18 compared with P0. Further,
the minimum frequency of nerve stimulation necessary to achieve tetanus
increases from a value of ~10 to 20 Hz from E18 to P0
(Martin-Caraballo and Greer, unpublished observations).
Thus, although the action potential durations are longer and the firing
frequencies are lower in embryonic PMNs compared with the neonate, the
diaphragmatic contractile properties develop along a similar trend,
allowing for tetanic contractions to occur at each developmental stage.
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ACKNOWLEDGMENTS |
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The authors thank Dr. W. E. Cameron for helpful comments. The free calcium concentration for the two solutions was estimated with software (Sol I. D.) written by E. A. Erter (Univ. of Washington, Seattle).
This work was funded by the Alberta Lung Association and the Medical Research Council of Canada. J. J. Greer is an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar and M. Martin-Caraballo was awarded an AHFMR Studentship.
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FOOTNOTES |
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Address for reprint requests: J. J. Greer, Dept. of Physiology, 513 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 July 1998; accepted in final form 6 November 1998.
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REFERENCES |
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