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The Journal of Neurophysiology Vol. 87 No. 5 May 2002, pp. 2398-2407
Copyright ©2002 by the American Physiological Society
Instituto de Neurociencias-Consejo Superior de Investigaciones Científicas, Universidad Miguel Hernández, San Juan de Alicante 03550, Spain
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cabanes, Carmen,
Mikel López
de Armentia,
Félix Viana, and
Carlos Belmonte.
Postnatal Changes in Membrane Properties of Mice Trigeminal
Ganglion Neurons.
J. Neurophysiol. 87: 2398-2407, 2002.
Intracellular recordings from neurons in the mouse
trigeminal ganglion (TG) in vitro were used to characterize changes in membrane properties that take place from early postnatal stages (P0-P7) to adulthood (>P21). All neonatal TG neurons had uniformly slow conduction velocities, whereas adult neurons could be separated according to their conduction velocity into A
and C neurons. Based
on the presence or absence of a marked inflection or hump in the
repolarization phase of the action potential (AP), neonatal neurons
were divided into S- (slow) and F-type (fast) neurons. Their passive
and subthreshold properties (resting membrane potential, input
resistance, membrane capacitance, and inward rectification) were nearly
identical, but they showed marked differences in AP amplitude, AP
overshoot, AP duration, rate of AP depolarization, rate of AP
repolarization, and afterhyperpolarization (AHP) duration. Adult TG
neurons also segregated into S- and F-type groups. Differences in their
mean AP amplitude, AP overshoot, AP duration, rate of AP
depolarization, rate of AP repolarization, and AHP duration were also
prominent. In addition, axons of 90% of F-type neurons and 60% of
S-type neurons became faster conducting in their central and peripheral
branch, suggestive of axonal myelination. The proportion of S- and
F-type neurons did not vary during postnatal development, suggesting
that these phenotypes were established early in development. Membrane
properties of both types of TG neurons evolved differently during
postnatal development. The nature of many of these changes was linked
to the process of myelination. Thus myelination was accompanied by a
decrease in AP duration, input resistance
(Rin), and increase in membrane
capacitance (C). These properties remained constant in unmyelinated
neurons (both F- and S-type). In adult TG, all F-type neurons with
inward rectification were also fast-conducting A, suggesting that
those F-type neurons showing inward rectification at birth will evolve
to F-type A neurons with age. The percentage of F-type neurons
showing inward rectification also increased with age. Both F- and
S-type neurons displayed changes in the sensitivity of the AP to
reductions in extracellular Ca2+ or substitution
with Co2+ during the process of maturation.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Neurons of dorsal root (DRG) and
cranial sensory ganglia in adult mammals constitute a heterogeneous
population in terms of transduction properties of their sensory endings
and the quality of the sensation evoked by their activation. They also
differ in a number of morphological and functional characteristics,
such as size (Lawson 1979
; Ramón-Cajal
1899), degree of myelination of the peripheral axon
(Harper and Lawson 1985b), immunocytochemical properties
of the soma (Alvarez et al. 1991; Dodd and
Jessell 1985; Lawson and Waddel 1991),
neuropeptide content (McCarthy and Lawson 1989;
Quartu et al. 1992), and passive and active membrane properties of the cell body (Gallego and Eyzaguirre
1978; Harper and Lawson 1985a; Koerber et
al. 1988; Liu and Simon 1994; López de Armentia et al. 2000; Ritter and Mendell
1992; Rose et al. 1986; Villière
and McLachlan 1996; Waddell and Lawson 1990).
Electrophysiological characteristics of primary sensory neurons are
determined by the expression of various types of ion channels in their
membrane (Gallego 1983; Roy and Narahashi
1992; Scroggs and Fox 1992; Scroggs et
al. 1994) and are presumably associated with the encoding
capacity and the transducing properties of the neuron's peripheral
nerve endings (Belmonte and Gallego 1983; Koerber
et al. 1988).
Some specific characteristics of adult primary sensory neurons, such as
somatic size and myelination, are different at the time of birth,
experiencing further development during the early postnatal period
(Coggeshall et al. 1994; Lawson 1979;
Peters and Muir 1959). This also seems to be the case
for some of the electrophysiological properties of the soma membrane
(Fedulova et al. 1991; Fitzgerald and Fulton
1992; Spitzer 1979). Fulton (1987) suggested that at the time of birth, when myelination
had not yet take place, prospective C fibers already exhibited the long-duration action potential (AP) and a prolonged
afterhyperpolarization (AHP) characteristic of mature C neurons. In
contrast, putative A neurons were more diverse in terms of their
membrane properties: some of them were distinct electrophysiologically
from C-fiber neurons, whereas others also displayed relatively
long-lasting APs and AHPs, although they showed differences with
putative C neurons in other active and passive membrane properties.
The changes taking place in electrophysiological properties of
mammalian primary sensory neurons during early postnatal maturation are
largely unknown. To our knowledge, studies in the trigeminal ganglion
have not been undertaken. This is the period when peripheral receptor
organs become innervated (Belford and Killackey 1980) and the modality of peripheral endings is being defined
(Fitzgerald 1987). It also corresponds with the period
of peripheral axonal myelination (Friede and Samorajski
1968). Knowledge on the development of their properties may
contribute to explain the mechanisms underlying the establishment of
functional heterogeneity of mature primary sensory neurons.
In the present work, passive and active membrane properties of neonatal
mice trigeminal ganglion (TG) neurons were analyzed and compared with
those of adult animals. We report marked changes in their membrane and
firing properties, some linked to the process of myelination.
Preliminary results have been reported in abstract form (Cabanes
et al. 1999).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In vitro trigeminal ganglion preparation
Mice (129/Sv × C57BL/6 strain) of different postnatal ages (P1-P50) were deeply anesthetized by an intraperitoneal injection of pentobarbitone sodium (90 mg/kg, Euta-Lender, Madrid) and perfused through the heart with cold, oxygenated physiological saline containing (in mM): 128 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 1 NaH2PO4, 16 NaHCO3, and 5 glucose. The animals were killed by decapitation, and their heads placed inside a glass dish filled with ice-cold physiological saline where the TG was dissected free of surrounding tissues with its central and maxillary roots attached. After removal, the TG was transferred to a chamber filled with ice-cold physiological saline, oxygenated with a mixture of 95% O2-5% CO2. After careful cleaning under a dissecting microscope, the TG was pinned to the silicone elastomer (Sylgard)-coated bottom of a small (200 µl volume) recording chamber. The TG was perfused continuously (5-7 ml/min) with oxygenated physiological saline at room temperature (20-25°C).
The ganglion was illuminated tangentially with a fine (1 mm 
) fiber
optic light source (F-O-Lite, WPI, Sarasota, FL) that produced a
Nomarski-like image of the ganglion surface, viewed through the optics
(×20 objective, 0.35 NA, and 19.9 mm working distance) of an
Optiphot-2 upright microscope (Nikon, Tokyo, Japan). Single cells were
impaled with a glass microelectrode. The cut ends of the central and
peripheral (maxillary) roots of the TG nerve were inserted into tight
suction electrodes for pulsed electrical stimulation, using a nerve
stimulator (model S48) coupled to a photoelectric stimulus isolation
unit that delivered constant current pulses (model PSIU6) both from
Grass-Telefactor (West Warwick, RI). To calculate central and
peripheral conduction velocities (CV), the distance between the tip of
the suction electrode attached to the central or maxillary root and the
microelectrode tip was measured, and this value was divided by the
latency of the antidromic and orthodromic AP, respectively (Fig.
1). Distances were estimated with the
help of an eyepiece micrometer scale in neonatal TG and with a
calibrated ruler in adult ganglia.
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Electrical recording
Cells were impaled with borosilicate glass microelectrodes (1.0 mm OD and 0.5 mm ID, Clark Electromedical Instruments) filled with 3 M
KCl. Electrodes resistance ranged between 80 and 110 M
in adult
ganglia and between 110 and 180 M in neonatal ganglia. The
higher-resistance electrodes in neonates were required to minimize
impalement injury. Recordings were obtained using an Axoclamp-1A
amplifier (Axon Instruments, Union City, CA). Voltage and current
records were stored on videotape and/or digitized (sampling rate 5-28
kHz) with a 1401 A-D converter (Cambridge Electronic Design, Cambridge,
UK) and stored in a PC. Data were accepted only if the evoked AP had an
amplitude of 
60 mV, measured from resting membrane potential to the
peak overshoot.
Parameters measured and data analysis
The following electrophysiological parameters were measured:
resting membrane potential (Vm), input
resistance (Rin), and membrane time
constant (
). Rin was calculated
from the slope of the peak current-voltage (I-V)
relationship, following injection of hyperpolarizing current pulses
(duration, 100-150 ms; Fig. 1, C and D). The
value of was obtained from the single exponential fit to the onset
phase of a small hyperpolarizing voltage response. The membrane
capacitance (C) was estimated using the relation: = Rin × C. A rectification index in the
voltage response to hyperpolarizing pulses was calculated according to
the relationship: [(Rin at peak) 
(Rin at steady
state)/(Rin at peak)]*100. Neurons showing a rectification index higher that 5% for current pulses that
reached a peak voltage between 100 and 120 mV were considered as
"rectifying" neurons. The following parameters of the AP were measured (Fig. 1, A and B): AP amplitude, AP
overshoot, AP duration at 50% amplitude, AP repolarization time at
90% of amplitude, AP maximum rate of rise (dV/dt
max), AP maximum rate of fall (dV/dt min),
amplitude of the afterhyperpolarization (AHP), and duration of the AHP
at 50% of maximal amplitude. The pattern of AP discharge to long
(100-150 ms) depolarizing current pulses was also analyzed (Fig.
1C). Neurons were tested at a current intensity value twice AP threshold.
Records were analyzed using a commercial software package (Cambridge Electronic Design) and custom built in-house software. Data are presented as means ± SE. Statistical comparisons were made using Student's t-test for means when the distribution was normal and Mann-Whitney Rank Sum Test (MWR) when the distribution was not normal. We used the Z test for comparison of proportions (Sigmastat 2.0; Jandel Scientific Software, Erkrath, Germany). One-way ANOVA was used to analyze possible differences between subpopulations of adult neurons.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intracellular recordings were obtained from 172 TG neurons of 33 mice. According to the age of the animal, neurons were separated in two groups: neonatal neurons (ages P0-P7; n = 37) and adult neurons (ages P21-P50; n = 135).
Functional types of neurons
NEONATAL NEURONS. The conduction velocity (CV) of neonatal TG neurons was low with a mean of 1.1 ± 0.14 m/s (range from 0.27 to 3 m/s, n = 18) in the peripheral axon and 1.2 ± 0.16 m/s in the central CV (n = 29), possibly reflecting a lack of myelination at this stage. The distributions of axonal CVs were unimodal (Fig. 2A). It should be pointed out that peripheral and central branches of excised TG were very short in neonatal animals (<3 mm and 2 mm, respectively), hampering a reliable estimate of the CV.
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; Yoshida and Matsuda
1979): neurons with a hump or inflection in the repolarizing
phase of the AP (S-type neurons, Fig.
3A) and neurons without a hump
in the repolarizing phase of the AP (F-type neurons, Fig.
3B). The differentiated records of the AP (Fig. 3,
A and B, bottom) were used to evidence the
presence or absence of the hump, which appeared as an additional negative component in the time course of the AP derivative. The chance
of encountering either type of AP was about equal in neonatal TG, 51%
(19/37) of the neurons were of the F-type, whereas 49% (18/37) were
S-type neurons.
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0.05)
than F neurons, but the estimates of average input resistance and
membrane capacitance were similar in the two groups. These data suggest
that size differences are not marked between both classes of neonatal
neurons. The rectification index was also similar (Table 2).
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ADULT NEURONS.
In contrast to neonatal TG neurons, peripheral and central CV values of
adult TG neurons showed a broader distribution (ranging from 0.24 to 8 m/s) that extended into the range typical of myelinated axons (Fig.
2B). For individual neurons, a good correlation
(R2 = 0.69) was found between their
central and peripheral CVs. The slope of this relationship was close to
0.53, the peripheral CV being always faster (data not shown). Neurons
with peripheral CV <1.4 m/s or central CV <1 m/s were classified as
unmyelinated (C neurons), whereas those with peripheral CV >1.4 m/s or
central CV >1 m/s were considered thin myelinated (A neurons).
Seventy percent (95/135) of adult TG neurons were classified as A
and 30% (40/135) as C neurons.
). Figure 4
summarizes graphically the changes in peripheral CV of F- and S-type
neurons during postnatal development. The mean peripheral CV of all
F-type neonatal neurons was 1.3 ± 0.24 ms (n = 10). During postnatal development most F-type neurons became faster
conducting. Thus 90% of adult F-type neurons had CVs in the A range
(mean: 4.6 ± 0.38 m/s, n = 21 peripheral CV and
3.5 ± 0.24 m/s, n = 31 central CV). The remaining 10% had CVs in the C range (1.2 ± 0.1 m/s, n = 2 peripheral CV and 0.6 ± 0.11, n = 4 central CV;
Fig. 7A). In contrast, adult S-type neurons belong in nearly
equal proportion to the A (58%, 48/83) or the C neuron (42%,
35/83) group (Fig. 6A).
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range. There was a weak negative
linear correlation between AP duration and axonal CV
(r2 = 0.23) when considering all
subtypes of cells but this correlation disappeared within each of the
individual subgroups of Fig. 5.
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Changes in electrophysiological properties with age
The percentage of S- and F-type neurons was compared between neonatal and adult mice. Neither the percentage of S-type neurons (49% in neonatal, 61% in adult) nor of F-type neurons (51% in neonatal, 39% in adult) changed significantly during postnatal development (P = 0.365, Z test), suggesting that these phenotypes are already established and invariant at the early postnatal period. However, some properties of the AP changed in S- and F-type neurons during development (see following text).
ACTION POTENTIAL AND PASSIVE MEMBRANE PROPERTIES.
S-type neurons.
Figure 6A shows the
changes taking place in central and peripheral CV of S-type neurons
during postnatal development. The mean peripheral CV of type S neonatal
neurons was 0.9 ± 0.15 m/s (n = 8). In
adults, this population segregated into two subgroups with different
CVs: a group in the A range (4.0 ± 0.4 m/s peripheral CV,
n = 23 and 2.5 ± 0.22 m/s central CV,
n = 34) and a group in the C range (0.5 ± 0.1 ms peripheral CV, n = 9 and 0.4 ± 0.05 central CV, n = 26). The mean CV of adult S-type
A neurons was significantly different from the mean CV of S neurons
in neonates (P < 0.001). In contrast, the mean CV
of adult S-type C neurons was not different (P = 0.065; Table 1).
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neurons.
Differences in AP duration between both groups were significant
(P < 0.05, Fig. 6B; Table 1). The
increased AP duration was due primarily to an increase in the
repolarization duration (Fig. 6B). The maximal rate of
AP depolarization also increased in A neurons, remaining constant in
C neurons. In contrast, the maximal rate of AP repolarization increased
in both groups, particularly in A cells (Fig. 6C;
Table 1). During postnatal development, the AHP in S-type neurons also
increased in duration. This change was highly significant in C neurons
(P < 0.001) and did not reach the significant
level in A neurons (P = 0.203; Fig.
6D; Table 1).
We also examined changes in passive electrical properties. As shown in
Table 2, changes were more pronounced in neurons that became
myelinated. Thus mean Rin in S neurons did
not change significantly in the subgroup of adult C neurons but was
markedly reduced in the A group (P < 0.001;
Table 2). Moreover, was slower in both, A and C. The mean
capacitance (C) was significantly higher in adult A neurons
(P < 0.05; Table 2) but did not change
significantly with age in C neurons.
F-type neurons.
Figure 7A shows the changes
taking place in central and peripheral CV of F-type neurons during
maturation. As already mentioned, almost all F-type increased their CV
postnatally. The changes in the AP and AHP of F-type neurons during
maturation are summarized in Fig. 7. The AP duration in the majority
(those in the A range) of F-type neurons decreased with age
(P < 0.001, Fig. 7B; Table 1). This
reduction was explained by a faster rate of AP depolarization and
repolarization (P < 0.001; Fig. 7C; Table
1). In contrast to S-type neurons, the AHP duration in F-type neurons
did not change significantly during postnatal development, particularly in A neurons, which represent a large majority of this population of
TG cells (Fig. 7D; Table 1).
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neurons but remained similar in adult C neurons (Table
2). However, the average and membrane capacitance of A F-type
neurons was higher (P < 0.05) than in neonatal F-type neurons.
INWARD RECTIFICATION.
About one-third (11/35) of neonatal neurons displayed time-dependent
inward rectification in response to the injection of a negative current
pulse. This rectification is presumably due to activation of the
Ih current (Mayer and Westbrook
1983; Scroggs et al. 1994). Inward rectification
was abolished by 1 mM extracellular Cs+ (data not
shown). The incidence of inward rectification among F- and S-type
neonatal neurons was similar (26% of F neurons, 37% of S neurons,
P = 0.714, Z test). The size of this
rectification, computed as a rectification index (see
METHODS), was also similar (22 ± 4.6% for F neurons,
29 ± 9.5% for S neurons, P = 0.898, Z
test; Table 2).
F-type neurons while none of the C F-type neurons had this property
(Table 2). In the case of S-type neurons, these percentages were 56%
for A neurons and only 18% for C neurons (P = 0.001, Z test).
DISCHARGE PATTERN.
TG neurons responded to the intracellular injection of a long (100-150
ms) depolarizing current pulse either with a single AP (phasic
response) or with a repetitive discharge of APs (tonic response).
Phasic responses were present in 75% (18/24) of neonatal neurons
tested, whereas the rest responded tonically. The proportion of
neonatal F- and S-type neurons exhibiting a tonic response was similar
(4/14 F neurons; 2/10 S neurons; P = 0.981, Z test). Tonic neurons were more excitable than phasic
neurons because they required lower current values to fire APs. In
tonic neurons, the rheobase values were 0.3 ± 0.18 nA
(n = 4, Rin = 194 ± 45 M) for F-type neurons and 0.17 ± 0.17 nA
(n = 2, Rin = 265 ± 195 M) for S-type neurons. In phasic neurons, the thresholds were
0.9 ± 0.58 nA (n = 10, Rin = 124 ± 27 M) and
0.9 ± 0.19 nA (n = 8, Rin = 170 ± 45 M) for F- and
S-type neurons, respectively (P < 0.05, t-test). These differences in threshold were very clearly linked to the discharge pattern and not to the shape of the action potential because mean threshold currents were not different
(P = 0.78) in F-type neurons (0.7 ± 0.15 nA,
n = 14) compared with S-type neurons (0.8 ± 0.12 nA, n = 10).
in guinea pig. Only 8% (5/63) of adult neurons tested fired
repetitively in response to long depolarizing pulses. All neurons
firing tonically were C neurons of the S type that lacked IR in
response to hyperpolarizing pulses. In marked contrast to the neonate,
all adult F-type neurons discharged phasically. As was the case for
neonatal neurons, threshold currents required to evoke an AP were lower
in tonic compared with phasic cells (0.4 ± 0.49 nA,
Rin = 206 ± 36 M,
n = 5 vs. 1.0 ± 0.63 nA,
Rin = 101 ± 10 M,
n = 67; P < 0.05). Although the
proportion of neurons discharging tonically was higher in neonatal than
in adult neurons (6/24 vs. 5/63, respectively) differences among both
groups did not reach the significance level (P = 0.074, Z test).
EFFECTS OF LOW CA2+ ON THE AP. During development, a marked change in the sensitivity of the AP to reductions in external Ca2+ concentrations ([Ca2+]e) and to application of Co2+ were noticed. These results are summarized in Fig. 8 for the different types of neurons. In neonatal TG neurons, somatic APs of both S- (n = 2) and F-type (n = 6) neurons were highly sensitive to reductions in [Ca2+]e. In all neurons tested (n = 8), the AP amplitude decreased significantly on reduction in [Ca2+]e (82 ± 1.7 mV in control vs. 40 ± 11.3 mV in 250 µM Ca2+; P < 0.05, t-test; Fig. 8, A and C). The sensitivity of the AP to low external Ca2+ appears to be restricted to the first postnatal week because no effect was observed in neurons tested at age P10 (n = 3, data not shown). In contrast to results in immature neurons, low [Ca2+]e had no effect on the AP (and AHP) of adult type-F neurons (n = 7; Fig. 8B). In adult S-type neurons (n = 3), the mean duration of the AP was shortened slightly (1.3 ± 0.4 vs. 1.0 ± 0.1 ms, P = 0.484) due to a reduction of the hump but the mean amplitude (97 ± 2.0 vs. 100 ± 2.2 mV, P = 0.400, MWR test) was also unaffected (Fig. 8D).
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(n = 3). In adult mice, the effects of
Co2+ substitution on the AP were different
depending on the type of neuron. In F-type neurons (n = 20, all A neurons), 2.5 mM Co2+ did not
produce changes in Vm, AP, or AHP
properties (Fig. 8F; AP amplitude: 80 ± 2.9 vs.
77 ± 4.7 mV in cobalt, P = 0.451; AP duration:
0.6 ± 0.1 vs. 0.7 ± 0.1, P = 0365, MWR
test). In S-type neurons (2 C and 21 A), the AP duration and the AHP
duration were significantly reduced with Co2+
(1.5 ± 0.2 vs. 1.1 ± 0.2 ms and 26.8 ± 8.8 vs.
7.1 ± 1.8 ms, respectively, P < 0.05), but the
AP amplitude was unaffected (94 ± 1.9 vs. 92 ± 2.9 mV; Fig.
8H). In these neurons, Rin
and were not modified by Co2+
(n = 15).
TTX SENSITIVITY OF THE AP.
APs evoked by nerve stimulation (peripheral or central branch) were
always abolished by bath application of TTX (100 nM) in neonatal
(n = 11) and adult (n = 12) TG neurons
(data not shown). Additionally, in 9 of 11 neonatal neurons (type S or
F), TTX blocked the AP evoked by depolarizing current pulses injected
into the soma (Fig. 9A). The
AP of the two remaining neurons was resistant to TTX. One of these
neurons exhibited tonic activity, which persisted in TTX (Fig.
9B). In adult TG neurons, soma APs were totally blocked by
bath application of TTX in 8 of 12 neurons, all of them A (4 F-type
and 4 S-type neurons; Fig. 9C). In three additional A
neurons (all F type), the somatic AP was blocked only partially (not
shown). Finally, in one neuron (C type), the AP was slightly reduced by
100 nM TTX (Fig. 9D).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This report describes in detail changes in the electrophysiological properties of cranial primary sensory neurons of mice during postnatal development. To prevent alterations in cellular phenotype that may take place in sensory neurons when maturation is studied under culture conditions, a preparation consisting of acutely excised TG of mice superfused in vitro was employed.
The results show that at the moment of birth, TG neurons were already
electrophysiologically diverse. During the first 3 wk of postnatal
development, further changes in conduction velocity and passive and
active membrane properties take place, leading to an heterogeneous
population of unmyelinated and myelinated primary sensory neurons
characteristic of adult animals (Djouhri et al. 1998).
Furthermore, some of the electrophysiological changes appear to be
strongly linked to the process of myelination because they
developed only in those neurons that increased their axonal conduction
velocity. The functional variety of sensory neurons at birth suggests
that the specification of some properties takes place early, before
myelination but after TG neurons have reached their peripheral targets.
Previous morphological studies have shown that, at birth, most sensory
axons are unmyelinated but become myelinated during the first three
postnatal weeks (Peters and Muir 1959). In agreement with this and with previous electrophysiological studies in maturing DRG neurons (Fitzgerald 1987; Fulton
1987), conduction velocity of TG neonatal neurons was low and
increased with age to reach maximum values of ~8 m/s in the adult
animal. In vivo investigations in adult cat pulpal afferents have
reported the presence of fast-conducting A
fibers (Cadden et
al. 1983). We did not find trigeminal fibers in this conduction
range. This may be caused by species differences or to an experimental
bias: due to the small size of the peripheral root in the mouse
trigeminal nerve in vitro preparation (~10 mm), fast conducting
fibers could be easily lost, buried in the stimulus artifact.
The existence of different functional types of primary sensory neurons
in newborn rodents was suggested by the morphological studies of
Lawson (1979) and Coggeshall et al.
(1994). They described two subpopulations of neonatal DRG
neurons in the mouse and rat with different diameter and growing rates:
small-dark cells that arrested their soma growth already at P10 and
larger-lighter cells that continued their development until P20.
Moreover, recordings from rat cutaneous primary afferents have shown
that at P0, primary sensory neurons already differ in the transduction
properties of their peripheral endings in spite of the absence of
differences in conduction velocity (Fitzgerald 1987).
The shape of the AP has been used to distinguish type S and F neurons
as two functional types of adult primary sensory neurons
(Belmonte and Gallego 1983; Gallego and
Eyzaguirre 1978; Koerber et al. 1988;
López de Armentia et al. 2000). In the present
work, S- and F-type neurons were found in the trigeminal ganglion at
birth in about equal numbers. This proportion did not vary during
postnatal development, indicating that the developmental mechanism
underlying AP shape specification are established early in development
and do not change appreciably throughout the maturation process.
However, the present work also shows that some membrane properties of F
and S neurons evolve differently postnatally, presumably due to a
variable expression of ion channels in the membrane (Fedulova et
al. 1991; Gallego and Eyzaguirre 1978).
A vast majority of F neurons in the adult trigeminal ganglion belong to
the A type, suggesting that most neonatal F neurons, which had
relatively slow conduction velocity values, became myelinated during
maturation. Their AP duration decreased during this process in parallel
to augmented rates of AP depolarization and repolarization perhaps due
to an enhanced expression of Na+ (Ogata
and Tatebayashi 1992) and K+ channels
(Seifert et al. 1999), respectively. Also, with
postnatal development Rin became
comparatively lower and membrane capacitance larger in F A neurons,
probably reflecting an increase in cell soma surface with maturation
(Lawson et al. 1974). In contrast, AHP amplitude and
duration was similar in neonatal and adult F neurons, which suggests
that in this type of neurons the ionic mechanisms supporting the AHP
were already established at the moment of birth. Less than 10% of
adult F neurons remained unmyelinated. These neurons seem to represent
a truly different population within F neurons and have been observed in
the guinea pig trigeminal ganglion also (Cabanes, unpublished data). In
adult animals, all F neurons showing inward rectification were
myelinated, which strongly suggests that F neurons displaying IR in
early stages of postnatal development will become F myelinated neurons
in the adulthood.
The present observations also indicate that the evolution of membrane
properties of S-type neurons with age is more heterogeneous than in F
neurons, and these differences were apparently associated with
myelination. Compared with neonatal neurons, mature S neurons conducting in the A range displayed a shorter duration AP
attributable to faster depolarization and repolarization rates. A
longer AHP was also noticeable in the group of adult S neurons. The
changes in AP could be associated to increased expression of
K+ currents during postnatal development in the
TG neurons that became myelinated. Also, the comparatively
reduced Rin together with the larger C
observed in adult S A neurons suggests that in the population of S
neurons that develop a myelin sheath, the soma membrane surface
increased during postnatal development as was the case for F A
neurons. Contrarily, S neurons that remained unmyelinated showed
similar Rin, C, and values with
age, suggesting that their cell body size stayed constant. In this
group of adult S neurons of the C type, the duration of the AP was
longer and with a more prominent hump than in neonatal cells. The AHP
was also more prolonged. Increases in AP duration have been reported in
DRG neurons of adult rats exposed to high nerve growth factor levels (Ritter and Mendell 1992). It could be speculated
that variations during development of AP duration in A and C neurons of the S type may be due to variable expression of trkA receptors in
each subpopulation of cells (Bennet et al. 1996;
Mu et al. 1993).
The marked changes in Ca2+ sensitivity of
TTX-sensitive APs in F- and S-type neurons during maturation suggests
that neither express their full complement of mature properties at
birth. At present, we are unable to assign a functional value to this
observation, but the new findings partially contradict the proposal of
Fitzgerald and Fulton (1992). According to these
authors, C fibers would not undergo any further postnatal maturation.
In unpublished experiments, we have found that changes in
Ca2+ sensitivity of the AP are regulated by the
expression of the neurokinin 1 receptor (Cabanes, unpublished results).
In our experiments, a high percentage of neonatal TG neurons had their
AP abolished by TTX. These data agree with those obtained by
Fedulova et al. (1991) in rat DRG, showing that at birth
most neurons already express TTX-sensitive sodium current. In contrast, others authors found TTX-resistant (TTXr) Na+
currents in ~40% of neonatal DRG neurons (Nobukuni and
Hideharu 1992), and Roy and Narahashi (1992)
reported that TTXr Na+ current expression was
strongly age dependent: it was 100% at ages <P5 and only 10% at ages
>P11. In this last study, the high percentage of TTXr
Na+ currents in young animals may be due to the
use of cultured neurons. In contrast, we and Fedulova et al.
(1991) performed experiments on acutely excised ganglia.
Altogether, the present data show that maturational processes in mouse primary sensory TG neurons were not completed at the moment of birth. The evolution with age of TG neurons electrophysiological properties indicates that myelination, occurring in most if not all F neurons and in a part of S neurons, tend to reduce AP duration in both types of cells. The acquisition of a myelin sheath was also accompanied by changes in those passive membrane properties associated with a parallel increase in the surface area of the soma. Changes in other membrane properties like AHP duration or discharge pattern did not appear to be directly correlated with the myelination process and may be linked to the development of a functional specialization of adult neurons. The variable stage of maturation of primary sensory neurons at birth should be taken into consideration when comparing results obtained in intact and cultured neonatal neurons with those of adult animals.
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ACKNOWLEDGMENTS |
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The authors thank E. Quintero and A. Vegara for excellent technical assistance. We are grateful to Drs. R. Gallego and E. de la Peña for critical comments on the manuscript.
This work was supported by Ministerio de Educación y Cultura and Fundación Navarro Trípodi.
Present address of M. López de Armentia: Div. of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia.
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FOOTNOTES |
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Address for reprint requests: C. Cabanes, Universidad Miguel Hernández, Instituto de Neurociencias-CSIC, Apartado 18, San Juan de Alicante 03550, Spain (E-mail: carmen.cabanes{at}umh.es).
Received 21 May 2001; accepted in final form 4 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Brain Res
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31-41, 1983[ISI][Medline].This article has been cited by other articles:
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 S. Yoshida and S. Matsumoto Effects of {alpha}-Dendrotoxin on K+ Currents and Action Potentials in Tetrodotoxin-Resistant Adult Rat Trigeminal Ganglion Neurons J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 437 - 445. [Abstract] [Full Text] [PDF]
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) and at steady state (
) of the same
neuron as in C. All examples in the figure correspond to
neonatal animals.
,
n = 29; mean conduction velocity (CV) = 1.2 ± 0.2 n/s] and peripheral
(
,
n = 18; mean CV = 1.1 ± 0.14 m/s) CVs in
neonatal mice TG. B: distribution of central
(
) clearly
different from neurons with AP <0.75 ms and CV >2 m/s (A
, the evolution of neurons conducting
in the C range; · · · , changes in neurons conducting in
the A
